The results of a number of studies have led to the conclusion that periconceptional folic acid supplementation reduces the risk of NTDs [5–8]. Among the responses to this research evidence were calls in the early 1990s for mandatory fortification of food with folic acid. It was argued that this public health intervention would address concerns about achieving population level compliance with recommendations to women to take vitamin supplements containing folic acid before becoming pregnant and in the first weeks of pregnancy. These concerns were borne out in several Canadian studies suggesting that many caregivers [18, 19] and women [20, 21] remained unaware of the relationship between folic acid and NTDs. More recent studies have shown an increase in knowledge about folic acid, but supplementation rates remain low [22–25].
In March 1996 the US Food and Drug Administration (FDA) announced that it would permit addition of folic acid to enriched flour and other enriched cereal grain products, and that this addition would be mandatory as of January 1998. The level of fortification was set at 0.14 mg folic acid per 100 g of cereal grain product. It was determined that at this level of fortification, the intake of folate (from all sources) for the target and the general population would be kept below 1,000 μg/day, which was deemed to be the safe upper limit. This level of fortification was estimated to increase the average daily intake of folic acid in women of childbearing age by about 100 μg . Subsequent to the US decision, Canada followed suit, permitting folic acid fortification at an equivalent level in December 1996 (addition of folic acid to white flour and enriched pasta and cornmeal at 0.15 mg folic acid per 100 g of flour and 0.20 mg folic acid per 100 g of pasta). In Canada, fortification became mandatory in November 1998.
Rate of NTDs
Our results show a highly significant drop in the rate of NTDs in Newfoundland, taking into account all identified affected pregnancies (live births, stillbirths and pregnancies terminated after a prenatal diagnosis of an NTD). The 78% (95% CI 65%–86%) reduction in the NTD rate after implementation of fortification is greater than the 18%–22% reduction predicted at current levels of fortification [27, 28], and greater than the 19% reduction in birth prevalence of NTDs reported in the US after mandatory fortification . The results in Newfoundland are closer to the 54% reduction (95% CI 34%–68%) in rate of NTDs reported in Nova Scotia after fortification . De Wals et al. observed a 32% reduction (95% CI 23%–41%) in NTDs in Quebec between 1992–97 and 1998–2000 . Ray et al.  analyzed maternal serum screening data for Ontario and observed a decline in NTD prevalence from 1.13 per 1,000 pregnancies before fortification to 0.58 per 1,000 pregnancies thereafter (prevalence ratio 0.52, 95% CI 0.40–0.67).
The large reduction in the rate of NTDs in Newfoundland may be due, at least in part, to the fact that Newfoundland had higher background rates of NTDs. This population may be more sensitive to the influence of folic acid. In a large-scale public health campaign in northern and southern China, periconceptional use of 400 μg/day folic acid supplements was associated with a reduction in NTD risk of 79% for women in northern China, where the baseline NTD rate was high and similar to that observed in Newfoundland. A lower risk reduction of 41% was observed in the southern region where the pre-campaign NTD rate was much lower .
The 65% increase in the proportion of women taking vitamin supplements containing folic acid, from 17% in Phase I to 28% in Phase II, suggests that an increasing trend in folic acid supplementation may have played a role in the declining NTD rate in Newfoundland. In this study it was not possible to determine the individual contribution of supplementation and fortification to the trend in NTDs.
The annual rate of NTDs in the pre-fortification period (1994–97) did not differ significantly from that of the pre-supplementation period (1991–93); this is true whether 1997 is excluded or included in the pre-fortification period. The increase in the rates of NTDs in 1995 and 1996 appears random and largely unexplainable. The changes in the NTD rates between 1994–1996 and 1991–1993 and between 1995–1997 and 1991–1993 were not statistically significant. In addition, our data do not show an obvious increase in the proportion of NTDs in terminated pregnancies during 1994 and 1996 (data available upon request).
Dietary intakes and blood folate levels
The questionnaire used in this dietary assessment was a modified Willett questionnaire , administered in a face-to-face interview with trained personnel. The Willett food frequency dietary questionnaire has been well validated  and proved easy to administer for this sample population.
The daily intake of naturally occurring folate among women aged 19–44 years in this study (average 248 μg/day in the Phase II sample) was similar to values found in other studies of women's diet [34, 35]. For seniors in Phase II, naturally occurring folate in the diet averaged 290 μg/day which was comparable to values found for persons age 49 and older in an Australian study . The dietary folic acid intake due to fortification did not exceed the Tolerable Upper Intake Level (UL) of 1,000 μg folic acid/day  for any of the participants (this UL for folic acid does not include naturally occurring folate). It is important to note that this part of the study excluded persons taking vitamin supplements containing folic acid. While it was not possible to estimate the proportion of people in the general Newfoundland population who may be consuming more than 1,000 μg/day of folic acid from fortification and supplementation combined, it is likely that this proportion is small. The average dietary intake and maximum intake of folic acid due to fortification were 70 μg/day and 235 μg/day, respectively, for women aged 19–44 years, and 74 μg/day and 219 μg/day, respectively, for seniors. The average folic acid dose in folic acid containing over-the-counter supplements marketed in Canada is about 350 μg/day (Health Canada unpublished information).
The results of this study provide strong evidence of improved blood folate status in women aged 19–44 years following mandatory fortification with folic acid. Women showed evidence of increased levels of serum and RBC folate and decreased levels of plasma HCY. These results are consistent with an earlier study examining the effect of fortification in the Framingham offspring study cohort .
Mandatory food fortification with folic acid has resulted in improvements in folate indices in seniors. Both mean serum folate and mean RBC folate increased following folic acid fortification (55% and 23%, respectively, Table 4). Consistent with this was a moderate decrease in mean plasma HCY levels among seniors by 1.3 μmol/L. Fortification of food with folic acid and an upward shift in blood folate levels is of benefit to the elderly population especially with regard to risk of cardiovascular disease. High levels of homocysteine are associated with both cerebrovascular and coronary heart disease [38–40].
Vitamin B12 status
There was a decline in the proportion of seniors with low vitamin B12 levels, and there was actually a slight increase in mean vitamin B12 levels. In vitamin B12 deficiency, plasma MMA is usually elevated. Plasma MMA is believed to be a better indicator of vitamin B12 status at the tissue level than serum vitamin B12 levels are. Our study showed no change in mean MMA levels nor increased proportion of elderly with high levels. In addition, there was no change in the indicators of anaemia (i.e., haemoglobin and MCV) in seniors post fortification in our study. Thus, these results show no evidence of a deterioration in vitamin B12 status among seniors. Furthermore, there is no evidence of improved folate status resulting in masking of the haematological manifestations of vitamin B12 deficiency among seniors as a group. There was no evidence of deteriorating vitamin B12 status among young women participants based on vitamin B12 measurements. The upward trend in plasma MMA levels and higher proportion of abnormal values among young women is being further evaluated. It is unlikely that this is a direct effect of folic acid fortification and this observation is not consistent with any known effects of folic acid on vitamin B12 status.
We have documented the rate of NTDs among live births, stillbirths and terminated pregnancies known to have an NTD. It was not possible to include NTDs that may have occurred in pregnancies that resulted in a spontaneous abortion or a termination that occurred for reasons other than a congenital anomaly.
This study, and other studies of fortification in Canada, are limited by the fact that there was no precise date when exposure to food fortified with folic acid began. The addition of folic acid to white flour and enriched pasta and cornmeal was permitted as of December 1996. Industry was switching to folic acid-containing enrichment premixes, especially towards the end of 1997, in anticipation of both US requirements for fortification as of January 1, 1998, and Canadian plans to implement mandatory fortification. Although this requirement did not come into force in Canada until late 1998, the Phase I (November 1997 to March 1998) subjects of our study may have consumed at least some food fortified with folic acid. This would result in an underestimate of improvements in blood folate status due to fortification, and might lead us to miss adverse effects on vitamin B12 status. On the other hand, the fact that we observed such marked improvements in blood folate status leads us to conclude that there was a real increase in exposure to folic acid through fortification, over the study period.
Another limitation of this study is the possible underestimation of folic acid intake due to fortification. Our calculations were based on the assumption that manufacturers are fortifying flour at the required level. It has been suggested that allowance for "overages" is resulting in higher amounts in the affected products . Also, for enriched pasta, the required level of fortification is from a minimum of 0.20 mg/100 g pasta to a maximum of 0.27 mg/100 g. In our calculations we assumed the minimum level of fortification.
We initially selected a random sample of subjects through random digit dialling, and asked eligible respondents for voluntary participation in the study. The reasonable level of response for the dietary questionnaire and blood sampling among rural and urban women aged 19–44 years suggests that with caution, we can generalize the results to all Newfoundland women of childbearing age. However, these findings may not be representative of the rest of Canada because of population differences in factors such as genetic background and dietary behaviour. These differences may also affect the generalizability of the NTD trend.
The sample response rate for the dietary questionnaire and blood sampling in seniors was approximately 45% both in Phase I and in Phase II. Many of the refusals to participate were due to illness of the eligible person. Furthermore, seniors residing in long term care settings were not included. Thus our sample population of seniors may be healthier than the general population age 65 and over in the province.