Xinyin Jiang

Maternal choline intake alters the epigenetic state of fetal cortisol-regulating genes in humans

The in utero availability of methyl donors, such as choline, may modify fetal epigenetic marks and lead to sustainable functional alterations throughout the life course. The hypothalamic‐pituitaryadrenal (HPA) axis regulates cortisol production and is sensitive to perinatal epigenetic programming. As an extension of a 12‐wk dose‐response choline feeding study conducted in third‐trimester pregnant women, we investigated the effect of maternal choline intake (930 vs. 480 mg/d) on the epigenetic state of cortisolregulating genes, and their expression, in placenta and cord venous blood. The higher maternal choline intake yielded higher placental promoter methylation of the cortisol‐regulating genes, corticotropin releasing hormone (CRH; P=0.05) and glucocorticoid receptor (NR3C1; P=0.002); lower placental CRH transcript abundance (P=0.04); lower cord blood leukocyte promoter methylation of CRH (P=0.05) and NR3C1 (P=0.04); and 33% lower (P=0.07) cord plasma cortisol. In addition, placental global DNA methylation and dimethylated histone H3 at lysine 9 (H3K9me2) were higher (P=0.02) in the 930 mg choline/d group, as was the expression of select placental methyltransferases. These data collectively suggest that maternal choline intake in humans modulates the epigenetic state of genes that regulate fetal HPA axis reactivity as well as the epigenomic status of fetal derived tissues.—Jiang, X., Yan, J., West, A. A., Perry, C. A., Malysheva, O. V., Devapatla, S., Pressman, E., Vermeylen, F., Caudill, M. A. Maternal choline intake alters the epigenetic state of fetal cortisol‐regulating genes in humans. FASEB J. 26, 3563–3574 (2012). www.fasebj.org

Maternal choline intake modulates maternal and fetal biomarkers of choline metabolism in humans

BACKGROUND In 1998 choline Adequate Intakes of 425 and 450 mg/d were established for nonpregnant and pregnant women, respectively. However, to our knowledge, no dose-response studies have been conducted to evaluate the effects of pregnancy or maternal choline intake on biomarkers of choline metabolism. OBJECTIVE We sought to quantify the effects of pregnancy and maternal choline intake on maternal and fetal indicators of choline metabolism. DESIGN Healthy pregnant (n = 26; 27 wk gestation) and nonpregnant (n = 21) women were randomly assigned to receive 480 or 930 mg choline/d for 12 wk. Fasting blood samples and placental tissue and umbilical cord venous blood were collected and analyzed for choline and its metabolites. RESULTS Regardless of the choline intake, pregnant women had higher circulating concentrations of choline (30%; P < 0.001) but lower concentrations of betaine, dimethylglycine, sarcosine, and methionine (13-55%; P < 0.001). Obligatory losses of urinary choline and betaine in pregnant women were ∼2-4 times as high (P ≤ 0.02) as those in nonpregnant women. A higher choline intake yielded higher concentrations of choline, betaine, dimethylglycine, and sarcosine (12-46%; P ≤ 0.08) in both pregnant and nonpregnant women without affecting urinary choline excretion. The higher maternal choline intake also led to a doubling of dimethylglycine in cord plasma (P = 0.002). CONCLUSION These data suggest that an increment of 25 mg choline/d to meet the demands of pregnancy is insufficient and show that a higher maternal choline intake increases the use of choline as a methyl donor in both maternal and fetal compartments. This trial was registered at clinicaltrials.gov as NCT01127022.

A higher maternal choline intake among third-trimester pregnant women lowers placental and circulating concentrations of the antiangiogenic factor fms-like tyrosine kinase-1 (sFLT1)

This study investigated the influence of maternal choline intake on the human placental transcriptome, with a special interest in its role in modulating placental vascular function. Healthy pregnant women (n=26, wk 26–29 gestation) were randomized to 480 mg choline/d, an intake level approximating the adequate intake of 450 mg/d, or 930 mg/d for 12 wk. Maternal blood and placental samples were retrieved at delivery. Whole genome expression microarrays were used to identify placental genes and biological processes impacted by maternal choline intake. Maternal choline intake influenced a wide array of genes (n=166) and biological processes (n=197), including those related to vascular function. Of special interest was the 30% down‐regulation (P=0.05) of the antiangiogenic factor and preeclampsia risk marker fms‐like tyrosine kinase‐1 (sFLT1) in the placenta tissues obtained from the 930 vs. 480 mg/d choline intake group. Similar decreases (P=0.04) were detected in maternal blood sFLT1 protein concentrations. The down‐regulation of sFLT1 by choline treatment was confirmed in a human trophoblast cell culture model and may be related to enhanced acetylcholine signaling. These findings indicate that supplementing the maternal diet with extra choline may improve placental angiogenesis and mitigate some of the pathological antecedents of preeclampsia.—Jiang, X., Bar, H. Y., Yan, J., Jones, S., Brannon, P. M., West, A. A., Perry, C. A., Ganti, A., Pressman, E., Devapatla, S., Vermeylen, F., Wells, M. T., and Caudill, M. A. A higher maternal choline intake among third‐trimester pregnant women lowers placental and circulating concentrations of the antiangiogenic factor fms‐like tyrosine kinase‐1 (sFLT1). FASEB J. 27, 1245–1253 (2013). www.fasebj.org

Folate Intake, Mthfr Genotype, and Sex Modulate Choline Metabolism in Mice

Choline and folate are interrelated in 1-carbon metabolism, mostly because of their shared function as methyl donors for homocysteine remethylation. Folate deficiency and mutations of methylenetetrahydrofolate reductase (MTHFR) reduce the availability of a major methyl donor, 5-methyltetrahydrofolate, which in turn may lead to compensatory changes in choline metabolism. This study investigated the hypothesis that reductions in methyl group supply, either due to dietary folate deficiency or Mthfr gene deletion, would modify tissue choline metabolism in a sex-specific manner. Mthfr wild type (+/+) or heterozygous (+/-) knockout mice were randomized to a folate-deficient or control diet for 8 wk during which time deuterium-labeled choline (d9-choline) was consumed in the drinking water (~10 μmol/d). Mthfr heterozygosity did not alter brain choline metabolite concentrations, but it did enhance their labeling in males (P < 0.05) and tended to do so in females (P < 0.10), a finding consistent with greater turnover of dietary choline in brains of +/- mice. Dietary folate deficiency in females yielded 52% higher (P = 0.027) hepatic glycerophosphocholine, which suggests that phosphatidylcholine (PtdCho) degradation was enhanced. Labeling of the hepatic PtdCho in d3 form was also reduced (P < 0.001) in females, which implies that fewer of the dietary choline-derived methyl groups were used for de novo PtdCho biosynthesis under conditions of folate insufficiency. Males responded to folate restriction with a doubling (P < 0.001) of hepatic choline dehydrogenase transcripts, a finding consistent with enhanced conversion of choline to the methyl donor, betaine. Collectively, these data show that several adaptations in choline metabolism transpire as a result of mild perturbations in folate metabolism, presumably to preserve methyl group homeostasis.

Maternal choline supplementation: a nutritional approach for improving offspring health?

The modulatory role of choline on the fetal epigenome and the impact of in utero choline supply on fetal programming and health are of great interest. Studies in animals and/or humans suggest that maternal choline supplementation during pregnancy benefits important physiologic systems such as offspring cognitive function, response to stress, and cerebral inhibition. Because alterations in offspring phenotype frequently coincide with epigenetic modifications and changes in gene expression, maternal choline supplementation may be a nutritional strategy to improve lifelong health of the child. Future studies are warranted to elucidate further the effect of choline on the fetal epigenome and to determine the level of maternal choline intake required for optimal offspring physiologic function.

Pregnancy alters choline dynamics: results of a randomized trial using stable isotope methodology in pregnant and nonpregnant women

BACKGROUND Although biomarkers of choline metabolism are altered by pregnancy, little is known about the influence of human pregnancy on the dynamics of choline-related metabolic processes. OBJECTIVE This study used stable isotope methodology to examine the effects of pregnancy on choline partitioning and the metabolic activity of choline-related pathways. DESIGN Healthy third-trimester pregnant (n = 26; initially week 27 of gestation) and nonpregnant (n = 21) women consumed 22% of their total choline intake (480 or 930 mg/d) as methyl-d9-choline for the final 6 wk of a 12-wk feeding study. RESULTS Plasma d9-betaine:d9-phosphatidylcholine (PC) was lower (P ≤ 0.04) in pregnant than in nonpregnant women, suggesting greater partitioning of choline into the cytidine diphosphate-choline (CDP-choline) PC biosynthetic pathway relative to betaine synthesis during pregnancy. Pregnant women also used more choline-derived methyl groups for PC synthesis via phosphatidylethanolamine N-methyltransferase (PEMT) as indicated by comparable increases in PEMT-PC enrichment in pregnant and nonpregnant women despite unequal (pregnant > nonpregnant; P < 0.001) PC pool sizes. Pregnancy enhanced the hydrolysis of PEMT-PC to free choline as shown by greater (P < 0.001) plasma d3-choline:d3-PC. Notably, d3-PC enrichment increased (P ≤ 0.011) incrementally from maternal to placental to fetal compartments, signifying the selective transfer of PEMT-PC to the fetus. CONCLUSIONS The enhanced use of choline for PC production via both the CDP-choline and PEMT pathways shows the substantial demand for choline during late pregnancy. Selective partitioning of PEMT-PC to the fetal compartment may imply a unique requirement of PEMT-PC by the developing fetus.

Low dietary folate and methylenetetrahydrofolate reductase deficiency may lead to pregnancy complications through modulation of ApoAI and IFN‐γ in spleen and placenta, and through reduction of methylation potential

SCOPE Genetic or nutritional disturbances in folate metabolism lead to hyperhomocysteinemia and adverse reproductive outcomes. Folate-dependent homocysteine remethylation is required for methylation reactions and may influence choline/betaine metabolism. Hyperhomocysteinemia has been suggested to play a role in inflammation. The goal of this study was to determine whether folate-related pregnancy complications could be due to altered expression of some inflammatory mediators or due to disturbances in methylation intermediates. METHODS AND RESULTS Pregnant mice with or without a deficiency of methylenetetrahydrofolate reductase (MTHFR) were fed control diets or folate-deficient (FD) diets; tissues were collected at embryonic day 14.5. FD decreased plasma phosphocholine and increased plasma glycerophosphocholine and lysophosphatidylcholine. Liver betaine, phosphocholine, and S-adenosylmethionine:S-adenosylhomocysteine ratios were reduced in FD. In liver, spleen, and placenta, the lowest levels of apolipoprotein AI (ApoAI) were observed in Mthfr(+/-) mice fed FD. Increased interferon-gamma (IFN-γ) was observed in spleen and placentae due to FD or Mthfr genotype. Plasma homocysteine correlated negatively with liver and spleen ApoAI, and positively with IFN-γ. CONCLUSION Low dietary folate or Mthfr deficiency during pregnancy may result in adverse pregnancy outcomes by altering expression of the inflammatory mediators ApoAI and IFN-γ in spleen and placenta. Disturbances in choline metabolism or methylation reactions may also play a role.

Choline intake influences phosphatidylcholine DHA enrichment in nonpregnant women but not in pregnant women in the third trimester

BACKGROUND Phosphatidylcholine (PC) produced via the S-adenosylmethionine-dependent phosphatidylethanolamine (PE) N-methyltransferase (PEMT) pathway is enriched with docosahexaenoic acid (DHA). DHA plays a critical role in fetal development and is linked to health endpoints in adulthood. It is unknown whether choline, which can serve as a source of S-adenosylmethionine methyl groups, influences PC-DHA or the PC:PE ratio in pregnant and nonpregnant women. OBJECTIVE This study tested whether choline intake affects indicators of choline-related lipid metabolism, including erythrocyte and plasma PC-DHA and PC:PE ratios, in pregnant women in the third trimester and nonpregnant women. DESIGN Pregnant (n = 26) and nonpregnant (n = 21) women consumed 480 or 930 mg choline/d and a daily DHA supplement for 12 wk. Blood was collected at baseline and at the midpoint and end of the study. PC-DHA was analyzed as the proportion of total PC fatty acids. RESULTS Pregnant women had greater (P = 0.002) PC-DHA concentrations than did nonpregnant women at baseline. The proportion of erythrocyte and plasma PC-DHA increased (P ≤ 0.002) in pregnant and nonpregnant women regardless of choline intake. However, in nonpregnant women, consumption of 930 mg choline/d led to greater (P < 0.001) erythrocyte PC-DHA and a more rapid increase (P < 0.001) in plasma PC-DHA. Lower (P = 0.001-0.024) erythrocyte and plasma PC:PE in pregnant women was not modified by choline intake. CONCLUSIONS A higher choline intake may increase PEMT activity, resulting in greater PC-DHA enrichment of the PC molecule in nonpregnant women. Increased production of PC-DHA during pregnancy indicates elevated PEMT activity and a higher demand for methyl donors. This trial was registered at clinicaltrials.gov as NCT01127022.

Vitamin B-12 Status Differs among Pregnant, Lactating, and Control Women with Equivalent Nutrient Intakes

BACKGROUND Limited data are available from controlled studies on biomarkers of maternal vitamin B-12 status. OBJECTIVE We sought to quantify the effects of pregnancy and lactation on the vitamin B-12 status response to a known and highly controlled vitamin B-12 intake. METHODS As part of a 10-12 wk feeding trial, pregnant (26-29 wk gestation; n = 26), lactating (5 wk postpartum; n = 28), and control (nonpregnant, nonlactating; n = 21) women consumed vitamin B-12 amounts of ∼8.6 μg/d [mixed diet (∼6 μg/d) plus a prenatal multivitamin supplement (2.6 μg/d)]. Serum vitamin B-12, holotranscobalamin (bioactive form of vitamin B-12), methylmalonic acid (MMA), and homocysteine were measured at baseline and study-end. RESULTS All participants achieved adequate vitamin B-12 status in response to the study dose. Compared with control women, pregnant women had lower serum vitamin B-12 (-21%; P = 0.02) at study-end, whereas lactating women had higher (P = 0.04) serum vitamin B-12 throughout the study (+26% at study-end). Consumption of the study vitamin B-12 dose increased serum holotranscobalamin in all reproductive groups (+16-42%; P ≤ 0.009). At study-end, pregnant (vs. control) women had a higher holotranscobalamin-to-vitamin B-12 ratio (P = 0.04) with ∼30% (vs. 20%) of total vitamin B-12 in the bioactive form. Serum MMA increased during pregnancy (+50%; P < 0.001) but did not differ by reproductive state at study-end. Serum homocysteine increased in pregnant women (+15%; P = 0.009) but decreased in control and lactating women (-16-17%; P < 0.001). Despite these changes, pregnant women had ∼20% lower serum homocysteine than the other 2 groups at study-end (P ≤ 0.02). CONCLUSION Pregnancy and lactation alter vitamin B-12 status in a manner consistent with enhanced vitamin B-12 supply to the child. Consumption of the study vitamin B-12 dose (∼3 times the RDA) increased the bioactive form of vitamin B-12, suggesting that women in these reproductive states may benefit from vitamin B-12 intakes exceeding current recommendations. This trial was registered at clinicaltrials.gov as NCT01127022.

Choline intakes exceeding recommendations during human lactation improve breast milk choline content by increasing PEMT pathway metabolites

Demand for the vital nutrient choline is high during lactation; however, few studies have examined choline metabolism and requirements in this reproductive state. The present study sought to discern the effects of lactation and varied choline intake on maternal biomarkers of choline metabolism and breast milk choline content. Lactating (n=28) and control (n=21) women were randomized to 480 or 930 mg choline/day for 10-12 weeks as part of a controlled feeding study. During the last 4-6 weeks, 20% of the total choline intake was provided as an isotopically labeled choline tracer (methyl-d9-choline). Blood, urine and breast milk samples were collected for choline metabolite quantification, enrichment measurements, and gene expression analysis of choline metabolic genes. Lactating (vs. control) women exhibited higher (P < .001) plasma choline concentrations but lower (P ≤ .002) urinary excretion of choline metabolites, decreased use of choline as a methyl donor (e.g., lower enrichment of d6-dimethylglycine, P ≤ .08) and lower (P ≤ .02) leukocyte expression of most choline-metabolizing genes. A higher choline intake during lactation differentially influenced breast milk d9- vs. d3-choline metabolite enrichment. Increases (P ≤ .03) were detected among the d3-metabolites, which are generated endogenously via the hepatic phosphatidylethanolamine N-methyltransferase (PEMT), but not among the d9-metabolites generated from intact exogenous choline. These data suggest that lactation induces metabolic adaptations that increase the supply of intact choline to the mammary epithelium, and that extra maternal choline enhances breast milk choline content by increasing supply of PEMT-derived choline metabolites. This trial was registered at clinicaltrials.gov as NCT01127022.