Characterization of lipoproteins and associated lipidome in very preterm infants: a pilot study.

BACKGROUND: Preterm birth is associated with higher risks of suboptimal neurodevelopment and cardiometabolic disease later in life. Altered maternal-fetal lipid supply could play a role in such risks. Our hypothesis was that very preterm infants born with very low birth weight (VLBW) have altered lipidome and apolipoprotein profiles, compared with term infants. METHODS: Seven mothers of VLBW infants born at <32 GA and 8 full-term mother-infant dyads were included. Cholesterol and triglycerides in lipoproteins were determined in maternal plasma and in the two blood vessels of the umbilical cord (vein (UV) and artery (UA)) following FPLC isolation. Apolipoprotein concentrations in lipoproteins and plasma lipidomic analysis were performed by LC-MS/MS. RESULTS: We found higher cholesterol and VLDL-cholesterol in UV and UA and lower apolipoprotein A-I in HDL2 in UV in preterm neonates. Phosphatidylcholine (PC) containing saturated and monounsaturated fatty acids and specific sphingomyelin species were increased in UV and UA, whereas PC containing docosahexaenoic acid (DHA) was reduced in UV of VLBW neonates. CONCLUSIONS: Lower DHA-PC suggests a lower DHA bioavailability and may contribute to the impaired neurodevelopment. Altered HDL-2, VLDL, and sphingomyelin profile reflect an atherogenic risk and increased metabolic risk at adulthood in infants born prematurely. IMPACT: Lower ApoA-I in HDL2, and increased specific sphingomyelin and phosphatidylcholine containing saturated and monounsaturated fatty acid could explain the accumulation of cholesterol in umbilical vein in VLBW preterm neonates. Decreased phosphatidylcholine containing DHA suggest a reduced DHA availability for brain development in VLBW preterm infants. Characterization of alterations in fetal lipid plasma and lipoprotein profiles may help to explain at least in part the causes of the elevated cardiovascular risk known in people born prematurely and may suggest that a targeted nutritional strategy based on the composition of fatty acids carried by phosphatidylcholine may be promising in infants born very early.

Maternal obesity causes fetal cardiac hypertrophy and alters adult offspring myocardial metabolism in mice.

Obesity in pregnant women causes fetal cardiac dysfunction and increases offspring cardiovascular disease risk, but its effect on myocardial metabolism is unknown. We hypothesized that maternal obesity alters fetal cardiac expression of metabolism-related genes and shifts offspring myocardial substrate preference from glucose towards lipids. Female mice were fed control or obesogenic diets before and during pregnancy. Fetal hearts were studied in late gestation (embryonic day (E) 18.5; term ≈ E21), and offspring were studied at 3, 6, 9 or 24 months postnatally. Maternal obesity increased heart weight and peroxisome proliferator activated receptor gamma (Pparg) expression in female and male fetuses and caused left ventricular diastolic dysfunction in the adult offspring. Cardiac dysfunction worsened progressively with age in female, but not male, offspring of obese dams, in comparison to age-matched control animals. In 6-month-old offspring, exposure to maternal obesity increased cardiac palmitoyl carnitine-supported mitochondrial respiration in males and reduced myocardial 18 F-fluorodeoxyglucose uptake in females. Cardiac Pparg expression remained higher in adult offspring of obese dams than control dams and was correlated with contractile and metabolic function. Maternal obesity did not affect cardiac palmitoyl carnitine respiration in females or 18 F-fluorodeoxyglucose uptake in males and did not alter cardiac 3 H-oleic acid uptake, pyruvate respiration, lipid content or fatty acid/glucose transporter abundance in offspring of either sex. The results support our hypothesis and show that maternal obesity affects offspring cardiac metabolism in a sex-dependent manner. Persistent upregulation of Pparg expression in response to overnutrition in utero might underpin programmed cardiac impairments mechanistically and contribute to cardiovascular disease risk in children of women with obesity. KEY POINTS: Obesity in pregnant women causes cardiac dysfunction in the fetus and increases lifelong cardiovascular disease risk in the offspring. In this study, we showed that maternal obesity in mice induces hypertrophy of the fetal heart in association with altered expression of genes related to nutrient metabolism. Maternal obesity also alters cardiac metabolism of carbohydrates and lipids in the adult offspring. The results suggest that overnutrition in utero might contribute to increased cardiovascular disease risk in children of women with obesity.

Spirulina liquid extract prevents metabolic disturbances and improves liver sphingolipids profile in hamster fed a high-fat diet.

PURPOSE: Metabolic syndrome is characterized by hyperglycemia, hyperlipemia and exacerbated oxidative stress. The aim of the study was to determine whether Spirulysat®, a Spirulina liquid extract (SLE) enriched in phycocyanin, would prevent metabolic abnormalities induced by high-fat diet. METHODS: The effect of acute SLE supplementation on postprandial lipemia and on triton-induced hyperlipidemia was studied in hamster fed control diet (C). The effect of chronic SLE supplementation on lipid content in plasma, liver and aorta, and on glycemia and oxidative stress was studied in hamster fed control (C) or high-fat diet (HF) for two weeks and then treated with SLE for two weeks (CSp and HFSp) or not (C and HF). RESULTS: The acute SLE supplementation lowered plasma cholesterol and non-esterified fatty acid concentrations after olive oil gavage (P < 0.05) in CSp, while no effect was observed on triglyceridemia. HFD increased plasma MDA, basal glycemia, triglyceridemia, total plasma cholesterol, VLDL, LDL and HDL cholesterol, ceramide, sphingomyelin and glucosylceramide content in liver in HF compared to C (P < 0.05). SLE did not affect SOD and GPx activities nor total antioxidant status in HFSp group but lowered glycemia, glucoceramide and cholesterol in liver and cholesterol in aorta compared to HF (P < 0.05). SLE also decreased HMGCoA and TGF-ß1 gene expression in liver (P < 0.05) and tended to lower G6Pase (P = 0.068) gene expression in HFSp compared to HF. CONCLUSION: Although 2-week SLE supplementation did not affect oxidative stress, it protected from hyperglycemia and lipid accumulation in liver and aorta suggesting a protective effect against metabolic syndrome.

Placental fatty acid transport across late gestation in a baboon model of intrauterine growth restriction.

KEY POINTS: Intrauterine growth restriction (IUGR) is associated with perinatal morbidity and increased risk of lifelong disease, including neurodevelopmental impairment. Fatty acids (FA) are critical for normal brain development, although their transport across the placenta in IUGR pregnancies is poorly understood. The present study used a baboon model of IUGR (maternal nutrient restriction, MNR) to investigate placental expression of FA transport and binding proteins, and to determine gestational age-related changes in maternal and fetal plasma FA concentrations. We found MNR to be associated with increased placental expression of FA binding and transport proteins in late gestation, with fetal plasma FA concentrations that were similar to those of control animals. The present study is the first to report a profile of fetal and maternal plasma FA concentrations in a baboon model of growth restriction with data that suggest adaptation of placental transport to maintain delivery of critically needed FA. ABSTRACT: Intrauterine growth restriction (IUGR) is associated with specific changes in placental transport of amino acids, folate and ions. However, little is known about placental fatty acid (FA) transport in IUGR. We hypothesized that placental FA transport proteins (FATP) and FA binding proteins (FABP) are up-regulated and fetal plasma FA concentrations are decreased at term in a baboon model of IUGR. Pregnant baboons were fed control or maternal nutrient restricted (MNR) diet (70% of control calories) from gestation day (GD) 30 (term 184 days). Plasma and placental samples were collected at GD120 (control n = 8, MNR n = 9), GD140 (control n = 6, MNR n = 7) and GD170 (control n = 6, MNR n = 6). Placentas were homogenized, and syncytiotrophoblast microvillous plasma membrane (MVM) and basal plasma membranes (BM) were isolated. Protein expression of FABP1, 3, 4 and 5 (homogenate) and FATP2, 4, and 6 (MVM, BM) was determined by Western blotting. FA content in maternal and umbilical vein plasma was measured by gas chromatography-mass spectrometry. Placental FABP1 and FABP5 expression was increased in MNR compared to controls at GD170, as was MVM FATP2 and FATP6 expression at GD140 and FATP2 expression at GD170. BM FATP4 and FATP6 expression was increased in MNR at GD140. Fetal plasma FA concentrations were similar in controls and MNR. These data suggest the adaptation of placental transport when aiming to maintain delivery of critically needed FAs for fetal growth and brain development.

Maternal obesity results in decreased syncytiotrophoblast synthesis of palmitoleic acid, a fatty acid with anti-inflammatory and insulin-sensitizing properties.

The fetus is dependent on delivery of fatty acids (FAs) by the syncytiotrophoblast, the transporting epithelium of the human placenta. Obese pregnant women have dyslipidemia; however, whether obesity impacts placental lipid transport and metabolism remains to be fully established. Palmitoleic acid (POA), an FA with anti-inflammatory and insulin-sensitizing properties, is synthesized from palmitic acid (PA) catalyzed by stearoyl-coenzyme A desaturase (SCD) activity. We hypothesized that the uptake and incorporation of FAs and POA synthesis are reduced in primary human trophoblasts (PHTs) isolated from pregnancies complicated by maternal obesity. Villous cytotrophoblasts were isolated from 7 placentas of obese [body mass index (BMI) = 37.5 ± 1.9] and 12 normal (BMI = 23.6 ± 0.6) mothers. FA uptake and incorporation were assessed using uniformly labeled (U[13C])-FA mixtures of PA, oleic acid (OA), linoleic acid, and docosahexaenoic acid. Cellular [13C] FAs were quantified both in total cellular lipids and in lipid classes by GC-MS. Uptake and incorporation of [13C] FAs in total cellular lipids were not different in PHTs isolated from obese mothers compared with normal mothers. Only the concentration of OA was increased in the triglyceride fraction (P < 0.05) if the mother was obese. We found an isotopic enrichment of POA after U[13C]-PA treatment, demonstrating SCD activity in PHT cells. Labeled POA content and the POA:PA ratio were significantly lower in PHTs isolated from placentas of obese mothers compared with normal, healthy controls. Decreased syncytiotrophoblast POA synthesis may contribute to insulin resistance and low-grade inflammation in the mother, placenta, or fetus (or a combination of the 3) in pregnancies complicated by obesity.-Ferchaud-Roucher, V., Barner, K., Jansson, T., Powell, T. L. Maternal obesity results in decreased syncytiotrophoblast synthesis of palmitoleic acid, a fatty acid with anti-inflammatory and insulin-sensitizing properties.

Maternal obesity leads to long-term altered levels of plasma ceramides in the offspring as revealed by a longitudinal lipidomic study in children.

BACKGROUND/OBJECTIVES: Maternal obesity is associated with increased risk of obesity and other symptoms of the metabolic syndrome in the offspring. Nevertheless, the molecular mechanisms and cellular factors underlying this enhanced disease susceptibility remain to be determined. Here, we aimed at identifying changes in plasma lipids in offspring of obese mothers that might underpin, and serve as early biomarkers of, their enhanced metabolic disease risk. SUBJECTS/METHODS: We performed a longitudinal lipidomic profiling in plasma samples from normal weight, overweight, and obese pregnant women and their children that participated in the Prenatal Omega-3 Fatty Acid Supplementation, Growth, and Development trial conducted in Mexico. At recruitment women were aged between 18 and 35 years and in week 18-22 of pregnancy. Blood samples were collected at term delivery by venipuncture from mothers and from the umbilical cord of their newborns and from the same infants at 4 years old under non-fasting conditions. Lipidomic profiling was done using ultra-performance liquid chromatography high-resolution mass spectrometry. RESULTS: Analysis of the lipidomic data showed that overweight and obese mothers exhibited a significant reduction in the total abundance of ceramides (Cer) in plasma, mainly of Cer (d18:1/20:0), Cer (d18:1/22:0), Cer (d18:1/23:0), and Cer (d18:1/24:0), compared with mothers of normal body weight. This reduction was confirmed by the direct quantification of these and other ceramide species. Similar quantitative differences in the plasma concentration of Cer (d18:1/22:0), Cer (d18:1/23:0), and Cer (d18:1/24:0), were also found between 4-year-old children of overweight and obese mothers compared with children of mothers of normal body weight. Noteworthy, children exhibited equal daily amounts of energy and food intake independently of the BMI of their mothers. CONCLUSIONS: Maternal obesity results in long-lasting changes in plasma ceramides in the offspring suggesting that these lipids might be used as early predictors of metabolic disease risk due to maternal obesity.

Alterations in placental long chain polyunsaturated fatty acid metabolism in human intrauterine growth restriction.

Fatty acids (FA) are critical for fetal brain development and are transferred across the placenta by membrane-bound FA transport proteins (FATP), translocases (FAT/CD36), and cytosolic binding proteins (FABP). The cytosolic protein perilipin-2 aids in neutral lipid storage within lipid droplets. Decreased placental nutrient transport is believed to contribute to intrauterine growth restriction (IUGR); however, IUGR placental lipid transport and metabolism are poorly understood. We hypothesized that protein expression of FATPs, FABPs, and perilipin-2 in human placenta is decreased and placental lipid content and incorporation into lipid classes are reduced in IUGR. Placental tissue of idiopathic IUGR (n=25) and gestational age-matched, appropriately grown for gestational age (AGA) fetuses (n=19) was collected. We determined protein expression of FABP4 and perilipin-2 in placental homogenate and FATPs (2, 4, 6, CD36) in syncytiotrophoblast microvillous plasma membrane (MVM) by Western blot. Lipid droplet area (Oil Red O stain) and cellular FA content (GC/MS) were measured in chorionic villous tissue. MVM expression of FATP6 and CD36 was significantly increased in IUGR. The concentrations of seven n-6 and n-3 species long chain polyunsaturated FAs (LCPUFA) were significantly increased in the triglyceride fraction in IUGR vs AGA placenta. In summary, MVM FATP6 and CD36 protein expression is increased and LCPUFA are preferentially routed toward cellular storage in TG in the IUGR placenta, possibly to protect against oxidative stress associated with cellular FA accumulation. We speculate that these changes may be caused by impaired efflux of FA across the fetal-facing syncytiotrophoblast basal plasma membrane in IUGR placenta.

Maternal protein restriction during gestation and lactation in the rat results in increased brain levels of kynurenine and kynurenic acid in their adult offspring.

Early malnutrition is a risk factor for depression and schizophrenia. Since the offspring of malnourished dams exhibit increased brain levels of serotonin (5-HT), a tryptophan-derived neurotransmitter involved in the pathophysiology of these mental disorders, it is believed that the deleterious effects of early malnutrition on brain function are due in large part to altered serotoninergic neurotransmission resulting from impaired tryptophan (Trp) metabolism. However, tryptophan is also metabolized through the kynurenine (KYN) pathway yielding several neuroactive compounds including kynurenic (KA), quinolinic (QA) and xanthurenic (XA) acids. Nevertheless, the impact of perinatal malnutrition on brain kynurenine pathway metabolism has not been examined to date. Here, we used ultra-performance liquid chromatography-tandem mass spectrometry for the simultaneous quantification of tryptophan and a set of seven compounds spanning its metabolism through the serotonin and kynurenine pathways, in the brain of embryos and adult offspring of rat dams fed a protein-restricted (PR) diet. Protein-restricted embryos showed reduced brain levels of Trp, serotonin and KA, but not of KYN, XA, or QA. In contrast, PR adult rats exhibited enhanced levels of Trp in the brainstem and cortex along with increased concentrations of 5-HT, kynurenine and XA. The levels of XA and KA were also increased in the hippocampus of adult PR rats. These results show that early protein deficiency induces selective and long-lasting changes in brain kynurenine metabolism. Given the regulatory role of KYN pathway metabolites on brain development and function, these changes might contribute to the risk of developing psychiatric disorders induced by early malnutrition.

Plasma Lipidome Analysis by Liquid Chromatography-High Resolution Mass Spectrometry and Ion Mobility of Hypertriglyceridemic Patients on Extended-Release Nicotinic Acid: a Pilot Study.

BACKGROUND: Plasma high triacylglycerols and low HDL-C concentration are associated with increased cardiovascular events. Extended-release nicotinic acid (ERN) was shown to reduce plasma triacylglycerols and total cholesterol but also to markedly increase high-density lipoprotein-cholesterol (HDL-C). No data on the effect of ERN on different species of triacylglycerol, cholesteryl ester, and phospholipids are available. In this study, we applied a nontargeted lipidomic approach to investigate the plasma and lipoproteins lipids profile of hypertriglyceridemic patients treated with ERN or a placebo in order to identify new lipids markers associated with this treatment. METHODS: Eight hypertriglyceridemic patients enrolled in a crossover randomized trial with ERN for 8 weeks and treated with 2 g/day of ERN or a placebo. Ultra-performance liquid chromatography (UPLC) coupled to high-resolution mass spectrometry (HRMS) was used in mass spectrometry energy mode (HRMSE) combined with ion mobility spectrometry to characterize the plasma and very low density lipoprotein (VLDL), low density lipoprotein (LDL) and high density lipoprotein (HDL) lipidome. The accuracy and precision of the method were validated on plasma samples. RESULTS: Compared to placebo, among 155 plasma lipids characterized using UPLC-ESI-HRMS, a multivariate analysis revealed a significant increase of lysophosphatidylcholine (LPC 20:5), a significant decrease of phosphatidylethanolamine (PE 16:0/22:3) and sphingomyelin (SM d18:1/22:0) and a decrease of triacylglycerol (TG 16:0/16:1/18:2) after ERN treatment. Analysis of these lipids in lipoproteins showed an increase of LPC (20:5) in HDL, a decrease of PE (16:0/22:3) in HDL and LDL, of SM (d18:1/22:0) in VLDL and LDL and of TG (16:0/16:1/18:2) in VLDL. CONCLUSION: This lipidomic strategy characterized new specific lipid markers likely to be involved in the effect of ERN on cardiovascular risk opening a new strategy to analyze randomized controlled with this treatment. TRIAL REGISTRATION: NCT01216956.

Multiplexed peptide analysis for kinetic measurements of major human apolipoproteins by LC/MS/MS.

A multiplexed assay was developed by MS to analyze, in a single run, six major human Apos involved in lipoprotein metabolism: ApoA-I, ApoA-II, ApoB100, ApoC-II, ApoC-III, and ApoE. This method was validated in vivo in six subjects who received a 14 h constant infusion of [5,5,5-(2)H3]L-leucine at 10 µM/kg/h. Plasma lipoprotein fractions were isolated from collected blood samples and were digested with trypsin. Proteotypic peptides were subsequently analyzed by LC/MS/MS. Enrichment measurement data were compared with those obtained by the standard method using GC/MS. The required time to obtain the LC/MS/MS data was less than that needed for GC/MS. The enrichments from both methods were correlated for ApoA-I (r = 0.994; P < 0.0001) and ApoB100 (r = 0.999; P < 0.0001), and the Bland-Altman plot confirmed the similarity of the two methods. Intra- and inter-assay variability calculated for the six Apos of interest did not exceed 10.7 and 12.5%, respectively, and kinetic parameters were similar and/or in agreement with previously reported data. Therefore, LC/MS/MS can be considered as a useful tool for human Apo kinetic studies using stable isotopes.

Comparison of gas chromatography-mass spectrometry and gas chromatography-combustion-isotope ratio mass spectrometry analysis for in vivo estimates of metabolic fluxes.

Gas chromatography-mass spectrometry (GC-MS) was compared with gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) for measurements of cholesterol (13)C enrichment after infusion of labeled precursor ([(13)C1,2]acetate). Paired results were significantly correlated, although GC-MS was less accurate than GC-C-IRMS for higher enrichments. Nevertheless, only GC-MS was able to provide information on isotopologue distribution, bringing new insights to lipid metabolism. Therefore, we assessed the isotopologue distribution of cholesterol in humans and dogs known to present contrasted cholesterol metabolic pathways. The labeled tracer incorporation was different in both species, highlighting the subsidiarity of GC-MS and GC-C-IRMS to analyze in vivo stable isotope studies.

Maternal and fetal tryptophan metabolism in gestating rats: effects of intrauterine growth restriction.

L-Tryptophan (L-Trp) is a precursor for serotonin (5-HT) and nicotinamide adenine dinucleotide (NAD) synthesis. Both 5-HT and NAD may impact energy metabolism during gestation given that recent studies have demonstrated that increased 5-HT production is crucial for increasing maternal insulin secretion, and that sirtuin, an NAD(+)-dependent protein deacetylase, regulates endocrine signaling. Infants born with intrauterine growth restriction (IUGR) are at a higher risk of metabolic disease once they reach adulthood. IUGR is associated with altered maternal-fetal amino acid transfer. Whether IUGR affects L-Trp metabolism in mother and fetus has not been fully elucidated. Recently, we developed an analytical method using stable isotope-labeled L-Trp to explore the metabolism of L-Trp and its main metabolites, L-kynurenine (L-Kyn), 5-HT and quinolinic acid (QA). In this study, dams submitted to dietary protein restriction throughout gestation received intravenous infusions of stable isotope-labeled (15)N2-L-Trp to determine whether L-Trp metabolism is affected by IUGR. Samples were obtained from maternal, fetal and umbilical vein plasma, as well as the amniotic fluid (AF), placenta and liver of the mother and the fetus after isotope infusion. We observed evidence for active L-Trp transfer from mother to fetus, as well as de novo synthesis of 5-HT in the fetus. Plasma 5-HT was decreased in undernourished mothers. In IUGR fetuses, maternal-fetal L-Trp transfer remained unaffected, but conversion to QA was impaired, implying that NAD production also decreased. Whether such alterations in tryptophan metabolism during gestation have adverse consequences and contribute to the increased risk of metabolic disease in IUGR remains to be explored.

Effects of Extended-Release Nicotinic Acid on Apolipoprotein (a) Kinetics in Hypertriglyceridemic Patients.

OBJECTIVE: To determine the mechanisms by which extended-release nicotinic acid reduces circulating lipoprotein (a) concentrations in hypertriglyceridemic patients. APPROACH AND RESULTS: Eight nondiabetic, obese male subjects (aged 48±12 years; body mass index, 31.2±1.8 kg/m(2)) with hypertriglyceridemia (triglycerides, 226±78 mg/dL) were enrolled in an 8 week, double blind, placebo-controlled cross-over study. At the end of each treatment phase, fasted subjects received a 10 µmol/L per kg bolus injection of [5,5,5-(2)H3]-l-Leucine immediately followed by constant infusion of [5,5,5-(2)H3]-l-Leucine (10 µmol L(-1) kg(-1) h(-1)) for 14 hours, and blood samples were collected. A liquid chromatography-tandem mass spectrometry method was used to study apolipoprotein (a) (Apo(a)) kinetics. The fractional catabolic rate of Apo(a) was calculated with a single compartmental model using the apolipoprotein B100 (ApoB100) containing very low density lipoprotein tracer enrichment as a precursor pool. Extended-release nicotinic acid decreased plasma triglycerides (-46%; P=0.023), raised high-density lipoprotein cholesterol (+20%; P=0.008), and decreased Apo(a) plasma concentrations (-20%; P=0.008). Extended-release nicotinic acid also decreased ApoB100 (22%; P=0.008) and proprotein convertase subtilisin/kexin type 9 (PCSK9, -29%; P=0.008) plasma concentrations. Apo(a) fractional catabolic rate and production rates were decreased by 37% (0.58±0.28 versus 0.36±0.19 pool/d; P=0.008) and 50% (1.4±0.8 versus 0.7±0.4 nmol/kg per day; P=0.008), respectively. CONCLUSIONS: Extended-release nicotinic acid treatment decreased Apo(a) plasma concentrations by 20%, production rates by 50%, and catabolism by 37%. ApoB100 and PCSK9 concentrations were also decreased by treatment, but no correlation was found with Apo(a) kinetic parameters.

Direct and maternal n-3 long-chain polyunsaturated fatty acid supplementation improved triglyceridemia and glycemia through the regulation of hepatic and muscle sphingolipid synthesis in offspring hamsters fed a high-fat diet.

PURPOSE: We recently reported that direct and maternal supplementation with n-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) alleviates the metabolic disturbances in adult hamster pups fed with a high-fat diet (HFD). In this study, we hypothesized that these results involved a perinatal modulating effect of sphingolipids by n-3 LC-PUFA. METHODS: We studied the effect of direct and maternal n-3 LC-PUFA supplementation on sphingolipid contents in liver and muscle, hepatic triglycerides (TG) secretion and glucose tolerance. Offspring male hamsters born from supplemented (Cω) or unsupplemented (C) mothers were subjected after weaning to a HFD during 16 weeks, without (Cω-HF or C-HF) or with direct supplementation with n-3 LC-PUFA (C-HFω). RESULTS: Direct supplementation decreased sphingosine, sphinganine and ceramides in liver and decreased sphingosine, sphinganine, sphingosine-1-phosphate (S1P) and ceramides in muscle in C-HFω compared to C-HF (p < 0.05). Maternal supplementation decreased C20 ceramide and lactosylceramide in liver and sphinganine, S1P and lactosylceramide in muscle (p < 0.05). This supplementation tended to decrease glucosylceramide in liver (p < 0.06) and muscle (p < 0.07) in Cω-HF compared to C-HF. Direct supplementation increased glucose tolerance and decreased hepatic TG secretion and hepatic gene expression levels of diacylglycerol O-acyltransferase 2 (DGAT2), sterol regulatory element-binding protein-1c (SREBP-1c), fatty acid synthase, stearoyl-CoA desaturase-1 (SCD1) and tumor necrosis factor α (TNFα). Maternal supplementation decreased basal glycemia and hepatic TG secretion. We observed a positive correlation between hepatic TG secretion and hepatic ceramide (p = 0.0059), and between basal glycemia and hepatic ceramide (p = 0.04) or muscle lactosylceramide contents (p = 0.001). CONCLUSION: We observed an improvement of lipids and glucose metabolism in hamster with n-3 LC-PUFA direct supplementation and a decrease in glycemia and hepatic TG secretion with maternal supplementation. These results are probably related to a decrease in both lipogenesis and sphingolipid contents in liver and muscle.

Transplant tolerance is associated with reduced expression of cystathionine-γ-lyase that controls IL-12 production by dendritic cells and TH-1 immune responses.

Antigen-activated T lymphocytes undergo an immune or tolerogeneic response in part according to the activation status of their antigen-presenting cells. However, factors controlling the activation of antigen-presenting cells are not fully understood. In this study, we demonstrate that immune tolerance after organ allotransplantation in the rat is associated with a repressed intragraft expression of several enzymes of the trans-sulfuration pathway, including cystathionine γ-lyase (CSE). The pharmacologic blockade of CSE with propargylglycine delayed heart allograft rejection and abrogated type IV hypersensitivity but did not modify antibody responses, and was associated with a selective inhibition of the TH-1 type factors T-bet, IL-12, and IFN-γ. IL-12 repression could also be induced by propargylglycine in vitro in monocytes and dendritic cells (DCs), a phenomenon not mediated by changes to nuclear factor-κ B or hydrogen sulfide but that occurred together with a modulation of intracellular cysteine content. Intracellular cysteine levels were predominantly controlled in DCs by CSE activity, together with extracellular import via the X(c)(-) transporter. Our results indicate that CSE plays a critical role in regulating IL-12 in monocytes and DCs and is down-modulated in transplant tolerance, presumably participating in the maintenance of the tolerant state.

Oleic acid stimulation of amino acid uptake in primary human trophoblast cells is mediated by phosphatidic acid and mTOR signaling.

Normal fetal development is critically dependent on optimal nutrient supply by the placenta, and placental amino acid transport has been demonstrated to be positively associated with fetal growth. Mechanistic target of rapamycin (mTOR) is a positive regulator of placental amino acid transporters, such as System A. Oleic acid (OA) has been previously shown to have a stimulatory role on placental mTOR signaling and System A amino acid uptake in primary human trophoblast (PHT) cells. We investigated the mechanistic link between OA and System A activity in PHT. We found that inhibition of mTOR complex 1 or 2, using small interfering RNA to knock down raptor or rictor, prevented OA-stimulated System A amino acid transport indicating the interaction of OA with mTOR. Phosphatidic acid (PA) is a key intermediary for phospholipid biosynthesis and a known regulator of the mTOR pathway; however, phospholipid biosynthetic pathways have not been extensively studied in placenta. We identified placental isoforms of acyl transferase enzymes involved in de novo phospholipid synthesis. Silencing of 1-acylglycerol-3-phosphate-O-acyltransferase-4, an enzyme in this pathway, prevented OA mediated stimulation of mTOR and System A amino acid transport. These data indicate that OA stimulates mTOR and amino acid transport in PHT cells mediated through de novo synthesis of PA. We speculate that fatty acids in the maternal circulation, such as OA, regulate placental functions critical for fetal growth by interaction with mTOR and that late pregnancy hyperlipidemia may be critical for increasing nutrient transfer to the fetus.

Synthesis of phospholipids in human placenta.

INTRODUCTION: Placental phospholipid synthesis is critical for the expansion of the placental exchange surface area and for production of signaling molecules. Despite their importance, it is not yet established which enzymes involved in the de novo synthesis and remodeling of placental phospholipids are expressed and active in the human placenta. METHODS: We identified phospholipid synthesis enzymes by immunoblotting in placental homogenates and immunofluorescence in placenta tissue sections. Primary human trophoblast (PHT) cells from term healthy placentas (n = 10) were cultured and exposed to 13C labeled fatty acids (16:0, 18:1 and 18:2 n-6, 22:6 n-3) for 2 and 24 h. Three phospholipid classes; phosphatidic acid, phosphatidylcholine, and lysophosphatidylcholine containing 13C fatty acids were quantified by Liquid Chromatography with tandem mass spectrometry (LC/MS-MS). RESULTS: Acyl transferase and phospholipase enzymes were detected in human placenta homogenate and primarily expressed in the syncytiotrophoblast. Three representative 13C fatty acids (16:0, 18:1 and 18:2 n-6) were incorporated rapidly into phosphatidic acid in trophoblasts, but 13C labeled docosahexaenoic acid (DHA; 22:6 n-3) incorporation was not detected. 13C DHA was incorporated into phosphatidylcholine. Lysophosphatidylcholine containing all four 13C labeled fatty acids were found in high abundance. CONCLUSIONS: Phospholipid synthesis and remodeling enzymes are present in the syncytiotrophoblast. 13C labeled fatty acids were rapidly incorporated into cellular phospholipids. 13C DHA was incorporated into phospholipids through the remodeling pathway rather than by de novo synthesis. These understudied pathways are highly active and critical for structure and function of the placenta.

In situ NMR spectroelectrochemistry for the structure elucidation of unstable intermediate metabolites.

In situ NMR spectroelectrochemistry is presented in this study as a useful hybrid technique for the chemical structure elucidation of unstable intermediate species. An experimental setting was designed to follow the reaction in real time during the experimental electrochemical process. The analysis of (1)H NMR spectra recorded in situ permitted us (1) to elucidate the reaction pathway of the electrochemical oxidation of phenacetin and (2) to reveal the quinone imine as a reactive intermediate species without using any trapping reaction. Phenacetin has been considered as hepatotoxic at high therapeutic amounts, which is why it was chosen as a model to prove the applicability of the analytical method. The use of 1D and 2D NMR experiments led to the elucidation of the major species produced from the oxidation process. We demonstrated that in situ NMR spectroelectrochemistry constitutes a fast way for monitoring unstable quinone imines and elucidating their chemical structures.

Acebutolol and alprenolol metabolism predictions: comparative study of electrochemical and cytochrome P450-catalyzed reactions using liquid chromatography coupled to high-resolution mass spectrometry.

A comparative study of the electrochemical conversion and the biotransformation performed by the cytochrome P450 (CYP450) obtained by rat liver microsomes has been achieved to elucidate the oxidation mechanism of both acebutolol and alprenolol. For this purpose, a wide range of reactions such as N-dealkylation, O-dealkoxylation, aromatic hydroxylation, benzyl hydroxylation, alkyl hydroxylation, and aromatic hydroxylation have been examined in this study, and their mechanisms have been compared. Most of the results of the electrochemical oxidation have been found to be in accordance with those obtained by incubating acebutolol and alprenolol in the presence of CYP450, i.e., N-dealkylation, benzyl hydroxylation, and O-dealkoxylation reactions catalyzed by liver microsomes were found to be predicted by the electrochemical oxidation. The difficulty for the electrochemical process to mimic both aromatic and alkyl hydroxylation reactions has also been discussed, and the hypothesis for the absence of aromatic hydroxylated and alkyl hydroxylated products, respectively, for alprenolol and acebutolol, under the anodic oxidation has been supported by theoretical calculation. The present study highlights the potential and limitation of coupling of electrochemistry-liquid chromatography-high-resolution mass spectrometry for the study of phase I and phase II reactions of acebutolol and alprenolol.

Fetal sex differences in placental LCPUFA ether and plasmalogen phosphatidylethanolamine and phosphatidylcholine contents in pregnancies complicated by obesity.

BACKGROUND: We have previously reported that maternal obesity reduces placental transport capacity for lysophosphatidylcholine-docosahexaenoic acid (LPC-DHA), a preferred form for transfer of DHA (omega 3) to the fetal brain, but only in male fetuses. Phosphatidylethanolamine (PE) and phosphatidylcholine (PC), have either sn-1 ester, ether or vinyl ether (plasmalogen) linkages to primarily unsaturated and monounsaturated fatty acids and DHA or arachidonic acid (ARA, omega 6) in the sn-2 position. Whether ether and plasmalogen PC and PE metabolism in placenta impacts transfer to the fetus is unexplored. We hypothesized that ether and plasmalogen PC and PE containing DHA and ARA are reduced in maternal-fetal unit in pregnancies complicated by obesity and these differences are dependent on fetal sex. METHODS: In maternal, umbilical cord plasma and placentas from obese women (11 female/5 male infants) and normal weight women (9 female/7 male infants), all PC and PE species containing DHA and ARA were analyzed by LC-MS/MS. Placental protein expression of enzymes involved in phospholipid synthesis, were determined by immunoblotting. All variables were compared between control vs obese groups and separated by fetal sex, in each sample using the Benjamini-Hochberg false discovery rate adjustment to account for multiple testing. RESULTS: Levels of ester PC containing DHA and ARA were profoundly reduced by 60-92% in male placentas of obese mothers, while levels of ether and plasmalogen PE containing DHA and ARA were decreased by 51-84% in female placentas. PLA2G4C abundance was lower in male placentas and LPCAT4 abundance was lower solely in females in obesity. In umbilical cord, levels of ester, ether and plasmalogen PC and PE with DHA were reduced by 43-61% in male, but not female, fetuses of obese mothers. CONCLUSIONS: We found a fetal sex effect in placental PE and PC ester, ether and plasmalogen PE and PC containing DHA in response to maternal obesity which appears to reflect an ability of female placentas to adapt to maintain optimal fetal DHA transfer in maternal obesity.

Docosahexaenoic acid (DHA) is a critical omega 3 long chain polyunsaturated fatty acid (LCPUFA) for fetal brain development. We have recently reported that maternal obesity reduces placental transport capacity for LysophosPhatidylCholine-DHA (LPC-DHA), a preferred form for transfer of DHA to the fetal brain, but only in male fetuses. Other important lipids, the plasmalogen phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are considered DHA reservoirs, but its roles in the maternal­fetal unit are largely unexplored. We examined these lipid species in maternal and fetal circulation and in placental tissue to uncover potential novel roles for ether and plasmalogen lipids in the regulation of placenta delivery of these vital nutrients in pregnancies complicated by obesity depending of fetal sex. We demonstrated for the first time, that female fetuses of obese mothers decrease placental ether and plasmalogen PE containing DHA and arachidonic acid (ARA, omega 6), and show a high fetal­placental adaptability and placental reserve capacity that can maintain the PC-LCPUFA synthesis and the transfer of these crucial species to the fetus to preserve brain development. Our study also demonstrated that male fetuses, in response to maternal obesity, reduce the placental ester PC species containing DHA and ARA and reduce the ether and plasmalogen PE reservoir of DHA and ARA in fetal circulation. Our findings support a fetal sex effect in placental ester, ether and plasmalogen PE and PC containing DHA in response to maternal obesity which appears to reflect an ability of female placentas to adapt to maintain optimal fetal DHA transfer in maternal obesity.