Altered materno-fetal transfer of 13C-polyunsaturated fatty acids in obese pregnant women.

BACKGROUND & AIMS: Maternal obesity at conception is considered a major predictor of offspring obesity. This could by driven at least in part by an altered placental fat transfer. However, the pathophysiological mechanisms involved are not fully understood. We investigated the in vivo materno-fetal transfer of fatty acids (FAs) in obese pregnant women using stable isotopes. METHODS: Ten obese and ten normo-weight pregnant women (control) received orally a bolus of 13C-labeled FAs 12 h before elective caesarean section: oleic acid (13C-OA), linoleic acid (13C-LA) and docosahexaenoic acid (13C-DHA). Maternal blood samples were collected at -12 (basal), -8, -4, -2, 0 h relative to the time of cesarean section. At the time of birth, arterial and venous cord bloods as well as placental tissue were collected. FAs composition was determined by gas-liquid chromatography and isotopic enrichment by gas chromatography-combustion-isotope ratio mass spectrometry. RESULTS: Maternal plasma insulin and placental weight tended to higher values in obese pregnant women although they did not present serum hyperlipidemia. Higher concentrations of 13C-LA and 13C-DHA were found in non-esterified FAs fraction in maternal plasma of obese mothers. The ratio of placental uptake for 13C-LA and 13C-DHA was lower in obese women compared to normal weight pointing toward a limited capacity of FA placental transfer, especially of essential FAs. Maternal insulin was associated to this lower placenta/maternal plasma ratio for both 13C-LA (R = -0.563, P = 0.012) and 13C-DHA (R = -0.478, P = 0.033). In addition, the ratio cord/maternal plasma of 13C-LA was significantly lower in obese women compared to controls. CONCLUSIONS: In conclusion, obese mothers without hyperlipidemia showed a reduced materno-fetal transfer of polyunsaturated FAs which could affect fetal development. This affect dietary recommendation for obese pregnant women. TRIAL REGISTRY NUMBER: ISRCTN69794527.

In vivo kinetic study of materno-fetal fatty acid transfer in obese and normal weight pregnant women.

KEY POINTS: Placental structure and function can be modified as a result of maternal obesity affecting materno-fetal fatty acids (FA) transport. We report for the first time, in humans and in vivo, the kinetics of placental FA transfer in normo-weight and in normolipemic obese pregnant women using stable isotopes. The administration of different tracer FA with similar behaviour to the mother at different time points allows the collection of kinetic information on materno-fetal transfer of FA despite only one sample of placenta and cord can be collected per subject. Computational modelling showed a good fit to the data when considering all maternal plasma lipid classes but not when based only on non-esterified FA. The novel approach using multiple tracer FA administration combined with computational modelling shows a consistent time course of placental tracer FA and predicted total FA accumulation. ABSTRACT: We analyse for the first time the in vivo materno-fetal kinetic transfer of fatty acids (FA) labelled with stable isotopes in control and obese (OB) pregnant women. Labelled FA with a similar metabolism (stearic acid: 13 C-SA; palmitic acid: 13 C-PA; oleic acid: 13 C-OA) were orally administered at -4 h, -8 h and -12 h, respectively prior to elective caesarean section to 10 pregnant women with a body mass index >30 (OB) and 10 with a body mass index in the range 20-25 (NW). Placenta, venous and arterial cord blood were collected obtaining a wide range of FA enrichments. A combined experimental and computational modelling analysis was applied. FA fractional synthesis rate (FSR) in placenta was 11-12% h-1 . No differences were observed between NW and normo-lipidemic OB. It was not possible to estimate FA FSR in cord blood with this oral bolus dose approach. Computational modelling demonstrated a good fit to the data when all maternal plasma lipid classes were included but not with modelling based only on the non-esterified FA fraction. The estimated materno-fetal 13 C-FA transfer was ∼1%. In conclusion, our approach using multiple 13 C-FA tracers allowed us to estimated FSR in placental/maternal plasma but not in fetal/maternal compartments. Computational modelling showed a consistent time course of placental 13 C-FA transfer and predicted total fetal FA accumulation during the experiment. We conclude that, in addition to non-esterified FA fraction in the maternal circulation, maternal plasma very low-density lipoprotein and other lipoproteins are important contributors to placental FA transfer to the fetus.

A new key player in VEGF-dependent angiogenesis in human hepatocellular carcinoma: dimethylarginine dimethylaminohydrolase 1.

BACKGROUND: Anti-angiogenic therapies, targeting VEGF, are a promising treatment for hepatocellular carcinoma (HCC). To enhance this potential therapy, identification of novel targets in this pathway is of major interest. Nitric oxide (NO) plays a crucial role in VEGF-dependent angiogenesis. NO production depends on arginine as substrate and asymmetric dimethylarginine (ADMA) as inhibitor. Dimethylarginine dimethylaminohydrolase 1 (DDAH-1) catabolizes ADMA and therefore regulates NO and VEGF expression. This study unravels additional mechanisms to improve VEGF targeting therapies. METHODS: The expression of DDAH-1 was examined in HCC specimen and non-tumorous background liver of 20 patients undergoing liver resection. Subsequently, arginine/ADMA balance, NO production, and VEGF expression were analyzed. The influence of hypoxia on DDAH-1 and angiogenesis promoting factors was evaluated in HepG2 cells and primary human hepatocytes. RESULTS: DDAH-1 expression was significantly induced in primary HCC tumors compared to non-tumorous background liver. This was associated with an increased arginine/ADMA ratio, higher NO formation, and higher VEGF expression in human HCC compared to non-tumorous liver. Hypoxia induced DDAH-1, iNOS, and VEGF expression in a time-dependent manner in HepG2 cells. CONCLUSIONS: Our results indicate that DDAH-1 expression is increased in human HCC, which is associated with an increase in the arginine/ADMA ratio and enhanced NO formation. Hypoxia may be an initiating factor for the increase in DDAH-1 expression. DDAH-1 expression is associated with promotion of angiogenesis stimulating factor VEGF. Together, our findings for the first time identified DDAH-1 as a key player in the regulation of angiogenesis in human HCC, and by understanding this mechanism, future therapeutic strategies targeting VEGF can be improved.

Early micronutrient supplementation protects against early stress-induced cognitive impairments.

Early-life stress (ES) impairs cognition later in life. Because ES prevention is problematic, intervention is needed, yet the mechanisms that underlie ES remain largely unknown. So far, the role of early nutrition in brain programming has been largely ignored. Here, we demonstrate that essential 1-carbon metabolism-associated micronutrients (1-CMAMs; i.e., methionine and B vitamins) early in life are crucial in programming later cognition by ES. ES was induced in male C57Bl/6 mice from postnatal d (P)2-9. 1-CMAM levels were measured centrally and peripherally by using liquid chromatography-mass spectroscopy. Next, we supplemented the maternal diet with 1-CMAM only during the ES period and studied cognitive, neuroendocrine, neurogenic, transcriptional, and epigenetic changes in adult offspring. We demonstrate that ES specifically reduces methionine in offspring plasma and brain. Of note, dietary 1-CMAM enrichment during P2-9 restored methionine levels and rescued ES-induced adult cognitive impairments. Beneficial effects of this early dietary enrichment were associated with prevention of the ES-induced rise in corticosterone and adrenal gland hypertrophy did not involve changes in maternal care, hippocampal volume, neurogenesis, or global/Nr3c1-specific DNA methylation. In summary, nutrition is important in brain programming by ES. A short, early supplementation with essential micronutrients can already prevent lasting effects of ES. This concept opens new avenues for nutritional intervention.-Naninck, E. F. G., Oosterink, J. E., Yam, K.-Y., de Vries, L. P., Schierbeek, H., van Goudoever, J. B., Verkaik-Schakel, R.-N., Plantinga, J. A., Plosch, T., Lucassen, P. J., Korosi, A. Early micronutrient supplementation protects against early stress-induced cognitive impairments.

Accurate measurement of the essential micronutrients methionine, homocysteine, vitamins B6, B12, B9 and their metabolites in plasma, brain and maternal milk of mice using LC/MS ion trap analysis.

Methionine, homocysteine, vitamins B6, B12, B9, and their metabolites are crucial co-factors and substrates for many basic biological pathways including one-carbon metabolism, and they are particularly important for brain function and development and epigenetic mechanisms. These are essential nutrients that cannot be synthesized endogenously and thus need to be taken in via diet. A novel method was developed that enables simultaneous assessment of the exact concentrations of these essential micronutrients in various matrices, including maternal milk, plasma, and brain of neonatal mice. The protocol for analysis of these components in the various matrices consists of a cleanup step (i.e. lipid extraction followed by protein precipitation) combined with a liquid chromatography mass spectrometry (LC/MS) ion trap method with high sensitivity and selectivity (SRM mode). This novel method enables the measurement of these essential nutrients with good recoveries (69-117%), and high intra-day (<10%) and high intra-day precision (defined as <15% for compounds with an isotopologue and <20% for compounds without an isotopologue as internal standard) in plasma, maternal milk, and brain of mice at low and high levels. In addition, lower limits of quantitation (LOQ) were determined for the various matrices in the range for methionine (700-2000nmol/L), homocysteine (280-460-nmol/L), vitamins B6 (5-230nmol/L), B12 (7-11nmol/L), B9 (20-30nmol/L). Degradation of vitamins and oxidation of homocysteine is limited to a minimum, and only small sample volumes (30µL plasma, 20mg brain and maternal milk) are needed for simultaneous measurement. This method can help to understand how these nutrients are transferred from mother to offspring via maternal milk, as well as how these nutrients are absorbed by the offspring and eventually taken up in various tissues amongst the brain in preclinical and clinical research settings. Therefore the method can help to explore critical periods in lactating mothers and developing offspring.

Intravenous glutamine supplementation enhances renal de novo arginine synthesis in humans: a stable isotope study.

BACKGROUND: Arginine plays a role in many different pathways in multiple cell types. Consequently, a shortage of arginine, caused by pathologic conditions such as cancer or injury, has the potential to disturb many cellular and organ functions. Glutamine is the ultimate source for de novo synthesis of arginine in humans via the intestinal-renal axis. Therefore, we hypothesized that parenteral glutamine supplementation may stimulate the interorgan pathway of arginine production. OBJECTIVES: The objectives were to quantify arginine production from its precursor glutamine and to establish the contribution of the kidneys to de novo synthesis of arginine in patients receiving intravenous supplementation of glutamine dipeptide during major abdominal surgery. DESIGN: Whole-body and renal metabolism of glutamine, citrulline, and arginine was assessed by stable isotope techniques in 7 patients receiving a perioperative supplement of intravenous alanyl-glutamine (0.5 g · kg(-1) · d(-1)). RESULTS: Plasma glutamine, citrulline, and arginine concentrations increased significantly in patients receiving intravenous glutamine dipeptide. At whole-body level, 91% of total citrulline turnover was derived from glutamine, whereas 49% of whole-body citrulline turnover was used for de novo synthesis of arginine. The kidneys were responsible for 75% of whole-body arginine production from citrulline. CONCLUSIONS: Glutamine and citrulline are important sources for de novo arginine synthesis. The kidneys are the main production site for endogenous arginine. After comparison of these results with previous similar studies, our data suggest that an intravenous glutamine supplement doubles renal arginine production from citrulline. This trial was registered at www.trialregister.nl as NTR2914.

A novel method for simultaneous measurement of concentration and enrichment of NO synthesis-specific amino acids in human plasma using stable isotopes and LC/MS ion trap analysis.

Stable isotope studies offer the opportunity to study the in-depth metabolic pathway of glutamine, citrulline, and arginine amino acids involved in NO synthesis. The use of multiple stable isotopes can be used to elucidate the exact transformation of glutamine to citrulline and arginine de novo synthesis. This novel method provides a purification step using cation exchange resin in combination with a rapid and easy derivatization procedure for a precise and robust measurement of the concentration and isotopic enrichments of NO synthesis-specific amino acids using a liquid chromatography mass spectrometry (LC/MS) ion trap system with high sensitivity and selectivity. The ethyl chloroformate derivatization procedure is beneficial in terms of robustness, velocity, simplicity, and derivative stability. In addition, the ethyl chloroformate derivatization can be performed at room temperature in an aqueous environment without incubation and the isolation of the derivatives from the reaction mixture also serves as a purification step. The concentration and enrichment of NO synthesis-specific amino acids as well as phenylalanine and tyrosine to determine protein turnover, were measured with good inter-day precision for the concentration (<7.4%) and enrichment (<12.7%) in plasma samples at low and high levels. The low limit of quantification was 0.2µmol/L for most of the amino acids and the purification method showed to have good recoveries between 78% and 98%. No ion-suppression was observed by post-column infusion experiments. In order to develop new nutritional strategies, this novel method can be used to support the elucidation of the effect of administration of specific supplements on the glutamine-citrulline-arginine pathway by using stable isotope studies.

Ultra-fast searching assists in evaluating sub-ppm mass accuracy enhancement in U-HPLC/Orbitrap MS data.

A strategy, detailed methodology description and software are given with which the mass accuracy of U-HPLC-Orbitrap data (resolving power 50,000 FWHM) can be enhanced by an order of magnitude to sub-ppm levels. After mass accuracy enhancement all 211 reference masses have mass errors within 0.5 ppm; only 14 of these are outside the 0.2 ppm error margin. Further demonstration of mass accuracy enhancement is shown on a pre-concentrated urine sample in which evidence for 89 (342 ions) potential hydroxylated and glucuronated DHEA-metabolites is found. Although most DHEA metabolites have low-intensity mass signals, only 11 out of 342 are outside the ±1 ppm error envelop; 272 mass signals have errors below 0.5 ppm (142 below 0.2 ppm). The methodology consists of: (a) a multiple internal lock correction (here ten masses; no identity of internal lock masses is required) to avoid suppression problems of a single internal lock mass as well as to increase lock precision, (b) a multiple external mass correction (here 211 masses) to correct for calibration errors, (c) intensity dependant mass correction, (d) file averaging. The strategy is supported by ultra-fast file searching of baseline corrected, noise-reduced metAlign output. The output and efficiency of ultra-fast searching is essential in obtaining the required information to visualize the distribution of mass errors and isotope ratio deviations as a function of mass and intensity. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-010-0230-y) contains supplementary material, which is available to authorized users.

Generic sample preparation combined with high-resolution liquid chromatography-time-of-flight mass spectrometry for unification of urine screening in doping-control laboratories.

A unification of doping-control screening procedures of prohibited small molecule substances--including stimulants, narcotics, steroids, beta2-agonists and diuretics--is highly urgent in order to free resources for new classes such as banned proteins. Conceptually this may be achieved by the use of a combination of one gas chromatography-time-of-flight mass spectrometry method and one liquid chromatography-time-of-flight mass spectrometry method. In this work a quantitative screening method using high-resolution liquid chromatography in combination with accurate-mass time-of-flight mass spectrometry was developed and validated for determination of glucocorticosteroids, beta2-agonists, thiazide diuretics, and narcotics and stimulants in urine. To enable the simultaneous isolation of all the compounds of interest and the necessary purification of the resulting extracts, a generic extraction and hydrolysis procedure was combined with a solid-phase extraction modified for these groups of compounds. All 56 compounds are determined using positive electrospray ionisation with the exception of the thiazide diuretics for which the best sensitivity was obtained by using negative electrospray ionisation. The results show that, with the exception of clenhexyl, procaterol, and reproterol, all compounds can be detected below the respective minimum required performance level and the results for linearity, repeatability, within-lab reproducibility, and accuracy show that the method can be used for quantitative screening. If qualitative screening is sufficient the instrumental analysis may be limited to positive ionisation, because all analytes including the thiazides can be detected at the respective minimum required levels in the positive mode. The results show that the application of accurate-mass time-of-flight mass spectrometry in combination with generic extraction and purification procedures is suitable for unification and expansion of the window of screening methods of doping laboratories. Moreover, the full-scan accurate-mass data sets obtained still allow retrospective examination for emerging doping agents, without re-analyzing the samples.

Multiresidue analysis of beta-agonists in bovine and porcine urine, feed and hair using liquid chromatography electrospray ionisation tandem mass spectrometry.

The use of beta-agonists as growth promoters in cattle breeding is forbidden in many countries for reasons of fair trade and consumer protection. In recent years the use of liquid chromatography (LC) tandem mass spectrometry (MS/MS) has been shown to be the method of choice for the control of beta-agonists. In this study an LC-MS/MS multiresidue analysis method is presented for trace analysis of 22 beta-agonists. A truly generic concept has been designed based on mixed-mode solid-phase extraction and positive electrospray ionisation LC-MS/MS operated in the multiple reaction monitoring mode. This method allows application to a wide variety of sample matrices such as urine, feed and hair, following minor modifications to the analysis procedure only. The method features fit-for-purpose sensitivity in urine as shown by CCalpha and CCbeta values of less than 0.2 and less than 0.5 microg/l respectively, for all beta-agonists studied (terbutaline and reproterol, less than 0.3 and less than 1.0 respectively). Similar but semiquantitative application to feed and hair showed CCbeta values of less than 10.0 and less than 5.0 microg/kg, respectively. A further simplification and improvement is demonstrated using Ultra Performance LC (UPLC) and fast-switching MS/MS. The successful validation of this method following the latest EU requirements and its application to real samples demonstrate that a new versatile tool has been achieved for veterinary control of beta-agonists.

[A pilot study of vitamin D in psychogeriatric patients: 82% is (severely) deficient]. / Vitamine D pilot onderzoek bij psychogeriatrische patiënten: 82% is (ernstig) deficiënt.

In a group of 34 psychogeriatric patients (mean age 79 years) the prevalence of hypovitaminosis D was found to be 82%, taking 30 nmol/l as cut-off for calcidiol. We found 47% of the whole group to be severely deficient having values lower than 20 nmol/l. Results of related test are presented and discussed. Patient were treated with oral calcium and vitamin D3 medication. Origin, presentation and risks of hypovitaminosis D, including muscle weakness and the aggravating role of low calcium intake, are discussed with special attention to psychogeriatric patients. Suppletion of vitamin D and calcium is suggested for this patient group.