A total of 86 women were approached; 62 of them consented and took part in the study. The mean age of the study participants was 31 y (range: 18–45 y). Results for the dietary assessments of women who became pregnant during the course of the study were retained, but all biochemical analysis of blood samples taken after conception were removed from the data set, because pregnancy can alter one-carbon metabolism (24
). Therefore, there were complete dietary intake assessments for 28 to 30 women for each of the 12-mo and fasted blood samples for 20 to 29 women per month. The average duration of participation in the study was 6 mo.
Dietary data were collected for a total of 706 d and included 1143 different recipes. The mean daily weight of food consumed (excluding water intake) was 1553 g (95% CI: 1489, 1619 g/d), with no significant variation over the year (P = 0.78). The overall mean energy intake was 1830 kcal/d (95% CI: 1712, 1816), with no significant variation over the year (P = 0.60). However, the dietary macronutrient composition did change across the year. The intake of fat (averaging 20.4% of the total energy intake), protein (11.7%), and carbohydrates (67.9%) varied significantly throughout the year, as shown in . Seasonal variation in body weights for all of the nonpregnant women in the study region exhibited seasonal variation as observed previously for this community, with a dramatic decrease during the rainy season (a period of heavy agricultural workload) and sustained weight gain during the dry season ().
FIGURE 2. Seasonal trends in energy and macronutrient intakes and weight. A: n dietary intake measurements were made in an average of 29 women × 12 mo × 2 d. 95% CIs are shown with bars, at 2-mo intervals (different by substance to avoid excessive (more ...)
The results of the autocorrelations to establish the reliability (precision of a single measurement or extrapolation back to zero lag) and stability (variation with time not related to seasonality) of the 48-h dietary intakes are shown in A. In addition, repeatability of measurements on 2 consecutive days ranged between 40.1% and 60.1%. CCVs are shown in . The dietary intakes of folate, riboflavin, vitamin B-6, and choline were significantly below the current international recommendations (), expressed as the Estimated Average Requirement (EAR). The EAR is the daily dietary intake of a nutrient expected to satisfy the needs of 50% of a population group. The dietary intakes surpassing these recommendations are also in and were <30% for all intakes except vitamin B-12 (100%) and methionine (98.9%).
FIGURE 3. Reliability and stability of dietary intakes and blood biomarker concentrations for substances under study. Black lines: solid (A and C, folate; B and D, methionine), dot (A and C, riboflavin; D, SAM), dash (A and C, vitamin B-12; D, SAH), dash-dot-dot (more ...)
Dietary intakes of one-carbon metabolites in Gambian women of reproductive age and international intake recommendations for women1
The monthly variation in dietary intakes, expressed as percentage deviation from the overall geometric mean, are illustrated in (A, B, C, and D). The annual variation in the intakes of folate (P = 0.0038), riboflavin (P = 0.016), and choline and betaine (P < 0.0001 for both) was statistically significant. The results show that the intakes of riboflavin and folate decreased over the rainy season until September, followed by an increase, and reached their highest intake toward the end of the dry season peak (May). Choline and betaine intakes were highest in September, with second peaks in December and April, respectively. Methionine, vitamin B-6, and B-12 intakes, conversely, did not vary significantly throughout the year (P > 0.05). Of those nutrient intakes that showed significant variation over the year, substantial differences between both seasons were also shown for betaine, folate, and riboflavin () but not for total choline. Riboflavin and folate were higher during the peak of the dry season (February to April; 12.1% and 14.9%, respectively), and betaine was higher during the peak of the rainy season (July to September; 32.4%).
FIGURE 4. Seasonal trends of methyl donors and cofactors as per dietary intake and blood biomarker concentrations expressed as a percentage of the geometric mean of all measurements (between July 2009 and June 2010). Black lines: solid (A and E, folate; C and G, (more ...)
During the 12 mo of study, 316 plasma samples were obtained for analyses. Fewer RBC samples (a total of 293) were prepared for analysis of riboflavin, because it was not always possible to obtain the second heparinized blood sample required. The autocorrelation calculations for biomarkers showed that, with the exception of SAH and dimethylglycine, all were stable (ie they correlate almost as well at 5 mo as at 1 mo lag) (B). Different degrees of reliability were apparent: the measurements of vitamins B-12 and active vitamin B-12 were highly reliable, whereas others, particularly SAH, showed poorer reliability (ie, the correlation was weak, even at 1 mo lag).
The geometric means and CCVs of biomarker concentrations for this group of Gambian women over the 1-y study period are shown in . The annual variation in blood biomarker concentrations, as a percentage variation from the geometric mean, are illustrated in (E, F, G, and H). All blood biomarkers showed large, significant annual variation (vitamin B-12, P = 0.03; all the other biomarkers, P < 0.001). However, the actual seasonal pattern of variation differed in timing and amplitude (magnitude of variation from the geometric mean, as indicated by the CCV) between the various biomarkers measured, with the highest seasonal variation for plasma folate, vitamin B-6, and the ratio of dimethylglycine to betaine. The status of plasma folate and vitamin B-6 was highest from May to June, at the end of the dry season, and was lowest from September to March (, E and H). Riboflavin status declined progressively from the beginning of the rainy season peak (July) until January and then improved again. Betaine reached its peak later in the rainy season (September; E). Plasma choline showed higher concentrations in November, whereas vitamin B-12 and methionine, in contrast with the other nutrients, remained relatively stable over the year (, respectively). Regarding the functional biomarkers, the SAM/SAH ratio was highest in July and lowest in March, and the seasonal fluctuation in the ratio was explained largely by the seasonal change in SAH. Plasma tHcy showed an opposite change; the lowest and highest plasma tHcy concentrations were found in July and April, respectively. Plasma dimethylglycine concentrations were highest between November and March, at which time the ratio of dimethylglycine to betaine reached the highest peak.
Mean concentrations of blood biomarkers throughout the year and by season1
As for dietary intake, the concentration differences between the peak of the rainy and dry seasons for each biomarker are shown in . Comparison of the biomarker concentrations according to these divisions showed that plasma SAM, betaine, and folate concentrations and SAM/SAH ratio were higher, whereas plasma SAH, tHcy, dimethylglycine, the ratio of dimethylglycine to betaine, vitamin B-12, and vitamin B-6 were lower in the rainy than in the dry season (). A lower EGRAC value during the rainy season denotes a less-deficient riboflavin status. Cysteine, choline, and methionine exhibited some variation between seasons, but this represented a difference between seasons of <1.5%. Differences among villages, possibly mediated by subtle differences in wealth and dietary habits within this generally homogeneous environment, was significant for intakes of vitamin B-12 (P = 0.0023) and betaine (P = 0.0024) and for plasma concentrations of choline (P value= 0.0039), folate (P = 0.0131), methionine (P = 0.0022) and the SAM/SAH ratio (P = 0.0038). However, the effect of village did not confound other effects.
Dietary intake and blood biomarkers
Next, the relations between the dietary intakes of the nutrients under study and their blood concentrations were regressed cross-sectionally to determine the predictive ability of dietary intake on blood biomarkers. Additional comparisons were made between selected nutrients to investigate interactions in specific pathways.
Positive associations between dietary intakes and respective plasma biomarkers were not observed for folate, choline (with total choline or any of its components), or methionine (data not shown). Significant positive associations were found between the dietary intake and blood biomarkers for vitamin B-6 (β: 0.166 nmol/L per mg/d; 95% CI: 0.017, 0.316), betaine (β: 0.037 μmol/L per mg/d; 95% CI: 0.006, 0.069), and riboflavin (β: −0.038 EGRAC points per mg/d; 95% CI: −0.064, −0.0112, where a higher EGRAC indicates greater deficiency and thus explained the negative coefficient). Dietary vitamin B-12 showed no significant association with plasma total vitamin B-12, but was positively associated with plasma active vitamin B-12 (β: 0.043 pmol/L per μg/d; 95% CI: 0.015, 0.071). Plasma concentrations of folate were negatively associated with the combined intake of choline and betaine (β: −0.214 μmol/L per mg/d; 95% CI: −0.297, −0.131). Plasma concentrations of choline and betaine were not, however, associated with folate intake, but were inversely associated with the intake of riboflavin [choline (β: −0.065 μmol/L per mg/d; 95% CI: −0.103, −0.028); betaine (β: −0.058, 95% CI: −0.096, 0.021)].
To explore the potential effect of the dietary intake of the selected nutrients on one-carbon metabolism more generally, we also determined the strength of the associations between dietary intake and functional blood biomarker status. No statistically significant associations were found between the plasma SAM/SAH ratio or plasma SAM concentration and the intake of any of the nutrients under study. Plasma SAH showed an association with vitamin B-12 intake (β = 0.039 nmol/L per μg/d; 95% CI: 0.006, 0.072). Plasma tHcy and dimethylglycine concentrations were correlated with the combined intake of choline (β = 0.121 μmol/L per mg/d; 95% CI: 0.060, 0.182) and betaine (β= 0.088 μmol/L per mg/d; 95% CI: 0.004, 0.172). As found for plasma choline and betaine, the plasma dimethylglycine was also inversely associated with the dietary intake of riboflavin (β = −0.086 μmol/L per mg/d; 95% CI: −0.152, −0.020).