Association of maternal vitamin B12 and folate levels in early pregnancy with gestational diabetes: a prospective UK cohort study (PRiDE study).

AIMS/HYPOTHESIS: The prevalence of gestational diabetes mellitus (GDM) is increasing worldwide in all ethnic groups. Low vitamin B12 and low/high folate levels may contribute to GDM risk, but there is conflicting evidence. Our aim is to assess the relationships of early pregnancy vitamin B12 and folate levels with the risk of GDM status at 26-28 weeks of gestation. METHODS: This was a prospective, multi-centre, multi-ethnic cohort study (n = 4746) in the UK. Participants who were eligible to be selectively screened as per the National Institute for Health and Care Excellence (NICE) criteria were included in the study. RESULTS: GDM prevalence was 12.5% by NICE and 14.7% by International Association of Diabetes and Pregnancy Study Groups (IADPSG) criteria. Folate deficiency (1.3%) was rare but B12 insufficiency (42.3% at <220 pmol/l) and folate excess (36.5%) were common in early pregnancy. Early pregnancy median B12 levels were lower, and folate levels higher, in women who were diagnosed with GDM at 26-28 weeks. B12 was negatively associated with fasting plasma glucose (1 SD: -0.06 mmol/l; 95% CI -0.04, -0.08; p < 0.0001) and 2 h plasma glucose levels (-0.07 mmol/l; 95% CI -0.02, -0.12; p = 0.004). Higher B12 was associated with 14.4% lower RR of IADPSG-GDM (0.856; 95% CI 0.786, 0.933; p = 0.0004) after adjusting for key confounders (age, parity, smoking status, ethnicity, family history, household income and folate status). Approximately half of this association was mediated through BMI. Folate was positively associated with 2 h plasma glucose levels (0.08 mmol/l; 95% CI 0.04, 0.13; p = 0.0005) but its relationship with fasting plasma glucose was U-shaped (quadratic ß: 0.011; p = 0.05). Higher folate was associated with 11% higher RR of IADPSG-GDM (adjusted RR 1.11; 95% CI 1.036, 1.182; p = 0.002) (age, parity, smoking status, ethnicity, family history, household income and B12 status). Although no interactions were observed for B12 and folate (as continuous variables) with glucose levels and GDM risk, a low B12-high folate combination was associated with higher blood glucose level and risk of IADPSG-GDM (adjusted RR 1.742; 95% CI 1.226, 2.437; p = 0.003). CONCLUSIONS/INTERPRETATION: B12 insufficiency and folate excess were common in early pregnancy. Low B12 and high folate levels in early pregnancy were associated with small but statistically significant changes in maternal blood glucose level and higher RR of GDM. Our findings warrant additional studies on the role of unmetabolised folic acid in glucose metabolism and investigating the effect of optimising early pregnancy or pre-conception B12 and folate levels on subsequent hyperglycaemia. TRIAL REGISTRATION: ClinicalTrials.gov NCT03008824.

Intracellular and Tissue Levels of Vitamin B12 in Hepatocytes Are Modulated by CD320 Receptor and TCN2 Transporter.

The liver mass constitutes hepatocytes expressing receptors for vitamin B12 (B12)-bound transporters in circulation. However, intrahepatic and circulating B12 interrelationship levels remain unclear. We assessed the intracellular B12 levels at various circulating B12 concentrations in human HepG2 cell-line and liver tissue levels of B12 in the C57BL/6 mouse model. In HepG2 cells treated with a range of B12 concentrations, the intracellular and circulatory B12 levels, transcript and protein levels of B12 receptor (CD320) and transporter (TCN2) were determined using immunoassays, qRT-PCR and Western blot, respectively. Similar assessments were done in plasma and liver tissue of C57BL/6 mice, previously fed a diet of either a high or low B12 (30.82 µg B12/kg and 7.49 µg B12/kg, respectively) for 8-10 weeks. The physiological B12 status (0.15-1 nM) resulted in increased levels of intracellular B12 in HepG2 cells compared to supraphysiological levels of B12 (>1 nM). Gene and protein expression of CD320 and TCN2 were also higher at physiological levels of B12. Progressively increasing extracellular B12 to supraphysiological levels led to relative decreased levels of intracellular B12, lower expression of gene and protein levels of CD320 and TCN2. Similar results were observed in liver tissue from mice fed on a low B12 diet verses high B12 diet. These findings suggest that unlike supraphysiological B12, physiological levels of B12 in the extracellular media or circulation accelerates active transport of B12, and expression of CD320 and TCN2, resulting in higher relative uptake of B12 in hepatocytes.

Vitamin B12 deficiency and altered one-carbon metabolites in early pregnancy is associated with maternal obesity and dyslipidaemia.

Vitamin B12 (B12) is a micronutrient essential for one-carbon (1C) metabolism. B12 deficiency disturbs the 1C cycle and alters DNA methylation which is vital for most metabolic processes. Studies show that B12 deficiency may be associated with obesity, insulin resistance and gestational diabetes; and with obesity in child-bearing women. We therefore hypothesised that the associations between B12 deficiency, BMI and the metabolic risk could be mediated through altered 1C metabolites in early pregnancy. We explored these associations in two different early pregnancy cohorts in the UK (cohort 1; n = 244 and cohort 2; n = 60) with anthropometric data at 10-12 weeks and plasma/serum sampling at 16-18 weeks. B12, folate, total homocysteine (tHcy), methionine, MMA, metabolites of 1C metabolism (SAM, SAH) and anthropometry were measured. B12 deficiency (< 150 pmol/l) in early pregnancy was 23% in cohort 1 and 18% in cohort 2. Regression analysis after adjusting for likely confounders showed that B12 was independently and negatively associated with BMI (Cohort 1: ß = - 0.260, 95% CI (- 0.440, - 0.079), p = 0.005, Cohort 2: (ß = - 0.220, 95% CI (- 0.424, - 0.016), p = 0.036) and positively with HDL cholesterol (HDL-C) (ß = 0.442, 95% CI (0.011,0.873), p = 0.045). We found that methionine (ß = - 0.656, 95% CI (- 0.900, - 0.412), p < 0.0001) and SAH (ß = 0.371, 95% CI (0.071, 0.672), p = 0.017) were independently associated with triglycerides. Low B12 status and alteration in metabolites in 1C metabolism are common in UK women in early pregnancy and are independently associated with maternal obesity and dyslipidaemia. Therefore, we suggest B12 monitoring in women during peri-conceptional period and future studies on the pathophysiological relationship between changes in 1C metabolites and its association with maternal and fetal outcomes on larger cohorts. This in turn may offer potential to reduce the metabolic risk in pregnant women and their offspring.

Reduced GLP-1 Secretion at 30 Minutes After a 75-g Oral Glucose Load Is Observed in Gestational Diabetes Mellitus: A Prospective Cohort Study.

Glucagon-like peptide 1 (GLP-1) levels may be reduced in type 2 diabetes, but whether a similar impairment exists in gestational diabetes mellitus (GDM) has not been established. We studied this in a prospective cohort study of pregnant women (n = 144) during oral glucose tolerance test (OGTT). GLP-1, glucose, and insulin were sampled at 30-min intervals during a 2-h 75-g OGTT, and indices of insulin secretion and sensitivity were calculated. In a nested case-control study, women with GDM (n = 19) had 12% lower total GLP-1 secretion area under the curve (AUC) compared with control subjects matched for age, ethnicity, and gestational age (n = 19), selected from within the lowest quartile of glucose120 min values in our cohort. GDM had lower GLP-1 response in the first 30 min (19% lower GLP-130 min and 17% lower AUC0-30 min) after adjustment for possible confounders. Their glucose levels began to diverge at 30 min of the OGTT with increasing insulin levels, and by 120 min, their insulin levels were three times higher. In a secondary cohort of 57 women that included "high-normal" glucose120 min values, low GLP-1 AUC0-30 min was independently associated with lower indices of insulin secretion and sensitivity. In conclusion, we have observed that women with GDM have lower GLP-1 response at 30 min of an OGTT and hyperglycemia at 120 min despite significant hyperinsulinemia.

Vitamin B12 Status among Pregnant Women in the UK and Its Association with Obesity and Gestational Diabetes.

BACKGROUND: To evaluate vitamin B12 and folate status in pregnancy and their relationship with maternal obesity, gestational diabetes mellitus (GDM), and offspring birthweight. METHODS: A retrospective case-control study of 344 women (143 GDM, 201 no-GDM) attending a district general hospital and that had B12 and folate levels measured in the early 3rd trimester was performed. Maternal history including early pregnancy body mass index (BMI) and neonatal data (birthweight, sex, and gestational age) was recorded for all subjects. RESULTS: 26% of the cohort had B12 levels <150 pmol/L (32% vs. 22% in the two groups respectively, p < 0.05) while 1.5% were folate deficient. After adjusting for confounders, 1st trimester BMI was negatively associated with 3rd trimester B12 levels. Women with B12 insufficiency had higher odds of obesity and GDM (aOR (95% CI) 2.40 (1.31, 4.40), p = 0.004, and 2.59 (1.35, 4.98), p = 0.004, respectively), although the latter was partly mediated by BMI. In women without GDM, the lowest quartile of B12 and highest quartile of folate had significantly higher adjusted risk of fetal macrosomia (RR 5.3 (1.26, 21.91), p = 0.02 and 4.99 (1.15, 21.62), p = 0.03 respectively). CONCLUSION: This is the first study from the UK to show that maternal B12 levels are associated with BMI, risk of GDM, and additionally may have an independent effect on macrosomia. Due to the increasing burden of maternal obesity and GDM, longitudinal studies with B12 measurements in early pregnancy are needed to explore this link.

Simultaneous detection of five one-carbon metabolites in plasma using stable isotope dilution liquid chromatography tandem mass spectrometry.

Disturbance in one-carbon (1-C) cycle occurs due to nutritional deficiencies (vitamin B12/folate) or specific genetic polymorphisms. This leads to altered levels of key 1-C metabolites such as SAM (s-adenosyl methionine), SAH (s-adenosyl homocysteine), methionine, homocysteine and MMA (methyl malonic acid). These 1-C metabolites are determinants of cellular methylation potential and epigenetic modifications of DNA which impairs metabolic pathways in several pathological diseases and developmental programming. Though methods were able to measure these analytes only independently, none of the methods detect simultaneously. Therefore we developed a method to measure these five 1-C metabolites in a single run using liquid chromatography tandem mass spectrometry (LC-MS/MS). We used stable isotopes dilution LC-MS/MS to measure the 1-C metabolites in human plasma. Blood samples were collected from pregnant women (n=30) at early gestation in the ongoing, multicentre, prospective PRiDE study. Linearity exhibited across the calibration range for all the analytes with the limit of detection (LOD) of 1.005nmol/l for SAM, 0.081nmol/l for SAH, 0.002µmol/l for methionine, 0.046µmol/l for homocysteine and 3.920nmol/l for MMA. The average recovery for SAM was 108%, SAH-110%, methionine-97%, homocysteine-91% and MMA-102%. The inter-assay CV for SAM was 7.3, SAH-5.6%, methionine-3.5%, homocysteine-7.0% and MMA-4.0%. The intra-assay CV for SAM was 8.7%, SAH-4.7%, methionine-5.4%, homocysteine-8.1% and MMA-6.1%. Pregnant women at early gestation with low B12 levels had significantly higher homocysteine, MMA, lower levels of methionine, SAM and SAM:SAH ratio and higher triglycerides. We developed a simple and rapid method to simultaneously quantify 1-C metabolites such as SAM, SAH, methionine, homocysteine and MMA in plasma by stable isotope dilution LC-MS/MS which would be useful to elucidate the epigenetic mechanisms related in the gene-nutrient interactions.