Head group specificity in the requirement of phosphatidylcholine biosynthesis for very low density lipoprotein secretion from cultured hepatocytes

We have demonstrated that hepatic very low density lipoprotein (VLDL) secretion requires active phosphatidylcholine (PC) synthesis via either the CDP-choline pathway or phosphatidylethanolamine (PE) methylation pathway (Yao, Z., and Vance, D.E. (1988) J. Biol. Chem. 263, 2998-3004). In the present work, the head group specificity of phospholipid synthesis required for lipoprotein secretion was investigated in cultured hepatocytes isolated from choline-deficient rats. When N-monomethylethanolamine (0.1 mM) or N,N-dimethylethanolamine (0.1 mM) was added to the culture medium, the cells synthesized correspondingly phosphatidylmonomethylethanolamine (PMME) or phosphatidyldimethylethanolamine (PDME). However, the synthesis of PDME could correct the impaired VLDL secretion only to a limited extent, whereas the synthesis of PMME inhibited VLDL secretion. Although dimethylethanolamine did not promote VLDL secretion as well as choline, dimethylethanolamine altered the increased triacylglycerol synthesis in the choline-deficient cells as effectively as choline. Supplementation of the culture medium with ethanolamine (0.1 mM) had little effect on cellular PE or PC levels, nor was normal VLDL secretion resumed. However, the amounts of cellular PC and PE were both decreased when the medium was supplemented with N-monomethylethanolamine or N,N-dimethylethanolamine. These results suggest that the choline head group moiety of PC is specifically required for normal VLDL secretion and cannot be replaced with ethanolamine, monomethylethanolamine, or dimethylethanolamine. In addition, the impaired VLDL secretion from the choline-deficient hepatocytes could also be corrected by supplementation of betaine (0.2 mM) and homocysteine (0.2 mM), indicating the utilization of a methyl group from betaine for PC formation via methylation of PE.     

Molecular distinction of phosphatidylcholine synthesis between the CDP-choline pathway and phosphatidylethanolamine methylation pathway.

In addition to the CDP-choline pathway for phosphatidylcholine (PC) synthesis, the liver has a unique phosphatidylethanolamine (PE) methyltransferase activity for PC synthesis via three methylations of the ethanolamine moiety of PE. Previous studies indicate that the two pathways are functionally different and not interchangeable even though PC is the common product of both pathways. This study was designed to test the hypothesis that these two pathways produce different profiles of PC species. The PC species from these two pathways were labeled with specific stable isotope precursors, D9-choline and D4-ethanolamine, and analyzed by electrospray tandem mass spectrometry. Our studies revealed a profound distinction in PC profiles between the CDP-choline pathway and the PE methylation pathway. PC molecules produced from the CDP-choline pathway were mainly comprised of medium chain, saturated (e.g. 16:0/18:0) species. On the other hand, PC molecules from the PE methylation pathway were much more diverse and were comprised of significantly more long chain, polyunsaturated (e.g. 18:0/20:4) species. PC species from the methylation pathway contained a higher percentage of arachidonate and were more diverse than those from the CDP-choline pathway. This profound distinction of PC profiles may contribute to the different functions of these two pathways in the liver.     

Phosphatidylcholine Supply to Peroxisomes of the Yeast Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, phosphatidylcholine (PC), the major phospholipid (PL) of all organelle membranes, is synthesized via two different pathways. Methylation of phosphatidylethanolamine (PE) catalyzed by the methyl transferases Cho2p/Pem1p and Opi3p/Pem2p as well as incorporation of choline through the CDP (cytidine diphosphate)-choline branch of the Kennedy pathway lead to PC formation. To determine the contribution of these two pathways to the supply of PC to peroxisomes (PX), yeast mutants bearing defects in the two pathways were cultivated under peroxisome inducing conditions, i.e. in the presence of oleic acid, and subjected to biochemical and cell biological analyses. Phenotype studies revealed compromised growth of both the cho20Δopi3Δ (mutations in the methylation pathway) and the cki1Δdpl1Δeki1Δ (mutations in the CDP-choline pathway) mutant when grown on oleic acid. Analysis of peroxisomes from the two mutant strains showed that both pathways produce PC for the supply to peroxisomes, although the CDP-choline pathway seemed to contribute with higher efficiency than the methylation pathway. Changes in the peroxisomal lipid pattern of mutants caused by defects in the PC biosynthetic pathways resulted in changes of membrane properties as shown by anisotropy measurements with fluorescent probes. In summary, our data define the origin of peroxisomal PC and demonstrate the importance of PC for peroxisome membrane formation and integrity.     

Biosynthesis of phosphatidylcholine in bacteria

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes and can be synthesized by either of two pathways, the methylation pathway or the CDP-choline pathway. Many prokaryotes lack PC, but it can be found in significant amounts in membranes of rather diverse bacteria and based on genomic data, we estimate that more than 10% of all bacteria possess PC. Enzymatic methylation of phosphatidylethanolamine via the methylation pathway was thought to be the only biosynthetic pathway to yield PC in bacteria. However, a choline-dependent pathway for PC biosynthesis has been discovered in Sinorhizobium meliloti. In this pathway, PC synthase, condenses choline directly with CDP-diacylglyceride to form PC in one step. A number of symbiotic (Rhizobium leguminosarum, Mesorhizobium loti) and pathogenic (Agrobacterium tumefaciens, Brucella melitensis, Pseudomonas aeruginosa, Borrelia burgdorferi and Legionella pneumophila) bacteria seem to possess the PC synthase pathway and we suggest that the respective eukaryotic host functions as the provider of choline for this pathway. Pathogens entering their hosts through epithelia (Streptococcus pneumoniae, Haemophilus influenzae) require phosphocholine substitutions on their cell surface components that are biosynthetically also derived from choline supplied by the host. However, the incorporation of choline in these latter cases proceeds via choline phosphate and CDP-choline as intermediates. The occurrence of two intermediates in prokaryotes usually found as intermediates in the eukaryotic CDP-choline pathway for PC biosynthesis raises the question whether some bacteria might form PC via a CDP-choline pathway.     

Turnover of phosphatidylcholine in Saccharomyces cerevisiae. The role of the CDP-choline pathway

The regulation of phosphatidylcholine degradation as a function of the route of phosphatidylcholine (PC) synthesis and changing environmental conditions has been investigated in the yeast Saccharomyces cerevisiae. In the wild-type strains studied, deacylation of phosphatidylcholine to glycerophosphocholine is induced when choline is supplied to the culture medium and, also, when the culture temperature is raised from 30 to 37 degrees C. In strains bearing mutations in any of the genes encoding enzymes of the CDP-choline pathway for phosphatidylcholine biosynthesis (CKI1, choline kinase; CPT1, 1, 2-diacylglycerol choline phosphotransferase; PCT1, CTP:phosphocholine cytidylyltransferase), no induction of phosphatidylcholine turnover and glycerophosphocholine production is seen in response to choline availability or elevated temperature. In contrast, the induction of phosphatidylcholine deacylation does occur in a strain bearing mutations in genes encoding enzymes of the methylation pathway for phosphatidylcholine biosynthesis (i.e. CHO2/PEM1 and OPI3/PEM2). Whereas the synthesis of PC via CDP-choline is accelerated when shifted from 30 to 37 degrees C, synthesis of PC via the methylation pathway is largely unaffected by the temperature shift. These results suggest that the deacylation of PC to GroPC requires an active CDP-choline pathway for PC biosynthesis but not an active methylation pathway. Furthermore, the data indicate that the synthesis and turnover of CDP-choline-derived PC, but not methylation pathway-derived PC, are accelerated by the stress of elevated temperature.     

Phosphatidylcholine and choline homeostasis

Phosphatidylcholine (PC) is made in mammalian cells from choline via the CDP-choline pathway. Animals obtain choline primarily from the diet or from the conversion of phosphatidylethanolamine (PE) to PC followed by catabolism to choline. The main fate of choline is the synthesis of PC. In addition, choline is oxidized to betaine in kidney and liver and converted to acetylcholine in the nervous system. Mice that lack choline kinase (CK) alpha die during embryogenesis, whereas mice that lack CKbeta unexpectedly develop muscular dystrophy. Mice that lack CTP:phosphocholine cytidylyltransferase (CT) alpha also die during early embryogenesis, whereas mice that lack CTbeta exhibit gonadal dysfunction. The cytidylyltransferase beta isoform also plays a role in the branching of axons of neurons. An alternative PC biosynthetic pathway in the liver uses phosphatidylethanolamine N-methyltransferase to catalyze the formation of PC from PE. Mice that lack the methyltransferase survive but die from steatohepatitis and liver failure when placed on a choline-deficient diet. Hence, choline is an essential nutrient. PC biosynthesis is required for normal very low density lipoprotein secretion from hepatocytes. Recent studies indicate that choline is recycled in the liver and redistributed from kidney, lung, and intestine to liver and brain when choline supply is attenuated.     

Specificity and rate of human and mouse liver and plasma phosphatidylcholine synthesis analyzed in vivo

Phosphatidylcholine (PC) synthesis by the direct cytidine diphosphate choline (CDP-choline) pathway in rat liver generates predominantly mono- and di-unsaturated molecular species, while polyunsaturated PC species are synthesized largely by the phosphatidylethanolamine-N-methyltransferase (PEMT) pathway. Although altered PC synthesis has been suggested to contribute to development of hepatocarcinoma and nonalcoholic steatohepatitis, analysis of the specificity of hepatic PC metabolism in human patients has been limited by the lack of sensitive and safe methodologies. Here we incorporated a deuterated methyl-D(9)-labled choline chloride, to quantify biosynthesis fluxes through both of the PC synthetic pathways in vivo in human volunteers and compared these fluxes with those in mice. Rates and molecular specificities of label incorporated into mouse liver and plasma PC were very similar and strongly suggest that label incorporation into human plasma PC can provide a direct measure of hepatic PC synthesis in human subjects. Importantly, we demonstrate for the first time that the PEMT pathway in human liver is selective for polyunsaturated PC species, especially those containing docosahexaenoic acid. Finally, we present a multiple isotopomer distribution analysis approach, based on transfer of deuterated methyl groups to S-adenosylmethionine and subsequent sequential methylations of PE, to quantify absolute flux rates through the PEMT pathway that are applicable to studies of liver dysfunction in clinical studies.     

The two biosynthetic routes leading to phosphatidylcholine in yeast produce different sets of molecular species. Evidence for lipid remodeling

Phosphatidylcholine (PC), a major lipid class in the membranes of eukaryotes, is synthesized either via the triple methylation of phosphatidylethanolamine (PE) or via the CDP-choline route. To investigate whether the two biosynthetic routes contribute differently to the steady-state profile of PC species, i.e., PC molecules with specific acyl chain compositions, the pools of newly synthesized PC species were monitored by labeling Saccharomyces cerevisiae with deuterated precursors of the two routes, (methyl-D3)-methionine and (D13)-choline, respectively. Electrospray ionization tandem mass spectrometry (ESI-MS/MS) revealed that the two PC biosynthetic pathways yield different sets of PC species, with the CDP-choline route contributing most to the molecular diversity. Moreover, yeast was shown to be capable of remodeling PC by acyl chain exchange at the sn-1 position of the glycerol backbone. Remodeling was found to be required to generate the steady-state species distribution of PC. This is the first study demonstrating a functional difference between the two biosynthetic routes in yeast.     

Synthesis of phosphatidylcholine by rat lung during choline deficiency

The effect of choline deficiency on the de novo pathway for phosphatidylcholine (PC) synthesis in the lung was investigated in rats fed a washed soy protein (lipotrophic) diet deficient in choline and methionine for 2-3 wk. Lungs from lipotrophic rats showed a decreased content of choline and choline-phosphate (P less than 0.05) compared with control but no change in content of cytidine 5'-diphosphocholine or PC. Isolated perfused lungs from lipotrophic rats were evaluated for choline and fatty acid utilization for PC synthesis. Lipotrophic lungs perfused with 5 microM [14C-methyl]-choline chloride showed increased incorporation into PC while there was no significant effect at saturating levels of choline (100 microM). There was increased incorporation of [1-14C]-palmitic acid into PC and diglyceride and increased incorporation of D-[U-14C]glucose into fatty acids of PC. Increased choline and glucose incorporation was not due to alteration of intracellular specific activity of these substrates. This study indicates the utilization of choline and fatty acid for PC synthesis is stimulated as a result of choline deficiency while lung CDP-choline concentration is maintained, possibly through regulation of choline phosphate cytidyl transferase activity. These mechanisms compensate for decreased choline availability to maintain the PC content of lungs.     

Choline Deficiency in Cultured Adrenal Medullary Cells: Effect on Phosphatidylcholine Biosynthesis

The effect of choline deficiency on the composition and biosynthesis of the major membrane phospholipids was examined in adrenal medullary cells maintained in suspension cultures. The amount and proportions of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in these cells were not affected by the removal of choline from the culture media. However, the rate of biosynthesis of choline at the phosphatide level by the stepwise methylation of PE increased twofold within 24 h after choline was removed from the culture media, while ethanolamine incorporation into PE was increased by 50%. In contrast, the rate of incorporation of labeled choline into PC, presumably via CDP-choline, was virtually identical in cells that had been preincubated in the presence or absence of 1 mM choline. These results demonstrate that cultured cells of neural origin are capable of compensating for lack of exogenous choline by forming choline at the phosphatide level through the sequential methylation of PE. The hypolipidemic drug, DH-990, when added to the culture media, inhibited conversion of phosphatidylmonomethylethanolamine (PME) to PC, but had no effect on the N-methylation of PE. This differential effect indicates that the initial N-methylation of PE is catalyzed by an enzyme that is distinguishable from the enzyme(s) catalyzing the conversion of PME to PC.     

Comparative study of the effects of short- and long-term ethanol treatment and alcohol withdrawal on phospholipid biosynthesis in rat hepatocytes

This study describes the effects of short- and long-term ethanol treatment and withdrawal on the biosynthesis of the phospholipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in hepatocytes isolated from rats, using isotopically labelled choline and ethanolamine as exogenous precursors. Our results demonstrate that short-term ethanol consumption increases the incorporation of exogenous polar bases into PC and PE, whereas long-term ethanol administration provokes a differential effect in both PC and PE biosynthesis via cytidine diphosphate derivatives (CDP-derivatives), decreasing PC synthesis and increasing the biosynthesis of PE. We suggest that the increased biosynthesis of PE after ethanol treatment results from changes in lipogenic substrates produced as a consequence of ethanol metabolism, whilst the specific inhibition of PC biosynthesis seems to be a consequence of alterations of enzymes involved in the CDP-choline pathway. With regard to the influence of ethanol on PE methylation to give PC, our results demonstrate that ethanol activates this pathway in short-term, as well as chronic ethanol treatment. Ethanol withdrawal returns the activity of the PC and PE pathways to control levels. The alterations in the biosynthesis of the main phospholipids, PC and PE, demonstrated in this study could be of a great physiological interest in determining the pathology of alcoholism.     

神经节苷脂GM_3对人白血病J6－2细胞磷脂酰胆碱代谢的影响

神经节苷脂ＧＭ３诱导人单核样白血病Ｊ６－２细胞沿单核／巨噬细胞途径分化．在ＧＭ３诱导分化同时,Ｊ６－２细胞磷脂代谢发生了显著变化．采用（（３２）Ｐ）Ｐｉ、［ＧＨ３－３Ｈ］胆碱和［ＣＨ３－３Ｈ］ＳＡＭ参入实验对ＧＭ３影响Ｊ６－２细胞ＰＣ代谢的机制进行了初步的探讨．ＧＭ３促进［（３２）Ｐ］Ｐｉ参入Ｊ６－２细胞ＰＣ;抑制［ＣＨ３－３Ｈ］胆碱参入ＰＣ及ＰＣ合成的前体磷酸胆碱及ＣＤＰ－胆碱;ＧＭ３促进［ＣＨ３－３Ｈ］ＳＡＭ参入ＰＣ,但抑制［ＣＨ３－３Ｈ］ＳＡＭ参入ＰＣ合成的前体胆碱、磷酸胆碱和ＣＤＰ－胆碱．上述结果提示,ＧＭ３抑制Ｊ６－２细胞ＰＣ合成的ＣＤＰ－胆碱途径,促进ＰＣ合成的ＰＥ甲基化途径．     

Novel pathway for phosphatidylcholine biosynthesis in bacteria associated with eukaryotes

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes and can be synthesised by either of two pathways, the CDP-choline pathway or the methylation pathway. Many prokaryotes lack PC, but it can be found in significant amounts in membranes of distantly related bacteria such as Rhizobacteria and Spirochetes. Enzymatic methylation of phosphatidylethanolamine via the methylation pathway was thought to be the only biosynthetic pathway to yield PC in bacteria. However, a novel choline-dependent pathway for PC biosynthesis has been discovered in Sinorhizobium meliloti. In this pathway, a novel enzymatic activity, PC synthase, condenses choline directly with CDP-diacylglyceride to form PC in one step. Surprisingly, genomes of some pathogens (Pseudomonas aeruginosa, Borrelia burgdorferi and Legionella pneumophila) contain genes similar to the sinorhizobial gene for phosphatidylcholine synthase. We, therefore, suggest that the new PC synthase pathway is present in a number of bacteria displaying symbiotic or pathogenic associations with eukaryotes and that the eukaryotic host functions as the provider of choline for this pathway.     

Rhizobium meliloti mutants deficient in phospholipid N-methyltransferase still contain phosphatidylcholine.

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes. In addition to this structural function, PC is thought to play a major role in lipid turnover and signalling in eukaryotic systems. In prokaryotes, only some groups of bacteria, among them the members of the family Rhizobiaceae, contain PC. To understand the role of PC in bacteria, we have studied Rhizobium meliloti 1021, which is able to form nitrogen-fixing nodules on its legume host plants and therefore has a very complex phenotype. R. meliloti was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine, and potential mutants defective in phospholipid N-methyltransferase were screened by using a colony autoradiography procedure. Filters carrying lysed replicas of mutagenized colonies were incubated with S-adenosyl-L-[methyl-14C]methionine. Enzymatic transfer of methyl groups to phosphatidylethanolamine (PE) leads to the formation of PC and therefore to the incorporation of radiolabel into lipid material. Screening of 24,000 colonies for reduced incorporation of radiolabel into lipids led to the identification of seven mutants which have a much-reduced specific activity of phospholipid N-methyltransferase. In vivo labelling of mutant lipids with [14C]acetate showed that the methylated PC biosynthesis intermediates phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine are no longer detectable. This loss is combined with a corresponding increase in the potential methyl acceptor PE. These results indicate that PC biosynthesis via the methylation pathway is indeed blocked in the mutants isolated. However, mass spectrometric analysis of the lipids shows that PC was still present when the mutants had been grown on complex medium and that it was present in the mutants in wild-type amounts. In vivo labelling with [methyl-14C]methionine shows that in phospholipid N-methyltransferase-deficient mutants, the choline moiety of PC is not formed by methylation. These findings suggest the existence of a second pathway for PC biosynthesis in Rhizobium.     

Effect of interferon on phospholipid methylation by peripheral blood mononuclear cells

The effect of human interferon (IFN) preparations on the metabolic pathway leading to the synthesis of phosphatidylcholine (PC) by a stepwise addition of methyl groups to phosphatidylethanolamine (PE) was investigated in human peripheral blood mononuclear (PBMN) cells. An inhibition of the synthesis of PC via this pathway was regularly observed with both alpha- (recombinant or natural) and beta-IFN. This inhibition was apparent within the first 5 min of treatment, reached its maximum between 15 min and 1 hr, and persisted at the same level until 6 hr, the last time point examined. Each of the transmethylated products of PE underwent a similar inhibition, as measured by the turnover rate of individual products. The intracellular pool of the methyl donors, methionine and S-adenosyl-methionine (SAM), was shown to be unaffected. The methyltransferase activity of IFN-pretreated cell extracts was unchanged. These findings support the hypothesis that IFN induces a functional change in phospholipid methylation at the level of organized membrane-bound phospholipid methyltransferase enzymes in intact cells.     

Nutrition and epigenetics: an interplay of dietary methyl donors, one-carbon metabolism and DNA methylation

DNA methylation is the most extensively studied mechanism of epigenetic gene regulation. Increasing evidence indicates that DNA methylation is labile in response to nutritional and environmental influences. Alterations in DNA methylation profiles can lead to changes in gene expression, resulting in diverse phenotypes with the potential for increased disease risk. The primary methyl donor for DNA methylation is S-adenosylmethionine (SAM), a species generated in the cyclical cellular process called one-carbon metabolism. One-carbon metabolism is catalyzed by several enzymes in the presence of dietary micronutrients, including folate, choline, betaine and other B vitamins. For this reason, nutrition status, particularly micronutrient intake, has been a focal point when investigating epigenetic mechanisms. Although animal evidence linking nutrition and DNA methylation is fairly extensive, epidemiological evidence is less comprehensive. This review serves to integrate studies of the animal in vivo with human epidemiological data pertaining to nutritional regulation of DNA methylation and to further identify areas in which current knowledge is limited.     

Human choline dehydrogenase: Medical promises and biochemical challenges

Human choline dehydrogenase (CHD) is located in the inner membrane of mitochondria primarily in liver and kidney and catalyzes the oxidation of choline to glycine betaine. Its physiological role is to regulate the concentrations of choline and glycine betaine in the blood and cells. Choline is important for regulation of gene expression, the biosynthesis of lipoproteins and membrane phospholipids and for the biosynthesis of the neurotransmitter acetylcholine; glycine betaine plays important roles as a primary intracellular osmoprotectant and as methyl donor for the biosynthesis of methionine from homocysteine, a required step for the synthesis of the ubiquitous methyl donor S-adenosyl methionine. Recently, CHD has generated considerable medical attention due to its association with various human pathologies, including male infertility, homocysteinuria, breast cancer and metabolic syndrome. Despite the renewed interest, the biochemical characterization of the enzyme has lagged behind due to difficulties in the obtainment of purified, active and stable enzyme. This review article summarizes the medical relevance and the physiological roles of human CHD, highlights the biochemical knowledge on the enzyme, and provides an analysis based on the comparison of the protein sequence with that of bacterial choline oxidase, for which structural and biochemical information is available.     

Diverse pathways of phosphatidylcholine biosynthesis in algae as estimated by labeling studies and genomic sequence analysis

Phosphatidylcholine (PC) is an almost ubiquitous phospholipid in eukaryotic algae and plants but is not found in a few species, for example Chlamydomonas reinhardtii. We recently found that some species of the genus Chlamydomonas possess PC. In the universal pathway, PC is synthesized de novo by methylation of phosphatidylethanolamine (PE) or transfer of phosphocholine from cytidine diphosphate (CDP)‐choline to diacylglycerol. Phosphocholine, the direct precursor to CDP‐choline, is synthesized either by methylation of phosphoethanolamine or phosphorylation of choline. Here we analyzed the mechanism of PC biosynthesis in two species of Chlamydomonas (asymmetrica and sphaeroides) as well as in a red alga, Cyanidioschyzon merolae. Comparative genomic analysis of enzymes involved in PC biosynthesis indicated that C. merolae possesses only the PE methylation pathway. Radioactive tracer experiments using [³²P]phosphate showed delayed labeling of PC with respect to PE, which was consistent with the PE methylation pathway. In Chlamydomonas asymmetrica, labeling of PC was detected from the early time of incubation with [³²P]phosphate, suggesting the operation of phosphoethanolamine methylation pathway. Genomic analysis indeed detected the genes for the phosphoethanolamine methylation pathway. In contrast, the labeling of PC in C. sphaeroides was slow, suggesting that the PE methylation pathway was at work. These results as well as biochemical and computational results uncover an unexpected diversity of the mechanisms for PC biosynthesis in algae. Based on these results, we will discuss plausible mechanisms for the scattered distribution of the ability to biosynthesize PC in the genus Chlamydomonas.     

Gestational intake of methyl donors and global LINE-1 DNA methylation in maternal and cord blood: Prospective results from a folate-replete population

Maternal diet affects offspring DNA methylation in animal models, but evidence from humans is limited. We investigated the extent to which gestational intake of methyl donor nutrients affects global DNA methylation in maternal and umbilical cord blood. Among mother-infant pairs in Project Viva, a folate-replete US population, we estimated maternal intakes of vitamin B12, betaine, choline, folate, cadmium, zinc and iron periconceptionally and during the second trimester. We examined associations of these nutrients with DNA methylation, measured as %5-methyl cytosines (%5mC) in Long Interspersed Nuclear Element-1 (LINE-1), in first trimester (n = 830) and second trimester (n = 671) maternal blood and in cord blood at delivery (n = 516). Cord blood methylation was higher for male than female infants {mean [standard deviation (SD)] 84.8 [0.6] vs. 84.4 [0.7]%}. In the multivariable-adjusted model, maternal intake of methyl donor nutrients periconceptionally and during the second trimester of pregnancy was not positively associated with first trimester, second trimester or cord blood LINE-1 methylation. Periconceptional betaine intake was inversely associated with cord blood methylation [regression coefficient = -0.08% (95% confidence interval (CI): -0.14,-0.01)] but this association was attenuated after adjustment for dietary cadmium, which itself was directly associated with first trimester methylation and inversely associated with cord blood methylation. We also found an inverse association between periconceptional choline [-0.10%, 95% CI: -0.17,-0.03 for each SD (~63 mg/d)] and cord blood methylation in males only. In this folate-replete population, we did not find positive associations between intake of methyl donor nutrients during pregnancy and DNA methylation overall, but among males, higher early pregnancy intakes of choline were associated with lower cord blood methylation.