Induction and Aggravation of the Endoplasmic-Reticulum Stress by Membrane-Lipid Metabolic Intermediate Phosphatidyl-N-Monomethylethanolamine

Phosphatidylcholine (PC) is produced via two distinct pathways in both hepatocytes and yeast, Saccharomyces cerevisiae. One of these pathways involves the sequential methylation of phosphatidylethanolamine (PE). In yeast cells, the methyltransferase, Cho2, converts PE to phosphatidylmonomethylethanolamine (PMME), which is further modified to PC by another methyltransferase, Opi3. On the other hand, free choline is utilized for PC production via the Kennedy pathway. The blockage of PC production is well known to cause endoplasmic reticulum (ER) stress and activate the ER-stress sensor, Ire1, to induce unfolded protein response (UPR). Here, we demonstrate that even when free choline is sufficiently supplied, the opi3Δ mutation, but not the cho2 Δ mutation, induces the UPR. The UPR was also found to be induced by CHO2 overexpression. Further, monomethylethanolamine, which is converted to PMME probably through the Kennedy pathway, caused or potentiated ER stress in both mammalian and yeast cells. We thus deduce that PMME per se is an ER-stressing molecule. Interestingly, spontaneously accumulated PMME seemed to aggravate ER stress in yeast cells. Collectively, our findings demonstrate the multiple detrimental effects of the low-abundance phospholipid species, PMME.

School-Aged Children in Vancouver, Canada Do Not Meet Dietary Choline Recommendations but Meet Recommendations for Folate and Vitamin B12

Objectives Nutrients such as choline, betaine, folate, and vitamin B12 are required for proper growth and development, but little is known about these nutrients in children. Our objective was to determine intakes and biomarkers of these nutrients in school-aged children (5 to 6 years old). Methods A cross-sectional study of healthy children (n = 285) recruited from Vancouver, Canada were assessed. Dietary information was collected using three 24-hour recalls. Nutrient intakes were estimated using the Canadian Nutrient File and USDA database for choline; supplement use was collected by questionnaire. Plasma biomarkers were quantified by mass spectrometry and immunoassay. Results Daily dietary intakes were (mean ± SD) choline 250 ± 96 mg, betaine 88 ± 41 mg, folate 335 ± 126 µg DFE, and vitamin B12 3.78 ± 2.71 µg. Top food sources were dairy and eggs (42–57%) for choline and vitamin B12, and cereal and grains (41–60%) for betaine and folate. Only 40% of children met the choline adequate intake (AI) recommendation for North America (≥250 mg/d); 81.5% met the European AI (≥170 mg/d). Plasma free choline, betaine, and dimethylglycine concentrations were (mean ± SD) 8.62 ± 2.13 µmol/L, 45.3 ± 13.7 µmol/L, 3.34 ± 1.03 µmol/L, respectively. Dietary choline intake and plasma free choline were not related. However, dietary choline intake was positively associated with plasma dimethylglycine. Less than 5% of children had inadequate folate and vitamin B12 intakes. More than half of the children (59.4%) were consuming a supplement containing B-vitamins, but none included choline or betaine. Some children (5.26%) had total folic acid intakes above the North American upper limit (UL; >400 µg/d); 10.90% had intakes above the European UL (>300 µg/d). Vitamin B12 intake was positively associated with plasma total vitamin B12 (mean ± SD; 594 ± 158 pmol/L) in an adjusted model (sex, age, total energy intake, and supplement use; p < 0.05). Conclusions Our findings suggest that many school-aged children are not meeting dietary choline recommendations. Some children may have excessive folic acid intakes. The impact of imbalanced methyl nutrient intakes during this active period of growth and development needs to be determined in the future. Funding Sources CIHR, NSERC, and BC Children's Hospital Research Institute.

Choline Content of Term and Preterm Infant Formulae Compared to Expressed Breast Milk—How Do We Justify the Discrepancies?

Choline/phosphatidylcholine concentrations are tightly regulated in all organs and secretions. During rapid organ growth in the third trimester, choline requirement is particularly high. Adequate choline intake is 17–18 mg/kg/day in term infants, whereas ~50–60 mg/kg/day is required to achieve fetal plasma concentrations in preterm infants. Whereas free choline is supplied via the placenta, other choline carriers characterize enteral feeding. We therefore quantified the concentrations and types of choline carriers and choline-related components in various infant formulae and fortifiers compared to breast milk, and calculated the supply at full feeds (150 mL/kg/day) using tandem mass spectrometry. Choline concentration in formula ranged from values below to far above that of breastmilk. Humana 0-VLB (2015: 60.7 mg/150 mL; 2020: 27.3 mg/150 mL), Aptamil-Prematil (2020: 34.7 mg/150 mL), Aptamil-Prematil HA (2020: 37.6 mg/150 mL) for preterm infants with weights < 1800 g, and Humana 0 (2020: 41.6 mg/150 mL) for those > 1800 g, comprised the highest values in formulae studied. Formulae mostly were rich in free choline or phosphatidylcholine rather than glycerophosphocholine and phosphocholine (predominating in human milk). Most formulae (150 mL/kg/day) do not supply the amounts and physiologic components of choline required to achieve fetal plasma choline concentrations. A revision of choline content in formulae and breast milk fortifiers and a clear declaration of the choline components in formulae is required to enable informed choices.

Plasma Free Choline Concentration Did Not Reflect Dietary Choline Intake in Early and Late Pregnancy: Findings from the APrON Study

Objectives Choline is a critical nutrient for fetal development. Pregnancy studies showed that most women have choline intakes below the adequate intake (AI) level of 450 mg/d. Research on plasma free choline as an indicator of dietary choline intake showed conflicting results to date. We sought to compare plasma free choline concentration between women with different choline intakes and to explore the association between plasma free choline and dietary choline intake in early (EP) and late pregnancy (LP). Methods This study included data and non-fasting plasma samples from pregnant women enrolled in the Alberta Pregnancy Outcomes and Nutrition (APrON) cohort study. EP (&lt;20 weeks of gestation) and LP (&gt;20 weeks of gestation) dietary choline intake was estimated using a 24-hr recall. Two categories of dietary choline intake were created: 1) low choline (LCI), i.e., choline intake in 1st quartile (Q) in EP, with these women having choline intake in 1st or 2nd Q in LP (n = 61); 2) high choline intake (HCI), i.e., choline intake in 4th Q in EP and in 3rd or 4th Q in LP (n = 46). Linear mixed-effects models were used to explore the association between plasma free choline and dietary choline intake across EP and LP, after adjustment for maternal age, ethnicity and weeks of gestation. Results Median (IQR) maternal age was 32 (30–35) y, and 80% were Caucasian. LCI was 101 (86–109) and 109 (93–127) mg/day in EP and LP, respectively, and HCI was 251 (223–286) and 212 (177–274) mg/day. Plasma free choline (μmol/L) did not differ between LCI and HCI at EP [LCI: 10.6 (9.03, 12.9); HCI: 11.7 (10.2, 13.8)] and LP [LCI: 11.7 (10.6, 12.7); HCI: 12.7 (10.7, 15.8)] (P &gt; 0.05, Wilcoxon rank-sum test). Per 10 mg of choline intake, plasma free choline increased by 0.34 (95%CI 0.12, 0.56) in those with LCI, and 0.18 (95%CI 0.050, 0.31) in women with HCI, across EP and LP after adjustment. Conclusions In this subgroup of pregnant women, plasma free choline concentration did not reflect differences in dietary choline intake in EP or LP. This may be explained by an overall low choline intake (&lt;AI) which would promote rapid tissue uptake of choline. The identification of a sensitive and dynamic biomarker for choline status is required. Funding Sources UBC Four Year Doctoral Fellowship, Canada Research Chair Program, CIHR NTE Grant FRN 160,942, Alberta Innovates for the APrON cohort.

Threshold Effects of Plasma Methyl Donor Nutrients Status on High Lactate-Metabolomics Signatures of Metastatic Tumors in Non-Small-Cell Lung Cancer Patients

Objectives Lung cancer is the leading cause of cancer-related mortality in the worldwide. In non-small-cell lung cancers (NSCLC), metabolic reprogramming of lactate metabolism has been proposed as a key player in cancer metastasis. Nutritional status of methyl donors was associated with increased risks of lung cancers, yet their roles in lactate metabolism and metastatic NSCLC development remains unclear. Methods The cross-sectional study recruited 100 NSCLC patients with selected 18 paired NSCLC tissues from National Taiwan University Hospital (NTUH), Taiwan. Plasma methyl donors levels (folate/free choline/betaine) of NSCLC patients and target metabolomics signatures of paired NSCLC tissues were analyzed by Liquid chromatography–mass spectrometry (LC/MS). Clinical data of NSCLC tissues were collected from the department of pathology in NTUH. Results Partial Least Squares Discriminant Analysis (PLSDA) revealed that metabolic signatures of tumor/non-tumor lung tissues were well segregated. The two major tumor metabolites signatures of metastatic NSCLC were lactate and glucose, the levels of which were high and low, respectively, with both metabolites displaying the two highest VIP scores. A correlation heatmap showed that tumor lactate levels were strongly associated with increased TCA metabolites (pyruvate, succinate, fumarate and malate) and anaplerotic amino acid levels (alanine and arginine), and inversely correlated with glycolytic metabolites (glucose and PEP). When stratifying plasma methyl donors status of 18 paired NSCLC tumors with metabolomics VIP scores, threshold levels of folate (≥6 ng/ml), free choline (≥9.78 µmol/L) and betaine (≥62.0 µmol/L) were associated with high lactate and low glucose signatures of NSCLC. In particular, plasma betaine was positively associated with tumor lactate (r = 0.51, P = 0.031); plasma lactate was positively associated with tumor glucose (r = 0.53, P = 0.023). Conclusions Our data demonstrate that metastatic NSCLCs were signified with high-lactate metabolizing-metabolomics fingerprints which were modified by plasma methyl donors’ status with a differential threshold effect. Funding Sources This study was supported by the three-year grant from the Ministration of Science and Technology, Taiwan, ROC.

Placenta Lactate Is Inversely Associated with Birth Weight: Threshold Effects of Maternal Methyl Donor Status on Fetal Lactate Metabolism in Taiwanese Mother-Newborn Pairs

Objectives We examined associations between maternal methyl donor status with lymphocytic epigenetic markers at third trimester, fetal fuel metabolism in placenta and neonatal birth weight (BW). Methods This study included seventy-eight healthy mother-newborn pairs with placenta samples and newborn growth data from the Taiwan Pregnancy-Newborn Epigenetics cohort. Maternal plasma methyl donors (folate/free choline/betaine) and lymphocytic epigenetic marks (DNA methylation of LINE1 and H19 imprinted gene) at the third trimester were measured. Placenta fuel metabolites (glucose/lactate/folate), expressions of glycolytic enzymes (hexokinase II: HK-II and lactate dehydrogenase: LDH) and the metabolic signaling AMP-activated protein kinase (AMPK) were assayed. Associations with BW were analyzed by multiple linear regression. Results Both maternal LINE1 methylation (β: −0.289, P = 0.044) and placenta lactate level (β: −0.262, P = 0.034) predicted neonatal BW variance. Maternal RBC folate level predicted LINE1 methylation (β: 0.300, P = 0.048). The significance of fetal lactate-prediction on neonatal BW variance was negated by adjustment for placenta glucose, maternal methyl donor status (folate/free choline/betaine), and maternal prepregnancy BMI. Further adjustment for maternal epigenetic marks (LINE1 and H19 methylation) resumed the prediction power (β: −0.366, P = 0.039). By stratification of maternal and fetal methyl donor status, the significant fetal lactate-BW prediction only displayed in mothers with high plasma folate levels (&gt;10.2 ng/mL) (β: −0.371, P = 0.037), low free choline levels (&lt;12.9 μM) (β: −0.444, P = 0.038) and low betaine levels (&lt;13.4 μM) (β: −0.536, P = 0.010). As maternal plasma folate significantly predicted placenta folate (β: 0.283, P = 0.014), high placenta folate levels (&gt;17.5 ng/g) markedly strengthen the fetal lactate-BW prediction (β: −0.657, P = 0.001), which coincided with higher expression of placenta AMPK levels. Conclusions Our data highlights the inverse association between placenta lactate and birth weight. Maternal third trimester and placental methyl donor status may affect neonatal birth weight variance through their threshold effects on fetal lactate metabolism. Funding Sources This study was supported by a grant from the Ministry of Science and Technology, Taiwan.

Choline Supplementation Mitigates the Adverse Effects of a High Folic Acid Maternal Diet on Food Intake Regulation in the Offspring

Objectives Folic acid (FA) intake by many women in North America is exceeding recommendations. We have shown that high maternal FA induces methylation-dependent programming of energy regulation associated with an obesogenic phenotype in adult rat offspring. However, it is unclear if this is a direct effect of high FA or due to an imbalance between FA and other methyl-nutrients (i.e., choline) in the 1-carbon cycle. Unlike FA, choline intake by women is below recommendations and is absent from most prenatal supplements, potentially affecting fetal development. The objective of this study was to examine the mechanisms and effects of choline content in high FA maternal diets on in-utero programming of energy regulation and later-life offspring phenotype. Methods Pregnant Wistar rats were fed an AIN-93 G diet with recommended FA and choline (1X, RFRC, control), or 5X-FA diet with choline at 0.5X-(HFLC), 1X-(HFRC), or 2.5X- (HFHC). In pups at birth, brain and liver 1-carbon metabolites, hypothalamic DNA methyltransferase (DNMT) activity and global DNA methylation (5-mC%) were measured. At weaning, one male pup/dam was fed the control diet and weekly weight-gain and food intake were recorded for 20 weeks. Results Offspring born to dams on the HFLC and HFRC, but not HFHC diet, had higher food intake (P &lt; 0.05) and weight-gain (P &lt; 0.01) than controls. In liver at birth, free choline was lower in HFHC than in HFLC pups, but betaine was unaffected. In contrast, in brains, betaine but not free choline concentrations, directly reflected the maternal choline diets. These results suggest that choline may modulate central food intake pathways via the methyl-donor betaine, warranting further investigation. Hypothalamic DNMT activity was highest (P &lt; 0.05) in HFLC pups but global methylation was not affected. Thus, gene expression by RNA sequencing and gene-specific methylation in the hypothalamus is in progress to elucidate the mechanisms underlying the observed phenotype. Conclusions Increased maternal choline mitigates the high FA diet induced increase in body weight and food intake in the adult offspring and results in tissue-specific changes in 1-carbon metabolism at birth. These findings have potential application to human health, providing support to optimize choline and FA intakes by women of childbearing age. Funding Sources CIHR-INMD.

Free Choline, but Not Phosphatidylcholine, Elevates Circulating Trimethylamine-N-oxide and This Response Is Modified by the Gut Microbiota Composition in Healthy Men

Objectives Trimethylamine-N-oxide (TMAO), a choline-derived gut microbiota-dependent metabolite, is a newly recognized risk marker for cardiovascular disease. However, the contributions of different forms of choline and gut microbiota composition on TMAO production are largely unknown. The objectives of this study were to: 1) compare acute TMAO response to meals containing free choline (choline bitartrate) versus fat-soluble choline (phosphatidylcholine) and 2) to determine the effects of gut microbiota composition on TMAO response. Methods In a controlled, double-blinded, cross-over study, healthy men (n = 37) were provided meals containing (i) 600 mg choline as choline bitartrate (free choline); (ii) 600 mg choline as phosphatidylcholine; or (iii) no choline control in a random order. Blood and urine samples were collected at baseline and throughout the 6-h study period; a one-time stool sample was collected at baseline. Results Compared to no choline and phosphatidylcholine, free choline yielded 295% higher plasma TMAO (P = 0.002) and 250% higher urinary TMAO (P = 0.01), with no difference in TMAO response between phosphatidylcholine and no choline. High-TMAO producers (those with ≥40% increase in urinary TMAO response to free choline) had significantly different beta-diversity measures (unweighted UniFrac; PERMANOVA P = 0.01) compared to low-TMAO producers (those with &lt;40% increase in TMAO response) but showed no difference in alpha-diversity. Analysis of Composition of Microbiomes (ANCOM) revealed that high-TMAO producers had more abundant lineages of Clostridium from Ruminococcaceae and Lachnospiraceae (in phylum Firmicutes) compared to low-TMAO producers (P &lt; 0.05 with the strength of the ANCOM test W = 11 and W = 8, respectively). Conclusions Given that the majority of choline in food is in the form of phosphatidylcholine, the absence of TMAO elevation with phosphatidylcholine counters arguments that dietary choline should be avoided for TMAO-producing characteristics. Further, development of individualized dietary recommendations based on the gut microbiota composition may be a more appropriate strategy to reduce risk of cardiovascular disease. Funding Sources This research was supported by the Utah Agricultural Experiment Station Seed Grants Program.

Degradation of poly(ethylene terephthalate) catalyzed by metal-free choline-based ionic liquids

Glycolysis of PET is a prospective way for degradation of PET to its monomer bis(hydroxyethyl)terephthalate (BHET) which can be polymerized again to form new qualified PET materials, and hence provides possibilities for a permanent loop recycling.