1. What is the Minimum Effective Dose of Folic Acid for Preventing NTDs? James L. Mills, M.D., M.S. Caroline Signore, M.D. NICHD

2. Minimum Effective Dose: Outline Why is this a difficult question to answer? How can we estimate how much folic acid is needed? Actual experience with fortification Conclusions

3. Minimum Effective Dose: Why is This a Difficult Question to Answer?

4. The Definitive Study Test reduced doses until NTD rates rise 400 µg  Protective 300 µg  Protective 200 µg  Protective 100 µg  Not Protective UNETHICAL

5. What do we Know About Effective Dose? Clinical trial doses—definitely work, but too high MRC, 1991:4 mg Czeizel, 1992:800 µg Case control study doses—presumed to work, but are lower doses effective? Most U.S. studies reported on 400 µg

6. Complication: Genetic Differences in Folate Metabolism MTHFR GenotypeMean RCF (µg/L) CC347 CT314 TT284* Molloy AM, et al., Lancet, 1997; *p<0.01

7. How Can We Estimate How Much Folic Acid is Needed?

8. Maternal Red Cell Folate vs. NTD Risk in Dublin Study Design Reviewed early pregnancy maternal RCF from nested case-control study of 84 cases NTD and 266 normal controls Logistic regression analysis to examine relationship between RCF and NTD risk Daly L, et al., JAMA, 1995

9. Maternal RCF vs. NTD Risk in Dublin Continuous dose- response relationship RCF < 150 ng/mL Risk  6.6/1000 RCF > 400 ng/mL Risk  0.8/1000 Daly L, et al., JAMA, 1995

10. Maternal RCF vs. NTD Risk in Dublin Curve continues down Risk  0.2/1000? Daly L, et al., JAMA, 1995

11. Maternal RCF vs. NTD Risk in Dublin Curve flattens out (genetic Mendelian defects) Risk  0.5/1000? Unable to confirm optimal RCF level, but 400 ng/mL is highly protective Daly L, et al., JAMA, 1995

12. How Much Folic Acid is Needed to Raise RCF to Protective Levels?

13. NICHD/Trinity College/Health Research Board Trial Study design Randomized, placebo-controlled trial 121 patients randomized to receive placebo, 100 µg, 200 µg, or 400 µg folic acid/d Compliance assessed by sign-in sheets over 6 mo. study period Daly S, et al., Lancet, 1997

14. Results GroupN Initial Median RCF µg/L Final Median RCF µg/L Median Change µg/L Placebo19335311-12 100 µg/d22309375*67 200 µg/d28311475*130 400 µg/d26350571*200 Daly S, et al. Lancet, 1997 * p < 0.001

15. RCF and NTD Risk Calculation Initial and post-treatment RCF values substituted into regression equation from L. Daly, et al. to derive estimated change in NTD risk Daly S, et al. Lancet, 1997

16. Results Treatment Group N Estimated Reduction in NTD Risk Placebo190 100 µg/d2222% 200 µg/d2841% 400 µg/d2647% Daly S, et al. Lancet, 1997

17. Minimum estimate (considering non-compliance): ~200 µg/d will decrease NTD rate by 41%

18. Literature Review and Calculation of Folic Acid Effect on Serum Folate Mathematical model based on trials of folic acid supplementation on plasma folate Calculated a dose-response equation Wald N, et al. Lancet, 2001.

19. Quantifying the Effect of Folic Acid Wald N, et al. Lancet, 2001.

20. Predicted plasma folate levels were far lower than actual levels in NHANES 1999-2000 The effects were inconsistent by age

21. Explanations Non-compliance would lead to an underestimation of effect Studies selected for the model had insufficient duration of exposure to reach stable serum folate levels, resulting in an underestimation of effect

22. Exposure Time Required to Reach Stable Folate Levels Larger doses of folic acid require a longer period to achieve steady serum or plasma concentration Quinlivan EP and Gregory JF, Am J Clin Nutr, 2003

23. Was Duration of Exposure Sufficient to Produce Stable Levels? Adequate Duration Inadequate Duration Mean Age 20-35 2 (33%)4 (67%) Mean Age 40-65 4 (50%) TOTAL 6 (43%)8 (57%)

24. Differences May be Due to Inadequate Time for Stabilization Wald N, et al. Lancet, 2001.

25. Studies used in Wald’s model led to an underestimation of folic acid effect on serum folate

26. What Are Current Exposure Levels?

27. Estimating Daily Folic Acid Intake After Fortification

28. How Has Fortification Affected Folate Levels?

29. Serum Folate Levels Pre- and Post-Fortification 4.6 4.8 12.6 5.4 8.2 6.1  171%  48%  117% 13.0 18.7 10.0

30. RCF Levels Pre- and Post-Fortification  65%  40% 104 94 160 233 264 327

31. Summary Fortification probably increases folic acid exposure by 200 µg/day or more Serum and red cell folate levels have risen dramatically (by 171% and 65%, respectively)

32. Actual Experience with Fortification

33. The Gold Standard How much has the additional 200 µg/day decreased NTD rates?

34. Estimates Vary Because of Ascertainment Problems Stevenson RE, et al., Pediatrics, 2000 Method of DetectionPercent Ultrasonography55% MSAFP25% AF AFP3% Delivery17%

35. Experience: Nova Scotia Population based study of all NTDs from live births, stillbirths, and terminations Compared pre- and post-fortification at 150 µg/100 g Total NTD incidence fell by 54% 2.58/1000  1.17/1000 Persad VL, et al., CMAJ, 2002

36. Estimated Effect Canada: fortification at 150 µg /100 g grain  50% reduction U.S. data with comparably ascertained cases are still not widely available

37. Conclusions

38. It is difficult to pinpoint the lowest effective dose of folic acid Data from Ireland indicate 200 µg/day will prevent at least 40% of NTDs

39. Conclusions Actual experience in Canada indicates estimated doses of 200+ µg/day will prevent 50% or more In the U.S., ~50% prevention may be the maximum, or ~70% prevention may be possible—have we “maxed out?”

40. Conclusions Given food fortification and supplement use in the U.S., many women will not need fortified OCs For women using supplements, fortified OCs might put them over the IOM’s recommended limit For other women, who do not take supplements and who eat less fortified food, fortified OCs could be beneficial