Baseline characteristics and folate markers
Table 1 shows the main characteristics of the participants according to their treatment allocation. The study groups did not differ in mean age, BMI, creatinine, liver function markers, glucose, or haemoglobin. The prevalence of smoking, use of anti-contraceptive hormones, physical activity, education level, and the number of women with children did not differ between the groups (Table 1).
The baseline concentrations of serum folate, RBC-folate, and plasma tHcy did not differ between the study groups (Table 2). Twelve of the 198 participants (6.1%) had RBC-folate in the deficiency range <340 nmol/L, while only 24 (12%) had RBC-folate concentrations ≥906 nmol/L at baseline. At baseline, the percentages of women with folate deficiency (in both groups 6.1%) and those with sufficient RBC-folate (10.1 vs. 14.1%; p = 0.384) were comparable in the 400 and 800 µg/day groups. The median alcohol consumption as reported in the 2 × 3 days food protocol was 2.6 g/day, with only five women exceeding 20 g/day. At baseline, no significant differences were found between women with alcohol intake above or below the median in serum folate, RBC-folate, or tHcy (data not shown).
Post intervention folate markers and their changes
After 4 weeks, the 800 µg/day group had significantly higher serum folate (mean = 52.3 vs. 37.0 nmol/L; p < 0.001) and RBC-folate (928 vs. 805 nmol/L; p < 0.001) than the 400 µg/day group (Table 2). The percentage of women who achieved RBC-folate concentrations in the desirable range (≥906 nmol/L) after 4 weeks was significantly higher in the 800 µg/day group compared with the 400 µg/day group (45.5 vs. 31.3%; p = 0.041). The ratio of RBC-folate/serum folate declined strongly in both study groups after 4 weeks of intervention with folate, but it was lower in the 800 µg/day group compared with the 400 µg/day group after 4 weeks (20.0 vs. 24.5; p < 0.001). The plasma concentrations of tHcy were lower in the 800 µg/day compared with the 400 µg/day group after 4 weeks (mean = 5.7 vs. 6.1 µmol/L, p = 0.043) (Table 2).
The concentrations of serum and RBC-folate continued to increase between 4 and 8 weeks, but remained higher in the 800 µg/day group as compared with the 400 µg/day group after 8 weeks (serum folate = 67.2 vs. 45.7 nmol/L; RBC-folate = 1218 vs. 1021 nmol/L; both p < 0.001). The ratio of RBC-folate/serum folate did not show any further changes from 4 to 8 weeks, suggesting that RBC-folate increased parallel to the increase of serum folate after the first 4 weeks. At 8 weeks, significantly more women in the 800 µg/day group achieved desirable RBC-folate concentrations compared with the 400 µg/day group (83.8 vs. 54.5%; p < 0.001). The difference in plasma tHcy between the groups was not significant after 8 weeks (5.6 vs. 5.9 µmol/L, in the 800 and 400 µg/day, respectively; p = 0.123) (Table 2).
The changes of serum folate after 4 weeks (mean = +33.9 vs. +20.2 nmol/L; p < 0.001) and 8 weeks (+48.9 vs. +29.0 nmol/L; p < 0.001) were significantly higher in the 800 µg/day group compared with the 400 µg/day. Similarly, the changes of RBC-folate after 4 weeks (+276 vs. +173 nmol/L) and 8 weeks (+566 vs. +389 nmol/L) were higher in the 800 µg/day (both p < 0.001). Plasma tHcy declined in both groups (−1.7 and −1.9 µmol/L in the 400 and 800 µg/day after 8 weeks of supplementation), but the changes were not significantly different between the groups (Table 2).
Supplemental Fig. 2 shows mean (95% confidence intervals, CI) of concentrations of serum folate, RBC-folate and tHcy according to the intervention and the duration of the treatment. The longitudinal changes of all markers from baseline to visit 2 or 3 were significant in both groups.
Changes of folate markers according to baseline RBC-folate
We studied the differences in folate markers and their changes between the study groups according to baseline RBC-folate (<, ≥590 nmol/L, the median of the study population) (Tables 3, 4). Women with low baseline RBC-folate had approximately 50% lower serum folate compared with women with RBC-folate ≥590 nmol/L. Mean baseline plasma tHcy was low in both subgroups but tended to be lower in the subgroup with RBC-folate concentrations ≥590 nmol/L compared with the group with RBC-folate below this limit (7.2 vs. 7.9 µmol/L, respectively; p = 0.098). tHcy was lowered after supplementation, but the changes were not different between the study arms (Tables 3, 4).
Women who started the trial with baseline RBC-folate levels <590 nmol/L (Fig. 1; Table 3), maintained lower levels until week 8 as compared with women who started with levels ≥590 nmol/L (Table 4). However, women with low baseline RBC-folate levels who received 800 µg/day for 8 weeks were more likely to achieve the desirable range compared with those in the 400 µg/day group (69.4 vs. 20.0%) (Table 3). Women with low RBC-folate at start had reached a mean RBC-folate of 598 nmol/L (none ≥906 nmol/L) after 4 weeks and a mean of 778 nmol/L (20%, ≥906 nmol/L) after 8 weeks (Fig. 1). In the subgroup of women with RBC-folate ≥590 nmol/L, 80% of the participants reached desirable folate concentrations within 4 weeks of supplementing 800 µg/day, while 63.3% reached similar RBC folate range within 4 weeks of supplementation with 400 µg/day (Table 4, Fig. 1).
In the 400 µg/day group, there was no significant correlation between baseline RBC-folate and delta RBC-folate at 4 weeks (Spearman correlation r = −0.05, p = 0.641) or that at 8 weeks (r = 0.04, p = 0.670). The changes of RBC-folate in the 800 µg/day group showed a weak, but significant negative correlation with baseline RBC-folate at 4 weeks (r = −0.22, p = 0.033), but not at 8 weeks (r = −0.10, p = 0.318). Thus, the supplemental dose and time on supplements had a stronger influence on the changes of RBC-folate compared with the influence of baseline RBC-folate concentrations.
RBC-folate as a function of serum folate under supplementation
The post-intervention concentrations of RBC-folate were stratified into ranges that differ by 100 nmol/L (Supplemental Figs. 3 and 4). The response of serum concentrations of folate to treatment was strong and concentrations were not proportional to RBC-folate levels after supplementation. At 8 weeks, serum folate ≥47.0 nmol/L in the 400 µg/day or ≥61.2 nmol/L in the 800 µg/day group corresponded to RBC-folate concentrations of >940 nmol/L.
Ten intolerance cases were documented and considered related to the study supplements (mostly gastrointestinal intolerability), but in only one case, the participant dropped out (in the 400 µg/day group). No serious adverse events (including no death) were reported and no differences in reported adverse events between the study groups.