Maternal and Cord Blood Folate Concentrations Are Inversely Associated with Fetal DNA Hydroxymethylation, but Not DNA Methylation, in a Cohort of Pregnant Canadian Women.

BACKGROUND: Aberrancies in fetal DNA methylation programming may modify disease susceptibility of the offspring. Maternal folate status has potential to alter fetal DNA methylation. OBJECTIVES: We examined the association of maternal and cord blood concentrations of folate and unmetabolized folic acid (UMFA), vitamin B-12, vitamin B-6, and choline with fetal DNA methylation and hydroxymethylation and assessed potential modifying effects of 38 fetal genetic variants in 22 genes. METHODS: Nutrient blood concentrations were measured in 368 pregnant women in early pregnancy (12-16 wk of gestation) and at delivery (37-42 wk of gestation) and in cord blood. DNA methylation and hydroxymethylation in cord blood mononuclear cells were quantified by LC-MS/MS. Pearson partial correlations were used to determine the association between individual nutrients and DNA methylation and hydroxymethylation. RESULTS: Serum and RBC folate and plasma UMFA concentrations (primary outcomes) in early pregnancy, at delivery, and in cord blood were not significantly associated with fetal DNA methylation. In contrast, maternal RBC folate in early pregnancy (r = -0.16, P = 0.04) and cord plasma UMFA (r = -0.23, P = 0.004) were inversely correlated with fetal DNA hydroxymethylation. Neither maternal and cord blood concentrations of other nutrients nor fetal genotypes (secondary outcomes) were significantly associated with fetal DNA methylation or hydroxymethylation. Infants born to mothers with RBC folate concentrations in the highest quartile and serum vitamin B-12 concentrations in the lowest quartile in early pregnancy had significantly lower fetal DNA methylation and higher birth weight compared with those born to mothers with lower RBC folate and higher serum vitamin B-12 concentrations (P = 0.01). CONCLUSIONS: Maternal and cord blood folate concentrations are associated with fetal DNA hydroxymethylation, but not DNA methylation, in a cohort of pregnant Canadian women. The observation that high folate and low vitamin B-12 maternal status in early pregnancy may be associated with decreased fetal DNA methylation and higher birth weight warrants further investigation. This trial was registered at clinicaltrials.gov as NCT02244684.

Serum Betaine and Dimethylglycine Are Higher in South Asian Compared with European Pregnant Women in Canada, with Betaine and Total Homocysteine Inversely Associated in Early and Midpregnancy, Independent of Ethnicity.

BACKGROUND: As a methyl donor required in the folate-vitamin B-12 independent remethylation of total homocysteine (tHcy) to methionine, betaine is critical for fetal development. Pregnant South Asian women living in Canada had a higher reported prevalence of low vitamin B-12 status compared with Europeans; betaine concentrations in this population are unknown. OBJECTIVES: We aimed to compare serum betaine concentrations between South Asian and European pregnant women, and to determine the relation between betaine and tHcy concentrations in early pregnancy. METHODS: A retrospective cohort study was conducted using biobanked serum samples of 723 apparently healthy pregnant women of South Asian (50%) and European ethnicity residing in British Columbia, Canada. Betaine, dimethylglycine (DMG), tHcy, and related metabolites were quantified in samples collected in the first (8-13 weeks of gestation) and second (14-20 weeks of gestation) trimesters. The relation between betaine and tHcy concentrations was assessed using a generalized regression model adjusted for weeks of gestation, ethnicity, prepregnancy BMI, maternal age, neonatal sex, parity, total vitamin B-12, folate, pyridoxal 5'-phosphate, and methionine concentrations. RESULTS: Median serum concentrations of betaine and its metabolite DMG were higher in South Asian women in the first (19.8 [IQR: 16.3-25.0] and 1.55 [IQR: 1.30-1.96] $\mu {\rm mol/L} $, respectively) and second trimesters (16.1 [IQR: 12.9-19.8] and 1.42 [IQR: 1.14-1.81] $\mu {\rm mol/L} $, respectively) compared with European women (17.6 [IQR: 13.7-22.6] and 1.38 [IQR: 1.12-1.77] $\mu {\rm mol/L} $, respectively) and (12.9 [IQR: 10.6-16.7] and 1.19 [IQR: 0.97-1.52] $\mu {\rm mol/L} $, respectively; all P values < 0.0001). Betaine was inversely associated with tHcy concentration (ß = -0.0208; 95% CI: -0.0341, -0.00742; P = 0.002). Additionally, total vitamin B-12 was associated with tHcy concentration (ß = -0.0312; 95% CI: -0.0401, -0.0224), after adjusting for confounding factors. CONCLUSIONS: Pregnant South Asian women residing in Canada had higher betaine and DMG concentrations, compared with women of European ethnicity, while betaine and total vitamin B-12 predicted tHcy independent of ethnicity. Our results emphasize the role of betaine, as methyl donor, in the remethylation of tHcy in a folate-replete population.

Reference intervals for serum total vitamin B12 and holotranscobalamin concentrations and their change points with methylmalonic acid concentration to assess vitamin B12 status during early and mid-pregnancy.

Background Maternal vitamin B12 (B-12) adequacy is important for maternal health and optimal fetal growth. However, pregnancy-specific cut-offs for B-12 biomarkers are lacking. Methods Reference intervals for serum total B-12, holotranscobalamin (holoTC) and methylmalonic acid (MMA) concentrations were calculated following CLSI EP28-A3c guidelines in 723 pregnant women of European (50%) and South Asian (50%) ethnicity, residing in British Columbia, Canada, at median (range) 11.4 (8.3-13.9) and 16.1 (14.9-20.9) weeks of gestation. Change point analyses described relationships between log serum MMA concentration with serum total B-12 and holoTC concentrations, assuming linear-linear relationships. Results The central 95% reference interval limits indicated that serum total B-12 <89.9 and <84.0 pmol/L, holoTC <29.5 and <26.0 pmol/L and MMA >371 and >374 nmol/L, in the first and second trimesters, respectively, may indicate B-12 deficiency in pregnant women. The lower limits of total B-12 and holoTC and the upper limits of MMA significantly differed by ethnicity in both trimesters. According to the change point analysis, total B-12 <186 and <180 pmol/L and holoTC <62.2 and <67.5 pmol/L in the first and second trimesters, respectively, suggested an increased probability of impaired intracellular B-12 status, with no difference between ethnicities. Conclusions We present novel reference limits and change points for B-12 biomarkers, which may be employed to identify possible B-12 deficiency in women during early and mid-pregnancy. Future research is needed to validate these cut-offs and determine the predictors and functional outcomes associated with impaired B-12 status in ethnically diverse populations.

Pregnant women of South Asian ethnicity in Canada have substantially lower vitamin B12 status compared with pregnant women of European ethnicity.

Maternal vitamin B12 (B12) status has been inversely associated with adverse pregnancy outcomes and positively with fetal growth and infant development. South Asians, Canada's largest ethnic minority, are prone to B12 deficiency. Yet, data are lacking on B12 status in South Asian pregnant women in North America. We sought to determine B12 status, using multiple biomarkers, in 1st and 2nd trimester pregnant women of South Asian and, for comparison, European ethnicity living in Vancouver, Canada. In this retrospective cohort study, total B12, holotranscobalamin (holoTC), methylmalonic acid (MMA), and total homocysteine concentrations were quantified in two routinely collected (mean gestational week: 11·5 (range 8·3-13·9) and 16·5 (range 14·9-20·9)), banked serum samples of 748 healthy pregnant South Asian (n 371) and European (n 377) women. South Asian pregnant women had significantly lower B12 status than European pregnant women at both time points, as indicated by lower serum total B12 and holoTC concentrations, and higher MMA concentrations (all P≤0·001). The largest difference, which was substantial (Cohen's d≥0·5), was observed in mean serum total B12 concentrations (1st trimester: 189 (95 % CI 180, 199) v. 246 (95 % CI 236, 257) pmol/l; 2nd trimester: 176 (95 % CI 168, 185) v. 226 (95 % CI 216, 236) pmol/l). Further, South Asian ethnicity was a significant negative predictor of B12 status during pregnancy. South Asian women living in Vancouver have substantially lower B12 status during early pregnancy. Future research identifying predictors and health consequences of this observed difference is needed to allow for targeted interventions.

South Asian Ethnicity Is Related to the Highest Risk of Vitamin B12 Deficiency in Pregnant Canadian Women.

Vitamin B12 (B12) adequacy during pregnancy is crucial for maternal health and optimal fetal development; however, suboptimal B12 status has been reported in pregnant Canadian women. Methylmalonic acid (MMA) is a sensitive indicator of B12 status. Since few studies have measured MMA during pregnancy in Canadian women, the objective of this study was to evaluate B12 status in pregnant women living in Metro Vancouver, using both plasma total B12 and MMA. We recruited a convenience sample of 320 pregnant women between 20 and 35 gestational weeks from local healthcare facilities. Plasma total B12 concentrations indicative of deficiency (<148 pmol/L) and suboptimal B12 status (148-220 pmol/L) were found in 18% and 33% of the women, respectively. Normal plasma MMA concentration (<210 nmol/L) was observed in 82% of all women. Gestational age was a strong predictor of plasma total B12 and MMA concentration, and South Asian ethnicity of B-12 deficiency and MMA concentrations. Overall, there was a high discrepancy between the prevalence of B12 inadequacy depending on the biomarker used. Independently, however, South Asian women were at particular risk for B12 deficiency, likely due to lower animal source food intake. Further study of this vulnerable group and performance testing of B12 biomarkers is warranted.

Low Serum Vitamin B-12 Concentrations Are Prevalent in a Cohort of Pregnant Canadian Women.

BACKGROUND: Among Canadian women of reproductive age, 5% and 20% have serum vitamin B-12 concentrations indicative of deficiency (<148 pmol/L) and marginal status (148-220 pmol/L), respectively. Given the association between suboptimal vitamin B-12 and adverse pregnancy outcomes, an understanding of vitamin B-12 status during pregnancy, and factors that influence it, is required. OBJECTIVE: This prospective analysis from the PREFORM (PREnatal FOlic acid exposuRe on DNA Methylation in the newborn infant) study investigated 1) vitamin B-12 status in a cohort of Canadian pregnant women and their newborns, 2) the association of maternal dietary vitamin B-12 intake with maternal and cord blood concentrations of vitamin B-12 and its biomarkers, and 3) the association of fetal genetic polymorphisms with cord blood concentrations of vitamin B-12 and its biomarkers. METHODS: In pregnant Canadian women (n = 368; mean ± SD age: 32 ± 5 y), vitamin B-12 intakes were assessed in early (0-16 wk) and mid- to late (23-37 wk) pregnancy. Serum vitamin B-12 and plasma total homocysteine (tHcy) and methylmalonic acid (MMA) in maternal blood at 12-16 wk of pregnancy and at delivery (28-42 wk) and in cord blood were measured and compared by using regression analyses. The associations of 28 fetal genetic variants in vitamin B-12 metabolism and cord blood vitamin B-12, tHcy, and MMA concentrations were assessed by using regression analysis, with adjustment for multiple testing. RESULTS: A total of 17% and 38% of women had deficient and 35% and 43% had marginal serum vitamin B-12 concentrations at 12-16 wk of pregnancy and at delivery, respectively. Only 1.9-5.3% had elevated MMA (>271 nmol/L), and no women had elevated tHcy (>13 µmol/L). Maternal dietary vitamin B-12 intake during pregnancy was either weakly associated or not associated with maternal and cord blood vitamin B-12 (r(2) = 0.17-0.24, P < 0.0008), tHcy (P = NS) and MMA (r(2) = 0.05-0.11, P < 0.001). Fetal genetic polymorphisms were not associated with cord blood concentrations of vitamin B-12 and its biomarkers. CONCLUSIONS: Deficient and marginal serum vitamin B-12 concentrations are prevalent in Canadian pregnant women with the use of traditional cutoffs, despite supplement use. Given the growing interest among women to adhere to a vegetarian diet that may be lower in vitamin B-12, and vitamin B-12's importance in pregnancy, the functional ramifications of these observations need to be elucidated. This trial was registered at clinicaltrials.gov as NCT02244684.

Reference interval of methylmalonic acid concentrations in dried blood spots of healthy, term newborns to facilitate neonatal screening of vitamin B12 deficiency.

OBJECTIVES: Unrecognized vitamin B12 (B-12) deficiency in early infancy can lead to poor development of the child. Methylmalonic acid (MMA) is the most specific functional biomarker of B-12 status. To facilitate timely diagnosis and treatment of neonatal B-12 deficiency, the objective of this study was to calculate a reference interval of MMA in dried blood spots (DBSs) of healthy, term newborns. DESIGN AND METHODS: MMA was quantified in 160 newborn DBSs, routinely collected for newborn screening, using LC-MS/MS. The reference interval of DBS MMA was calculated according to current guidelines (CLSI EP28-A3c). The effect of storage at room temperature on DBS MMA concentrations was determined. RESULTS: The mean DBS MMA concentration of 160 healthy, term newborns was 16.8pmol (95% CI: 15.9-17.6pmol)/8-mm spot. The reference interval (2.5th to 97.5th percentile) for MMA was 9.89 to 29.3pmol/8-mm spot (0.450 to 1.33µmol/L whole blood). DBS MMA concentrations correlated positively (p<0.005) with storage time at room temperature. DBS MMA concentrations of children with methylmalonic acidemia (n=4) were above the upper limit of the reference interval. CONCLUSIONS: This is the first study to present a reference interval for DBS MMA of healthy, term newborns utilizing a new highly sensitive MMA method. Analysis of DBS MMA collected during newborn screening may have the potential to identify newborns with acquired B-12 deficiency. Newborn DBS MMA concentrations increase with storage at room temperature, therefore, sample storage has to be monitored for evaluation of DBS MMA data.

High prevalence of suboptimal vitamin B12 status in young adult women of South Asian and European ethnicity.

Suboptimal vitamin B12 (B12) status has been associated with an increased risk of congenital anomalies, preterm birth, and childhood insulin resistance. South Asians - Canada's largest minority group - and women of reproductive age are vulnerable to B12 deficiency. This study aimed to assess the prevalence of and factors associated with B12 deficiency and suboptimal B12 status in a convenience sample of young adult women of South Asian and European descent in Metro Vancouver. We measured serum B12, holotranscobalamin, plasma methylmalonic acid, red blood cell and plasma folate, and hematologic parameters in 206 nonpregnant, healthy women aged 19-35 years. Categorization for B12 status adhered to serum B12 cutoffs for deficiency (<148 pmol/L) and suboptimal B12 status (148-220 pmol/L). We collected demographic, lifestyle, and dietary intake data and conducted genotyping for common genetic variants linked to B-vitamin metabolism. The prevalence of deficiency and suboptimal B12 status were 14% and 20%, respectively. Serum vitamin B12 concentrations were negatively associated with oral contraceptive use and first-generation immigrant status, and positively with dietary B12 intake and B12 supplement use. The prevalence of B12 inadequacy in this sample of highly educated women is higher than in the general Canadian population. In light of maternal and fetal health risks associated with B12 inadequacy in early-pregnancy, practitioners should consider monitoring B12 status before and during early pregnancy, especially in immigrants and women with low dietary B12 intakes including non-users of vitamin supplements.

Methylmalonic acid quantified in dried blood spots provides a precise, valid, and stable measure of functional vitamin B-12 status in healthy women.

Methylmalonic acid (MMA) is a sensitive and specific functional biomarker of vitamin B-12 status, commonly assessed in plasma or serum. Dried blood spots (DBSs) allow simpler and more cost-efficient blood sampling than plasma. To facilitate convenient testing for vitamin B-12 deficiency in large-scale surveys and in population groups from remote areas, we developed a method for MMA quantification in DBSs and tested its applicability as well as the long-term stability of MMA in DBSs at various temperatures. MMA was extracted from an 8-mm DBS punch with water:methanol (95:5, v:v) and methyl-d3-malonic acid as the internal standard. After sample cleanup by ultrafiltration and hexane extraction, MMA was quantified by using reversed-phase LC-tandem mass spectrometry. Extraction conditions were optimized to maximize the detection signal and achieve DBS extract concentrations above the lowest limit of quantification (signal-to-noise ratio ≥ 10) of 10 nmol/L. Recovery was between 93% and 96%. Intra- and interassay variation (CV%) for DBS MMA was 0.49% and 2.3%, respectively. Calibrators showed linearity (R(2) = 0.998) between 10 and 10,000 nmol/L. In 94 healthy women, MMA concentrations in DBS extract (min-max: 10.2-80.5 nmol/L) and plasma (min-max: 68-950 nmol/L) were correlated (ρ = 0.90) (P < 0.001). MMA concentrations in DBSs were stable at room temperature for 1 wk, in the refrigerator for 8 wk, and at -80°C for at least 1 y. This simple and robust method allows quantification of MMA in DBSs of healthy individuals. The linear relation between plasma and DBS MMA suggests that DBS MMA could predict plasma MMA, the current reference indicator for functional vitamin B-12 deficiency. With the advantages of minimally invasive specimen collection and no need for laborious blood processing steps, this method has the potential to be a reliable, convenient, and field-applicable alternative for assessment of vitamin B-12 status.