Precursor Of Phosphatidylcholine Research Articles

The Endoplasmic Reticulum-Plasma Membrane Tethering Protein Ice2 Controls Lipid Droplet Size via the Regulation of Phosphatidylcholine in Candida albicans.

Lipid droplets (LDs) are intracellular organelles that play important roles in cellular lipid metabolism; they change their sizes and numbers in response to both intracellular and extracellular signals. Changes in LD size reflect lipid synthesis and degradation and affect many cellular activities, including energy supply and membrane synthesis. Here, we focused on the function of the endoplasmic reticulum-plasma membrane tethering protein Ice2 in LD dynamics in the fungal pathogen Candida albicans (C. albicans). Nile red staining and size quantification showed that the LD size increased in the ice2Δ/Δ mutant, indicating the critical role of Ice2 in the regulation of LD dynamics. A lipid content analysis further demonstrated that the mutant had lower phosphatidylcholine levels. As revealed with GFP labeling and fluorescence microscopy, the methyltransferase Cho2, which is involved in phosphatidylcholine synthesis, had poorer localization in the plasma membrane in the mutant than in the wild-type strain. Interestingly, the addition of the phosphatidylcholine precursor choline led to the recovery of normal-sized LDs in the mutant. These results indicated that Ice2 regulates LD size by controlling intracellular phosphatidylcholine levels and that endoplasmic reticulum-plasma membrane tethering proteins play a role in lipid metabolism regulation in C. albicans. This study provides significant findings for further investigation of the lipid metabolism in fungi.

Sexual differentiation in human malaria parasites is regulated by competition between phospholipid metabolism and histone methylation.

For Plasmodium falciparum, the most widespread and virulent malaria parasite that infects humans, persistence depends on continuous asexual replication in red blood cells, while transmission to their mosquito vector requires asexual blood-stage parasites to differentiate into non-replicating gametocytes. This decision is controlled by stochastic derepression of a heterochromatin-silenced locus encoding AP2-G, the master transcription factor of sexual differentiation. The frequency of ap2-g derepression was shown to be responsive to extracellular phospholipid precursors but the mechanism linking these metabolites to epigenetic regulation of ap2-g was unknown. Through a combination of molecular genetics, metabolomics and chromatin profiling, we show that this response is mediated by metabolic competition for the methyl donor S-adenosylmethionine between histone methyltransferases and phosphoethanolamine methyltransferase, a critical enzyme in the parasite's pathway for de novo phosphatidylcholine synthesis. When phosphatidylcholine precursors are scarce, increased consumption of SAM for de novo phosphatidylcholine synthesis impairs maintenance of the histone methylation responsible for silencing ap2-g, increasing the frequency of derepression and sexual differentiation. This provides a key mechanistic link that explains how LysoPC and choline availability can alter the chromatin status of the ap2-g locus controlling sexual differentiation.

Cytidine 5'-Diphosphocholine Corrects Alveolar Type II Cell Mitochondrial Dysfunction in Influenza-infected Mice.

Development of acute respiratory distress syndrome (ARDS) in influenza A virus (IAV)-infected mice is associated with inhibition of ATII (alveolar type II) epithelial cell de novo phosphatidylcholine synthesis, and administration of the phosphatidylcholine precursor cytidine 5'-diphosphocholine (CDP-choline) attenuates IAV-induced acute respiratory distress syndrome in mice. We hypothesized inhibition of phosphatidylcholine synthesis would also impact the function of ATII cell mitochondria. To test this hypothesis, adult C57BL/6 mice of both sexes were inoculated intranasally with 10,000 pfu/mouse influenza A/WSN/33 (H1N1). Control mice were mock-infected with virus diluent. Mice were treated with saline vehicle or CDP-choline (100 μg/mouse i.p.) once daily from 1 to 5 days postinoculation (dpi). ATII cells were isolated by a standard lung digestion protocol at 6 dpi for analysis of mitochondrial function. IAV infection increased uptake of the glucose analog fludeoxyglucose F 18 by the lungs and caused a switch from oxidative phosphorylation to aerobic glycolysis as a primary means of ATII cell ATP synthesis by 6 dpi. Infection also induced ATII cell mitochondrial depolarization and shrinkage, upregulation of PGC-1α, decreased cardiolipin content, and reduced expression of mitofusin 1, OPA1, DRP1, complexes I and IV of the electron transport chain, and enzymes involved in cardiolipin synthesis. Daily CDP-choline treatment prevented the declines in oxidative phosphorylation, mitochondrial membrane potential, and cardiolipin synthesis resulting from IAV infection but did not fully reverse the glycolytic shift. CDP-choline also did not prevent the alterations in mitochondrial protein expression resulting from infection. Taken together, our data show ATII cell mitochondrial dysfunction after IAV infection results from impaired de novo phospholipid synthesis, but the glycolytic shift does not.

Choline Kinetics in Neonatal Liver, Brain and Lung-Lessons from a Rodent Model for Neonatal Care.

Choline requirements are high in the rapidly growing fetus and preterm infant, mainly serving phosphatidylcholine (PC) synthesis for parenchymal growth and one-carbon metabolism via betaine. However, choline metabolism in critical organs during rapid growth is poorly understood. Therefore, we investigated the kinetics of D9-choline and its metabolites in the liver, plasma, brain and lung in 14 d old rats. Animals were intraperitoneally injected with 50 mg/kg D9-choline chloride and sacrificed after 1.5 h, 6 h and 24 h. Liver, plasma, lungs, cerebrum and cerebellum were analyzed for D9-choline metabolites, using tandem mass spectrometry. In target organs, D9-PC and D9-betaine comprised 15.1 ± 1.3% and 9.9 ± 1.2% of applied D9-choline at 1.5 h. D9-PC peaked at 1.5 h in all organs, and decreased from 1.5–6 h in the liver and lung, but not in the brain. Whereas D9-labeled PC precursors were virtually absent beyond 6 h, D9-PC increased in the brain and lung from 6 h to 24 h (9- and 2.5-fold, respectively) at the expense of the liver, suggesting PC uptake from the liver via plasma rather than local synthesis. Kinetics of D9-PC sub-groups suggested preferential hepatic secretion of linoleoyl-PC and acyl remodeling in target organs. D9-betaine showed rapid turnover and served low-level endogenous (D3-)choline synthesis. In conclusion, in neonatal rats, exogenous choline is rapidly metabolized to PC by all organs. The liver supplies the brain and lung directly with PC, followed by organotypic acyl remodeling. A major fraction of choline is converted to betaine, feeding the one-carbon pool and this must be taken into account when calculating choline requirements.

Citicoline and COVID-19-Related Cognitive and Other Neurologic Complications.

With growing concerns about COVID-19’s hyperinflammatory condition and its potentially damaging impact on the neurovascular system, there is a need to consider potential treatment options for managing short- and long-term effects on neurological complications, especially cognitive function. While maintaining adequate structure and function of phospholipid in brain cells, citicoline, identical to the natural metabolite phospholipid phosphatidylcholine precursor, can contribute to a variety of neurological diseases and hypothetically toward post-COVID-19 cognitive effects. In this review, we comprehensively describe in detail the potential citicoline mechanisms as adjunctive therapy and prevention of COVID-19-related cognitive decline and other neurologic complications through citicoline properties of anti-inflammation, anti-viral, neuroprotection, neurorestorative, and acetylcholine neurotransmitter synthesis, and provide a recommendation for future clinical trials.

Choline restores respiration in Psd1-deficient yeast by replenishing mitochondrial phosphatidylethanolamine

Phosphatidylethanolamine (PE) is essential for mitochondrial respiration in yeast, Saccharomyces cerevisiae, whereas the most abundant mitochondrial phospholipid, phosphatidylcholine (PC), is largely dispensable. Surprisingly, choline (Cho), which is a biosynthetic precursor of PC, has been shown to rescue the respiratory growth of mitochondrial PE-deficient yeast; however, the mechanism underlying this rescue has remained unknown. Using a combination of yeast genetics, lipid biochemistry, and cell biological approaches, we uncover the mechanism by showing that Cho rescues mitochondrial respiration by partially replenishing mitochondrial PE levels in yeast cells lacking the mitochondrial PE-biosynthetic enzyme Psd1. This rescue is dependent on the conversion of Cho to PC via the Kennedy pathway as well as on Psd2, an enzyme catalyzing PE biosynthesis in the endosome. Metabolic labeling experiments reveal that in the absence of exogenously supplied Cho, PE biosynthesized via Psd2 is mostly directed to the methylation pathway for PC biosynthesis and is unavailable for replenishing mitochondrial PE in Psd1-deleted cells. In this setting, stimulating the Kennedy pathway for PC biosynthesis by Cho spares Psd2-synthesized PE from the methylation pathway and redirects it to the mitochondria. Cho-mediated elevation in mitochondrial PE is dependent on Vps39, which has been recently implicated in PE trafficking to the mitochondria. Accordingly, epistasis experiments placed Vps39 downstream of Psd2 in Cho-based rescue. Our work, thus, provides a mechanism of Cho-based rescue of mitochondrial PE deficiency and uncovers an intricate interorganelle phospholipid regulatory network that maintains mitochondrial PE homeostasis.

Phospholipid and LC-PUFA metabolism in Atlantic salmon (Salmo salar) testes during sexual maturation.

The importance of dietary lipids in male reproduction are not as well understood as in females, in which dietary lipids, such as phospholipids (PL) and associated fatty acids (FA), are important structural components of the eggs and provide energy for their offspring. In mammals, lipids are suggested to be important for spermatogenesis and to structural components of the spermatozoa that could improve fertilization rates. New knowledge of how lipids affect sexual maturation in male Atlantic salmon (Salmo salar), an important global aquaculture species, could provide tools to delay maturation and/or improve reproductive success. Therefore, changes in testicular composition of lipids and gene transcripts associated with spermatogenesis and lipid metabolism were studied in sexually maturing male salmon compared to immature males and females. An increase in total testis content of FA and PL, and a shift to higher PL composition was observed in maturing males, concomitant with increases in mRNA levels for genes involved in spermatogenesis, FA uptake and synthesis, and production of long chain-polyunsaturated fatty acids (LC-PUFA) and PL. A particularly interesting finding was elevated testis expression of acyl-CoA synthetase 4 (acsl4), and acyl-CoA thioesterase 2 (acot2), critical enzymes that regulate intra-mitochondrial levels of 20:4n-6 FA (arachidonic acid), which have been associated with improved cholesterol transport during steroidogenesis. This suggested that FA may have direct effects on sex steroid production in salmon. Furthermore, we observed increased testis expression of genes for endogenous synthesis of 16:0 and elongation/desaturation to 22:6n-3 (docosahexaenoic acid) in sexually maturing males relative to immature fish. Both of these FA are important structural components of the PL, phosphatidylcholine (PC), and were elevated concomitant with increases in the content of phosphatidic acid, an important precursor for PC, in maturing males compared to immature fish. Overall, this study suggests that, similar to mammals, lipids are important to spermatogenesis and serve as structural components during testicular growth and maturation in Atlantic salmon.

A patatin-like phospholipase functions during gametocyte induction in the malaria parasite Plasmodium falciparum.

Patatin-like phospholipases (PNPLAs) are highly conserved enzymes of prokaryotic and eukaryotic organisms with major roles in lipid homeostasis. The genome of the malaria parasite Plasmodium falciparum encodes four putative PNPLAs with predicted functions during phospholipid degradation. We here investigated the role of one of the plasmodial PNPLAs, a putative PLA2 termed PNPLA1, during blood stage replication and gametocyte development. PNPLA1 is present in the asexual and sexual blood stages and here localizes to the cytoplasm. PNPLA1-deficiency due to gene disruption or conditional gene-knockdown had no effect on intraerythrocytic growth, gametocyte development and gametogenesis. However, parasites lacking PNPLA1 were impaired in gametocyte induction, while PNPLA1 overexpression promotes gametocyte formation. The loss of PNPLA1 further leads to transcriptional down-regulation of genes related to gametocytogenesis, including the gene encoding the sexual commitment regulator AP2-G. Additionally, lipidomics of PNPLA1-deficient asexual blood stage parasites revealed overall increased levels of major phospholipids, including phosphatidylcholine (PC), which is a substrate of PLA2 . PC synthesis is known to be pivotal for erythrocytic replication, while the reduced availability of PC precursors drives the parasite into gametocytogenesis; we thus hypothesize that the higher PC levels due to PNPLA1-deficiency prevent the blood stage parasites from entering the sexual pathway.

54 Choline alters the pattern of DNA methylation and lipid content of pre-implantation bovine embryos

Choline is a methyl donor for DNA methylation and a precursor for phosphatidylcholine, which is the major phospholipid of cell membranes. Early embryonic development involves processes of DNA demethylation and remethylation as well as synthesis of new cell membranes. Addition of choline chloride (ChCl) to culture medium of embryos produced invitro increased birthweight of Brahman calves after embryo transfer. The objective was to determine whether the addition of ChCl to culture medium alters the pattern of DNA methylation and lipid content of pre-implantation embryos produced invitro. Embryos were incubated after fertilisation in BBH7 culture medium containing 0.0, 0.004, 1.3, or 1.8mM ChCl (adjusted with NaCl to maintain isotonicity). Concentrations were chosen to approximate free choline (0.004mM) and total choline (1.30mM) in plasma of cows at week 1 postpartum and concentration of total choline in plasma of cows fed rumen-protected choline (1.8mM). Cleavage and blastocyst rate (n=8 replicates) were evaluated at Days 3 and 7.5 post-insemination, respectively. Embryos ≥8 cells (range 8 to 24 cells; stages near embryonic genome activation) and expanded blastocysts were harvested at Day 3.75 (n=232) and 7.5 (n=204) to estimate global DNA methylation by immunostaining for 5-methyl-cytosine. Methylation of DNA was estimated by calculating the ratio of fluorescence for 5-methyl-cytosine to that of propidium iodide (DNA). Another group of expanded blastocysts was harvested (n=99) to estimate lipid content using Nile Red. Embryo development was analysed by GLIMMIX procedure and fluorescence data by GLM procedure of SAS (SAS Institute Inc.). The proportion of zygotes that cleaved after fertilisation (77.5±2.3, 78.1±2.3, 74.5±2.4, and 80.1±2.2% for 0.0, 0.004, 1.3, and 1.8mM ChCl; P=0.2736) and cleaved embryos that became blastocysts (37.8±4.4, 41.5±4.6, 42.8±4.6, and 39.6±4.4%; P=0.5764) was similar between treatments. The DNA methylation at both days was affected by treatment (P<0.001). At Day 3.75, 1.3mM choline reduced methylation and there were no effects of other concentrations (1.13a±0.03, 1.04a±0.03, 0.92b±0.03, and 1.13a±0.04; means with different superscripts differ at P<0.05). For blastocysts, in contrast, DNA methylation was increased for embryos treated with 1.3 and 1.8mM choline (0.98a±0.04, 1.04ab±0.03, 1.25c±0.03, and 1.11b±0.03). Lipid content in blastocysts was also affected by treatment (P=0.0139). In particular, lipid content was higher for embryos treated with 1.3 and 1.8mM choline (409.1a±54.3, 542.3ab±62.3, 651.3b±54.3, and 583.9b±55.0). In conclusion, addition of ChCl to culture medium altered DNA methylation in bovine pre-implantation embryos produced invitro in a manner dependent on developmental stage and choline concentration. Likewise, ChCl increased lipid content in the resultant blastocysts. Support was provided by the Red Larson Endowment.

Choline Supplementation in Cystic Fibrosis-The Metabolic and Clinical Impact.

Background: Choline is essential for the synthesis of liver phosphatidylcholine (PC), parenchymal maintenance, bile formation, and lipoprotein assembly to secrete triglycerides. In choline deficiency, the liver accretes choline/PC at the expense of lung tissue, thereby impairing pulmonary PC homoeostasis. In cystic fibrosis (CF), exocrine pancreas insufficiency results in impaired cleavage of bile PC and subsequent fecal choline loss. In these patients, the plasma choline concentration is low and correlates with lung function. We therefore investigated the effect of choline supplementation on plasma choline/PC concentration and metabolism, lung function, and liver fat. Methods: 10 adult male CF patients were recruited (11/2014–1/2016), and orally supplemented with 3 × 1 g choline chloride for 84 (84–91) days. Pre-/post-supplementation, patients were spiked with 3.6 mg/kg [methyl-D9]choline chloride to assess choline/PC metabolism. Mass spectrometry, spirometry, and hepatic nuclear resonance spectrometry served for analysis. Results: Supplementation increased plasma choline from 4.8 (4.1–6.2) µmol/L to 10.5 (8.5–15.5) µmol/L at d84 (p < 0.01). Whereas plasma PC concentration remained unchanged, D9-labeled PC was decreased (12.2 [10.5–18.3] µmol/L vs. 17.7 [15.5–22.4] µmol/L, p < 0.01), indicating D9-tracer dilution due to higher choline pools. Supplementation increased Forced Expiratory Volume in 1 second percent of predicted (ppFEV1) from 70.0 (50.9–74.8)% to 78.3 (60.1–83.9)% (p < 0.05), and decreased liver fat from 1.58 (0.37–8.82)% to 0.84 (0.56–1.17)% (p < 0.01). Plasma choline returned to baseline concentration within 60 h. Conclusions: Choline supplementation normalized plasma choline concentration and increased choline-containing PC precursor pools in adult CF patients. Improved lung function and decreased liver fat suggest that in CF correcting choline deficiency is clinically important. Choline supplementation of CF patients should be further investigated in randomized, placebo-controlled trials.

Natural variation in acyl editing is a determinant of seed storage oil composition

Seeds exhibit wide variation in the fatty acid composition of their storage oil. However, the genetic basis of this variation is only partially understood. Here we have used a multi-parent advanced generation inter-cross (MAGIC) population to study the genetic control of fatty acid chain length in Arabidopsis thaliana seed oil. We mapped four quantitative trait loci (QTL) for the quantity of the major very long chain fatty acid species 11-eicosenoic acid (20:1), using multiple QTL modelling. Surprisingly, the main-effect QTL does not coincide with FATTY ACID ELONGASE1 and a parallel genome wide association study suggested that LYSOPHOSPHATIDYLCHOLINE ACYLTRANSFERASE 2 (LPCAT2) is a candidate for this QTL. Regression analysis also suggested that LPCAT2 expression and 20:1 content in seeds of the 19 MAGIC founder accessions are related. LPCAT is a key component of the Lands cycle; an acyl editing pathway that enables acyl-exchange between the acyl-Coenzyme A and phosphatidylcholine precursor pools used for microsomal fatty acid elongation and desaturation, respectively. We Mendelianised the main-effect QTL using biparental chromosome segment substitution lines and carried out complementation tests to show that a single cis-acting polymorphism in the LPCAT2 promoter causes the variation in seed 20:1 content, by altering the LPCAT2 expression level and total LPCAT activity in developing siliques. Our work establishes that oilseed species exhibit natural variation in the enzymic capacity for acyl editing and this contributes to the genetic control of storage oil composition.

Disturbed phospholipid metabolism in serine biosynthesis defects revealed by metabolomic profiling

Serine biosynthesis defects are autosomal recessive metabolic disorders resulting from the deficiency of any of the three enzymes involved in de novo serine biosynthesis, specifically phosphoglycerate dehydrogenase (PGDH), phosphoserine aminotransferase (PSAT), and phosphoserine phosphatase (PSP). In this study, we performed metabolomic profiling on 4 children with serine biosynthesis defects; 3 with PGDH deficiency and 1 with PSAT deficiency. The evaluations were performed at baseline and with serine and glycine supplementation. Metabolomic profiling performed at baseline showed low phospholipid species, including glycerophosphocholine, glycerophosphoethanolamine, and sphingomyelin. All children had low serine and glycine as expected. Low glycerophosphocholine compounds were found in 4 children, low glycerophosphoethanolamine compounds in 3 children, and low sphingomyelin species in 2 children. Metabolic profiling with serine and glycine supplementation showed normalization of most of the low phospholipid compounds in the 4 children. Phospholipids are the major component of plasma and intracellular membranes, and phosphatidylcholine is the most abundant phospholipid of all mammalian cell types and subcellular organelles. Phosphatidylcholine is of particular importance for the nervous system, where it is essential for neuronal differentiation. The observed low phosphatidylcholine species in children with serine biosynthesis defects that improved after serine supplementation, supports the role of serine as a significant precursor for phosphatidylcholine. The vital role that phosphatidylcholine has during neuronal differentiation and the pronounced neurological manifestations in serine biosynthesis defects suggest that phosphatidylcholine deficiency occurring secondary to serine deficiency may have a significant contribution to the development of the neurological manifestations in individuals with serine biosynthesis defects.

Conformational changes in the di-domain structure of Arabidopsis phosphoethanolamine methyltransferase leads to active-site formation

Phosphocholine (pCho) is a precursor for phosphatidylcholine and osmoprotectants in plants. In plants, de novo synthesis of pCho relies on the phosphobase methylation pathway. Phosphoethanolamine methyltransferase (PMT) catalyzes the triple methylation of phosphoethanolamine (pEA) to pCho. The plant PMTs are di-domain methyltransferases that divide the methylation of pEA in one domain from subsequent methylations in the second domain. To understand the molecular basis of this architecture, we examined the biochemical properties of three Arabidopsis thaliana PMTs (AtPMT1-3) and determined the X-ray crystal structures of AtPMT1 and AtPMT2. Although each isoform synthesizes pCho from pEA, their physiological roles differ with AtPMT1 essential for normal growth and salt tolerance, whereas AtPMT2 and AtPMT3 overlap functionally. The structures of AtPMT1 and AtPMT2 reveal unique features in each methyltransferase domain, including active sites that use different chemical mechanisms for phosphobase methylation. These structures also show how rearrangements in both the active sites and the di-domain linker form catalytically competent active sites and provide insight on the evolution of the PMTs in plants, nematodes, and apicomplexans. Connecting conformational changes with catalysis in modular enzymes, like the PMT, provides new insights on interdomain communication in biosynthetic systems.

Nutritional intervention and neurodevelopmental outcome in infants with suspected cerebral palsy: the Dolphin infant double-blind randomized controlled trial.

To investigate whether docosahexaenoic acid (DHA), choline, and uridine-5-monophosphate (UMP) supplementation improves neurodevelopmental outcome in infants with suspected cerebral palsy (CP) versus a comparison group of children. Infants aged 1 to 18months with suspected CP were recruited from UK child development centres. Participants received daily treatment or control supplementation for 2years (double-blind randomized control design). Stratification was by age, sex, predominant pattern of motor involvement (four limbs or other), and visual impairment (or not). The primary outcome was the cognitive composite score of the Bayley Scales of Infant and Toddler Development, Third Edition (CCS-Bayley-III). Secondary outcomes included language composite and motor composite scores of the Bayley Scales of Infant and Toddler Development, Third Edition (Bayley-III). Forty infants were recruited; 35 began supplementation, 29 completed 1 to 2years' supplementation. The treatment group CCS-Bayley-III was non-significantly higher than the comparison group (mean 77.7 [SD 19.2] and 72.2 [SD 19.8] respectively, mean modelled difference 4.4 [-2.8, 11.6]). The treatment group language scores, but not motor scores, were non-significantly higher than for the comparison group. Most families found supplementation feasible. No statistically significant differences in neurodevelopmental outcome between the treatment and comparison groups were identified. Further investigation of neurodevelopmental outcome after supplementation with DHA, choline, and UMP of infants with suspected CP is warranted. This was the first trial of phosphatidylcholine precursor supplementation in infants with suspected cerebral palsy (CP). Families of infants with suspected CP found 2-year nutritional supplementation feasible. There was no statistically significant neurodevelopmental advantage for the treatment group versus the comparison group. However, treatment group cognitive and language advantage were of clinically meaningful magnitude.

Maternal Choline Supplementation Modulates Maternal and Fetal Choline Metabolism and Downregulates Inflammatory Gene Expression in a Mouse Model of Placental Insufficiency

A properly functioning placenta allows efficient transfer of nutrients from mother to fetus and promotes fetal growth. Decreased placental efficiency is commonly associated with intrauterine growth restriction, preeclampsia, and miscarriage. Choline is an essential nutrient that is a precursor for several molecules with crucial roles in fetal development: betaine, an osmolyte and methyl donor; acetylcholine, a neurotransmitter and signaling molecule; and phosphatidylcholine (PC), the most prevalent membrane phospholipid. We previously showed that maternal choline supplementation (MCS) in the Dlx3+/− mouse, a model of placental insufficiency, increases fetal growth in early gestation and lowers expression of pro‐angiogenic genes implicated in preeclampsia. To explore the mechanisms contributing to these effects, we measured choline metabolites in the maternal liver and placentas of Dlx3+/− female mice consuming 1X (control), 2X or 4X the recommended intake levels of choline during gestation. Additionally, we measured expression of inflammatory genes that are elevated in preeclampsia and indicative of placental dysfunction. Dams were sacrificed at embryonic days E10.5, E12.5, E15.5, and E18.5. Data were analyzed using linear mixed models with dam as a random factor. MCS increased betaine concentrations in the maternal liver at all four time points (p&lt;.01) and prevented a decline in maternal hepatic betaine stores in late pregnancy. 4X choline also increased phosphocholine, a precursor of PC, at E15.5 (p=.014), and decreased glycerophosphocholine, a product of PC breakdown, at E12.5 (p=.025). In the placenta, MCS increased betaine concentrations at E10.5 and 12.5 (p=.005 and .041, respectively). These data suggest that effects of MCS may be primarily driven by betaine, potentially through increased supply of methyl groups for one‐carbon reactions including DNA methylation. This is supported by data demonstrating widespread effects of choline on gene expression. In Dlx3+/− placentas, choline decreased (p&lt;0.05) expression of pro‐inflammatory genes Il1b (at E12.5, 15.5 and 18.5), Tnf‐α (at E12.5) and NfκB (at E15.5). Further studies are ongoing to identify downstream effects of increased betaine on placental DNA methylation and growth factor signaling pathways.Support or Funding InformationFunding provided by USDA‐NIFA 2012‐67017‐30176

Insights into the mechanisms underlying mercury-induced oxidative stress in gills of wild fish (Liza aurata) combining 1H NMR metabolomics and conventional biochemical assays

Oxidative stress has been described as a key pathway to initiate mercury (Hg) toxicity in fish. However, the mechanisms underlying Hg-induced oxidative stress in fish still need to be clarified. To this aim, environmental metabolomics in combination with a battery of conventional oxidative stress biomarkers were applied to the gills of golden grey mullet (Liza aurata) collected from Largo do Laranjo (LAR), a confined Hg contaminated area, and São Jacinto (SJ), selected as reference site (Aveiro Lagoon, Portugal). Higher accumulation of inorganic Hg and methylmercury was found in gills of fish from LAR relative to SJ. Nuclear magnetic resonance (NMR)-based metabolomics revealed changes in metabolites related to antioxidant protection, namely depletion of reduced glutathione (GSH) and its constituent amino acids, glutamate and glycine. The interference of Hg with the antioxidant protection of gills was corroborated through oxidative stress endpoints, namely the depletion of glutathione peroxidase and superoxide dismutase activities at LAR. The increase of total glutathione content (reduced glutathione+oxidized glutathione) at LAR, in parallel with GSH depletion aforementioned, indicates the occurrence of massive GSH oxidation under Hg stress, and an inability to carry out its regeneration (glutathione reductase activity was unaltered) or de novo synthesis. Nevertheless, the results suggest the occurrence of alternative mechanisms for preventing lipid peroxidative damage, which may be associated with the enhancement of membrane stabilization/repair processes resulting from depletion in the precursors of phosphatidylcholine (phosphocholine and glycerophosphocholine), as highlighted by NMR spectroscopy. However, the observed decrease in taurine may be attributable to alterations in the structure of cell membranes or interference in osmoregulatory processes. Overall, the novel concurrent use of metabolomics and conventional oxidative stress endpoints demonstrated to be sensitive and effective towards a mechanistically based assessment of Hg toxicity in gills of wild fish, providing new insights into the toxicological pathways underlying the oxidative stress.

Phosphatidylethanolamine Metabolism in Health and Disease.

Phosphatidylethanolamine (PE) is the second most abundant glycerophospholipid in eukaryotic cells. The existence of four only partially redundant biochemical pathways that produce PE, highlights the importance of this essential phospholipid. The CDP-ethanolamine and phosphatidylserine decarboxylase pathways occur in different subcellular compartments and are the main sources of PE in cells. Mammalian development fails upon ablation of either pathway. Once made, PE has diverse cellular functions that include serving as a precursor for phosphatidylcholine and a substrate for important posttranslational modifications, influencing membrane topology, and promoting cell and organelle membrane fusion, oxidative phosphorylation, mitochondrial biogenesis, and autophagy. The importance of PE metabolism in mammalian health has recently emerged following its association with Alzheimer's disease, Parkinson's disease, nonalcoholic liver disease, and the virulence of certain pathogenic organisms.

Altered Metabolic Profile in Congenital Lung Lesions Revealed by 1H Nuclear Magnetic Resonance Spectroscopy

Congenital lung lesions are highly complex with respect to pathogenesis and treatment. Large-scale analytical methods, like metabolomics, are now available to identify biomarkers of pathological phenotypes and to facilitate clinical management. Nuclear magnetic resonance (NMR) is a unique tool for translational research, as in vitro results can be potentially translated into in vivo magnetic resonance protocols. Three surgical biopsies, from congenital lung malformations, were analyzed in comparison with one control sample. Extracted hydrophilic metabolites were submitted to high resolution 1H NMR spectroscopy and the relative concentration of 12 metabolites was estimated. In addition, two-dimensional NMR measurements were performed to complement the results obtained from standard monodimensional experiments. This is one of the first reports of in vitro metabolic profiling of congenital lung malformation. Preliminary data on a small set of samples highlights some altered metabolic ratios, dealing with the glucose conversion to lactate, to the relative concentration of phosphatidylcholine precursors, and to the presence of myoinositol. Interestingly some relations between congenital lung lesions and cancer metabolic alterations are found.

Phosphatidylcholine-derived bolaamphiphiles via click chemistry.

The copper-catalyzed azide alkyne cycloaddition is employed to modify phosphatidylcholine precursors with sn-2 acyl chains containing terminal alkyne or azide groups. Although the reactions are conducted as biphasic dispersions, the yields are essentially quantitative. Bolaamphiphiles are formed by simply clicking together two phosphatidylcholine alkyne precursors to a central bisazide scaffold. The chemistry introduces polar 1,4-triazole units into the lipophilic region of the bilayer membrane, and the bolaamphiphiles do not form stable vesicles. [structure: see text].

Release of Free F2-isoprostanes from Esterified Phospholipids Is Catalyzed by Intracellular and Plasma Platelet-activating Factor Acetylhydrolases

F2-isoprostanes are produced in vivo by nonenzymatic peroxidation of arachidonic acid esterified in phospholipids. Increased urinary and plasma F2-isoprostane levels are associated with a number of human diseases. These metabolites are regarded as excellent markers of oxidant stress in vivo. Isoprostanes are initially generated in situ, i.e. when the arachidonate precursor is esterified in phospholipids, and they are subsequently released in free form. Although the mechanism(s) responsible for the release of free isoprostanes after in situ generation in membrane phospholipids is, for the most part, unknown, this process is likely mediated by phospholipase A2 activity(ies). Here we reported that human plasma contains an enzymatic activity that catalyzes this reaction. The activity associates with high density and low density lipoprotein and comigrates with platelet-activating factor (PAF) acetylhydrolase on KBr density gradients. Plasma samples from subjects deficient in PAF acetylhydrolase do not release F2-isoprostanes from esterified precursors. The intracellular PAF acetylhydrolase II, which shares homology to the plasma enzyme, also catalyzes this reaction. We found that both the intracellular and plasma PAF acetylhydrolases have high affinity for esterified F2-isoprostanes. However, the rate of esterified F2-isoprostane hydrolysis is much slower compared with the rate of hydrolysis of other substrates utilized by these enzymes. Studies using PAF acetylhydrolase transgenic mice indicated that these animals have a higher capacity to release F2-isoprostanes compared with nontransgenic littermates. Our results suggested that PAF acetylhydrolases play key roles in the hydrolysis of F2-isoprostanes esterified on phospholipids in vivo.