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International audience; N natural isotope abundance (NIA) is systematically higher in infants’ hair than in that of their mothers at birth. This study aimed to investigate this difference in plasma pools. We compared 15N NIA values for plasma amino acid (AA) pools (free + protein-bound) in the umbilical cord artery (UCA) and vein (UCV) and in the maternal vein (MV) at birth. This preliminary study included 7 mother–infant dyads. Whole plasma was treated (HCl) to hydrolyze protein. Following derivatization, AAs were separated using gas chromatography and compound-specific 15N NIA values were measured on-line using an isotope ratio monitoring mass spectrometer. 15N NIA plasma AA pools in the UCA and UCV were highly correlated to the MV, r2 > 0.89 and r2 > 0.88 (both P

International audience; The essential oils (EOs) obtained by hydro-distillation of fruits of Morinda lucida (Rubiaceae) growing in Cote d'Ivoire were investigated. The oil was analyzed and characterized by GC and GC-MS. Analyses of the EOs led to the identification and quantification of 45 constituents. Characterization of the EOs revealed the predominance of (E)-phytol (14.80 %) and n-octanol (6.19 %) followed by beta-caryophyllene (5.54 %) and 6,10,14-trimethyl pentandecan-2-one (5.48 %). The antibacterial activity of EOs from M.lucida was tested against three bacteria Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa using a disc-diffusion method and the minimal inhibitory concentration (MIC) values determined. The inhibition zones and MIC values were in the range of 11.6 to 24.3 mm and 32 to 256 mu g/mL, respectively. The EOs showed good antibacterial activity against S. aureus and P. aeruginosa. The volatile oils extracted from M lucida exhibited a higher activity than the antibiotics tested on P aeruginosa.

International audience; The natural isotope abundance in bulk organic matter or tissues is not a sufficient base to investigate physiological properties, biosynthetic mechanisms, and nutrition sources of biological systems. In fact, isotope effects in metabolism lead to a heterogeneous distribution of H-2, O-18, C-13, and N-15 isotopes in metabolites. Therefore, compoundspecific isotopic analysis (CSIA) is crucial to biological and medical applications of stable isotopes. Here, we review methods to implement CSIA for N-15 and C-13 from plant, animal, and human samples and discuss technical solutions that have been used for the conversion to CO2 and N-2 for IRMS analysis, derivatization and isotope effect measurements. It appears that despite the flexibility of instruments used for CSIA, there is no universal method simply because the chemical nature of metabolites of interest varies considerably. Also, CSIA methods are often limited by isotope effects in sample preparation or the addition of atoms from the derivatizing reagents, and this implies that corrections must be made to calculate a proper d- value. Therefore, CSIA has an enormous potential for biomedical applications, but its utilization requires precautions for its successful application.

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.

The seeds of Carapa procera are exploited extensively in West African ethnopharmacy for the treatment of several pathologies, including inflammation. They also are effective as insect antifeedants and as a mosquito repellent. With the aim of identifying bioactive principles, an ethyl acetate extract of the defatted seeds was made and fractionated. Two principle compounds were isolated. One of these, 5,6-dehydro-7-deacetoxy- 7-oxogedunin (1), while known from another genus of the Meliaceae, is newly identified from the genus Carapa and its X‑ray structure is described for the first time. In addition, 1 displayed strong anti-clonogenic activity at 10 μM. The other compound, mexicanolide (2), is known from this species and showed neither cytotoxicity nor anticlonogenicity. These differences in efficacy are discussed in relation to known structure-activity relationships of limonoids.

Natural (15)N abundance (δ(15)N) varies between individual amino acids (AAs). We hypothesized that δ(15)N of nontransaminating and essential AAs ("source" AAs, such as phenylalanine) present in animal tissues could be used as a marker of dietary origin, whereas δ(15)N of transaminating AAs ("trophic" AAs, such as glutamic acid) could give more detailed insights into animal feed efficiency. Two diets based on dehydrated Lucerne pellets were tested in growing lambs, which promoted different feed efficiencies. No dietary effects were noted on δ(15)N of any AAs analyzed in lamb muscle. In addition, δ(15)N of phenylalanine was unexpectedly similar to that of glutamic acid, suggesting that δ(15)N of AAs is significantly derived from the metabolism of the rumen microbiota and, thus, are not suited for diet authentication in ruminants. In contrast, the δ(15)N of transaminating AAs facilitates an improved prediction of animal feed efficiency compared to the classical isotopic bulk N analysis.

The leaves of Chromolaena odorata (Asteraceae) are exploited extensively in West and Central African ethnopharmacy for the treatment of a wide range of conditions, despite this being a non-native species established in the last 50 years. With the objective of seeking bioactive principles, the nonvolatile compounds, an ethanolic (80% v/v) extract was made and fractionated. From the hexane-soluble fraction, three compounds were isolated. Two of these, 5-hydroxy-7,4'-dimethoxyflavanone and 2'-hydroxy-4,4',5',6'-tetramethoxychalcone, have previously been identified in C. odorata leaves. The third was fully characterised spectroscopically and found to be 1,6-dimethyl-4-(1-methylethyl)naphthalene (cadalene), not previously isolated from the Asteraceae. All three compounds were tested for their cytotoxicity and anticancer properties. 2'-Hydroxy-4,4',5',6'-tetramethoxychalcone was found to be both cytotoxic and anticlonogenic at 20 µm in cell lines Cal51, MCF7 and MDAMB-468, and to act synergistically with the Bcl2 inhibitor ABT737 to enhance apoptosis in Cal51 breast cancer cells.

Metabolomic studies by NMR spectroscopy are increasingly employed for a variety of biomedical applications. A very standardized 1D proton NMR protocol is generally employed for data acquisition, associated with multivariate statistical tests. Even if targeted approaches have been proposed to quantify metabolites from such experiments, quantification is often made difficult by the high degree of overlap characterizing 1H NMR spectra of biological samples. Two-dimensional spectroscopy presents a high potential for accurately measuring concentrations in complex samples, as it offers a much higher discrimination between metabolite resonances. We have recently proposed an original approach relying on the 1H 2D INADEQUATE pulse sequence, optimized for fast quantitative analysis of complex metabolic mixtures. Here, the first application of the quantitative 1H 2D INADEQUATE experiment to a real metabonomic study is presented. Absolute metabolite concentrations are determined for different breast cancer cell line extracts, by a standard addition procedure. The protocol is characterized by high analytical performances (accuracy better than 1%, excellent linearity), even if it is affected by relatively long acquisition durations (15 min to 1 h per spectrum). It is applied to three different cell lines, expressing different hormonal and tyrosine kinase receptors. The absolute concentrations of 15 metabolites are determined, revealing significant differences between cell lines. The metabolite concentrations measured are in good agreement with previous studies regarding metabolic profile changes of breast cancer. While providing a high degree of discrimination, this methodology offers a powerful tool for the determination of relevant biomarkers. Copyright © 2012 John Wiley & Sons, Ltd.

Multi-element stable isotope ratios have been assessed as a means to distinguish between fermented cocoa beans from different geographical and varietal origins. Isotope ratios and percentage composition for C and N were measured in different tissues (cotyledons, shells) and extracts (pure theobromine, defatted cocoa solids, protein, lipids) obtained from fermented cocoa bean samples. Sixty-one samples from 24 different geographical origins covering all four continental areas producing cocoa were analyzed. Treatment of the data with unsupervised (Principal Component Analysis) and supervised (Partial Least Squares Discriminant Analysis) multiparametric statistical methods allowed the cocoa beans from different origins to be distinguished. The most discriminant variables identified as responsible for geographical and varietal differences were the δ15N and δ13C values of cocoa beans and some extracts and tissues. It can be shown that the isotope ratios are correlated with the altitude and precipitation conditions found in the different cocoa-growing regions.

Quantitative analysis of metabolic mixtures by H-1 1D NMR offers a limited potential for precise quantification of biomarkers, due to strong overlap between the peaks. Two-dimensional spectroscopy is a powerful tool to unambiguously and simultaneously measure a larger number of metabolite contributions. However, it is still rarely used for quantification, first because quantitative analysis by 2D NMR requires a calibration procedure due to the multi-impulsional nature of 2D NMR experiments, and above all because of the prohibitive experiment duration that is necessary to obtain such a calibration curve. In this work, we develop and evaluate a 2D H-1 INADEQUATE protocol for a fast determination of metabolite concentrations in complex mixtures. The 2D pulse sequence is carefully optimized and evaluated in terms of precision and linearity. Quantitative H-1 INADEQUATE 2D spectra of metabolic mixtures are obtained in 7 min with a repeatability better than 2% for metabolite concentrations as small as 100 mu M and an excellent linearity. The method described in this work allows a fast and precise quantification of metabolic mixtures, and it forms a promising tool for metabonomic studies. (c) 2010 Elsevier B.V. All rights reserved.

The measurement of the nitrogen isotope ratio of taurine (2-aminoethanesulphonic acid) in biological samples has a large number of potential applications. Taurine is a small water-soluble molecule which is notoriously difficult to analyze due to its polarity and functionality. A method is described which allows the determination of the natural abundance delta N-15 values of taurine and structural analogues, such as 3-amino-1-propanesulphonic acid (APSA), by isotope ratio mass spectrometry interfaced to gas chromatography (GC-irm-MS). The one-step protocol exploits the simultaneous derivatization of both functionalities of these aminosulphonic acids by reaction with triethylortho-acetate (TEOA). Conditions have been established which ensure quantitative reaction thus avoiding any nitrogen isotope fractionation during derivatization and workup. The differences in the delta N-15 values of derivatized and non-derivatized taurine and APSA all fall within the working range of 0.4 parts per thousand (-0.02 to 0.39 parts per thousand). When applied to four sources of taurine with various delta N-15 values, the method achieved excellent reproducibility and accuracy. The optimized method enables the determination of the natural abundance delta N-15 values of taurine over the concentration range 1.5-7.84 mu mol.mL(-1) in samples of biological origin. Copyright (C) 2010 John Wiley & Sons, Ltd.

BACKGROUND: Optimization of nitrogen (N) and sulfur (S) nutrition of wheat influences the bread-making quality of grain. This study was conducted to determine whether the application of N and S in winter wheat at anthesis stage during two field trials could influence the absorption and distribution of N and S in grain and flour bread-making quality RESULTS: The incorporation of 15N and 34S tracers in various plant parts (leaves, stems, spikes) was determined by isotopic ratio mass spectrometry coupled with an elemental analyser (EA-IRMS). The effects of foliar fertilization on percentage of 15N and 34S recovery in various plant parts, grain yield, flour protein content, and dough properties were investigated after a combination of treatments: N (urea), S (micronized elemental S) and NS (both urea and micronized elemental S). Nitrogen (70–80%) and S (35–55%) were quickly assimilated during the days following application. Sixty days after application, 42–68% N and 6–12% S were recovered in the grain. When N and S fertilizers were applied simultaneously, N and S recoveries (68% and 12%), N/S ratios (11–14), flour protein content (115 g kg−1) and dough strength (310 × 10−4 J), swelling (21.8 mmH2O) and extensibility (96.4 mm) were increased. CONCLUSION: A synergistic effect between the foliar applied N and S fertilizers appears to increase N and S assimilation in grain and may improve bread-making qualities. Copyright © 2007 Society of Chemical Industry

This review aims at providing an analysis of the various available reference methods in nuclear magnetic resonance, based on recent literature. This paper will provide a tool to help users in choosing the appropriate method depending on the NMR experiment, sample and targeted application. For each method, the crucial parameters that should be particularly considered to reach the accuracy objectives of quantitative NMR are described. After defining the main concepts, the principle, the analytical performance and the main recent applications of each method are described, and a critical discussion is finally provided. (C) 2013 Elsevier B.V. All rights reserved.

International audience; In the article (Romek et al. 2013) we reported the values of δ15N (‰) and δ13C (‰) obtained bySince exclusively breast-suckled infants obtain their nutrient only from their mother's milk, it might be anticipated that a correlation will exist between the (15)N/(14)N isotope ratios of amino acids of protein of young infants and those supplied by their mother. The work presented here aimed to determine whether amino nitrogen transfer from human milk to infant hair protein synthesized within the first month of life conserves the maternal isotopic signature or whether post-ingestion fractionation dominates the nitrogen isotope spectrum. The study was conducted at 1 month post-birth on 100 mother-infant pairs. Isotope ratios (15)N/(14)N and (13)C/(12)C were measured using isotope ratio measurement by Mass Spectrometry (irm-MS) for whole maternal milk, and infant hair and (15)N/(14)N ratios were also measured by GC-irm-MS for the N-pivaloyl-O-isopropyl esters of amino acids obtained from the hydrolysis of milk and hair proteins. The δ(15)N and δ(13)C (‰) were found to be significantly higher in infant hair than in breast milk (δ(15)N, P < 0.001; δ(13)C, P < 0.001). Furthermore, the δ(15)N (‰) of individual amino acids in infant hair was also significantly higher than that in maternal milk (P < 0.001). By calculation, the observed shift in isotope ratio was shown not to be accounted for by the amino acid composition of hair and milk proteins, indicating that it is not simply due to differences in the composition in the proteins present. Rather, it would appear that each pool-mother and infant-turns over independently, and that fractionation in infant N-metabolism even in the first month of life dominates over the nutrient N-content.

Microalgae appear to be one of the most promising sustainable resources as alternative crops for the production of renewable transport fuel. The exploitation of this bioresource requires, however, a fine monitoring of the culture conditions, for example by using more relevant control variables than usual macroscopic indicators (biomass or pigment estimation). In this proof of concept study, we propose to search potential biomarkers of progressive nitrogen regime culture conditions using an untargeted metabolomic approach based on LC-HRMS combined to a non-invasive analysis based on FTIR spectroscopy. One microalgae model was investigated i.e. Chlamydomonas reinhardtii to characterize the effect of progressive nitrogen regime in batch culture conditions on its metabolome. FTIR allowed assessing the intracellular macrometabolic perturbations, highlighting the over-accumulation of carbohydrates. LC-HRMS complemented the macromolecular information by revealing the dependence of microalgae metabotypes on nitrogen regime conditions tested for cells culture. Patterns of significantly modulated metabolites were also detected during those slight contrasted nitrogen regimes and interesting features were structurally elucidated. This included metabolites belonging to the pantothenate, branched chain and aromatic amino acids pathways. In the last step of this proof of concept study, amino acid targets proposed by metabolomic investigations were assessed on nitrogen-limited continuous culture on photobioreactors. This was performed to test the validity of proposed targets in real small-scale industrial production conditions. Results were very encouraging and suggested the possibility of using potentially relevant metabolites as intracellular biomarkers only (tryptophan) or as both intra and extracellular biomarkers (e.g. 2-methylbutyric acid and ketoleucine).

International audience; RATIONALE In isotope tracer experiments used in nutritional studies, it is frequently desirable both to determine the 15N/14N ratios of target compounds and to quantify these compounds. This report shows how this can be achieved in a single chromatographic run for protein amino acids using an isotope ratio mass spectrometer. METHODS Protein hydrolysis by acidic digestion was used to release amino acids, which were then derivatized as their N-pivaloyl-O-isopropyl esters. Suitable conditions for sample preparation were established for both hair and milk proteins. The N-pivaloyl-O-isopropyl esters of amino acids were separated by gas chromatography (GC) on a 60m ZB-WAX column linked via a combustion interface to an isotope ratio mass spectrometer. The 15N/14N ratios were obtained from the m/z 28, 29 and 30 peak intensities and the quantities from the Area All (Vs) integrated peak areas. RESULTS It is shown from a five-point calibration curve that both parameters can be measured reliably within the range of 1.0 to 2.0mg/mL for the major amino acids derived from the hydrolysis of human maternal milk or hair samples. The method was validated in terms of inter-day and inter-user repeatability for both parameters for 14 amino acids. The amino acid percentage composition showed a good correlation with literature values. The method was applied to determine the variability in a population of lactating mothers and their infants. CONCLUSIONS It has been established that 15N values can be simultaneously determined for a complex mixture of amino acids at variable concentrations. It is shown that the percentage composition obtained correlates well with that obtained by calculation from the protein composition or from literature values. This procedure should provide a significant saving in analysis time, especially in those cases where the GC run-time is necessarily long. It allows the satisfactory determination of the variation within a sample population. Copyright (c) 2013 John Wiley & Sons, Ltd.

Leptoderris fasciculata (Benth.) Dunn is a woody liana that grows in the tropical zone of West Africa, where it is exploited in traditional medicine for a number of uses. In the south-eastern Ivory Coast, it is applied to stop bleeding in women after childbirth or in cases of menorrhagia. The phytochemistry of this plant has received little previous attention. We report the isolation of 40,5,6,7-tetramethoxyisoflavone and dihydrochalcone, new as natural products, together with 10 known compounds from the leaves of L. fasciculata collected in the Ivory Coast. (C) 2013 Phytochemical Society of Europe. Published by Elsevier B. V. All rights reserved.

Summary Sulphur is a crucial microelement for plant metabolism, involved in response mechanisms to oxidative stress, C1 metabolism, electron transfer, secondary metabolism and post-translational peptide modifications. However, relatively little is known about sulphur transport and partitioning in plants. Although key steps of sulphur assimilation have been shown using labelling with radioactive 35S in past decades, the use of a natural tracer, allowing the examination of metabolic commitments and mass balance, is desirable. Sulphur stable isotopes (32S and 34S) have been proven to be useful for the investigation of the origin of sulphur atoms in geochemistry and for the detection of the origin of sulphur atoms. Nevertheless, their use for the study of primary sulphur metabolism has been impeded by our lack of knowledge of 32S/34S isotope fractionations and convenient methods for δ34S analyses. Here, we review documented 32S/34S isotope fractionations that may apply to sulphur metabolism, and explain how they should yield disparities amongst sulphur-containing plant compounds.

Two-dimensional nuclear magnetic resonance (2D NMR) forms a powerful tool for the quantitative analysis of complex mixtures such as samples of metabolic relevance. However, its use for quantitative purposes is far from being trivial, not only because of the associated experiment time, but also due to its subsequent high sensitivity to hardware instabilities affecting its precision. In this paper, an alternative approach is considered to measure absolute metabolite concentrations in complex mixtures with a high precision in a reasonable time. It is based on a ``multi-scan single shot'' (M3S) strategy, which is derived from the ultrafast 2D NMR methodology. First, the analytical performance of this methodology is compared to the one of conventional 2D NMR. 2D correlation spectroscopy (COSY) spectra are obtained in 10 min on model metabolic mixtures, with a precision in the 1-4% range (versus 5-18% for the conventional approach). The M3S approach also shows a better linearity than its conventional counterpart. It ensures that accurate quantitative results can be obtained provided that a calibration procedure is carried out. The M3S COSY approach is then applied to measure the absolute metabolite concentration in three breast cancer cell line extracts, relying on a standard addition protocol. M3S COSY spectra of such extracts are recorded in 20 min and give access to the absolute concentration of 14 major metabolites, showing significant differences between cell lines.

The coupling between an electrochemical cell (EC) and a mass spectrometer (MS) is a useful screening tool (EC-MS) to study the oxidative transformation pathways of various electroactive species. For that purpose, we showed that the EC-MS method, carried out in the presence and absence of isotope 18O labeled water leads not only to a fast identification of oxidation products but also leads to a fast elucidation of the mechanism pathway reaction. We examined herein the case of the electrochemical hydrolysis of activated aromatic ether. Acebutolol (β-blockers) was selected herein as model of activated aromatic ether, and its electrochemical oxidation was examined in both the presence and absence of isotope 18O labeled water. To elucidate electrochemical hydrolysis pathway reaction: O-dealkylation or O-dealkoxylation, our approach was used to prove its applicability. The electrochemical oxidation mechanism was then elucidated showing an O-dealkoxylation reaction. In addition, density functional theory (DFT) calculations fully support the experimental conclusions.

Metabolomic analysis of mammalian cells can be applied across multiple fields including medicine and toxicology. It requires the acquisition of reproducible, robust, reliable, and homogeneous biological data sets. Particular attention must be paid to the efficiency and reliability of the extraction procedure. Even though a number of recent studies have dealt with optimizing a particular protocol for specific matrices and analytical techniques, there is no universal method to allow the detection of the entire cellular metabolome. Here, we present a strategy for choosing extraction procedures from adherent mammalian cells for the global NMR analysis of the metabolome. After the quenching of cells, intracellular metabolites are extracted from the cells using one of the following solvent systems of varying polarities: perchloric acid, acetonitrile/water, methanol, methanol/water, and methanol/chloroform/water. The hydrophilic metabolite profiles are analysed using (1)H nuclear magnetic resonance (NMR) spectroscopy. We propose an original geometric representation of metabolites reflecting the efficiency of extraction methods. In the case of NMR-based analysis of mammalian cells, this methodology demonstrates that a higher portion of intracellular metabolites are extracted by using methanol or methanol/chloroform/water. The preferred method is evaluated in terms of biological variability for studying metabolic changes caused by the phenotype of four different human breast cancer cell lines, showing that the selected extraction procedure is a promising tool for metabolomic and metabonomic studies of mammalian cells. The strategy proposed in this paper to compare extraction procedures is applicable to NMR-based metabolomic studies of various systems.

International audience; A method is described whereby the concentrations and 2H isotope enrichment of glutathione (GSH) and cysteine can be simultaneously determined in a single gas chromatography–mass spectrometry run following derivatization as their N,S-ethoxycarbonyl methyl esters. Improvements of the derivatization protocol and the use of a short gas chromatography column combined with single-ion monitoring allow for rapid quantification of these parameters with acceptable precision and reproducibility (coefficient of variation less than 5%). The method makes possible their quantitative measurement in very small volumes (50 μL) of human umbilical cord blood, and is thus suitable for determining the cysteine and GSH concentrations and 2H isotope enrichments in blood samples from neonates or in other conditions in which sample size is restricted. It is shown that the fractional synthesis rate of human umbilical erythrocyte lysates in vitro is several-fold greater than that measured in vivo.

International audience; The depletion of glutathione (GSH) reported in very-low-birth-weight infants is implicated in several pathologies, especially if deficiency occurs during foetal development. The cause of this depletion is suggested to be modification of GSH turnover. To probe the role of GSH, a reliable non-invasive method adapted to very-low-birth-weight infants is required. In this paper, we report the preparation of the N,S-ethoxycarbonyl methyl ester derivatives of GSH and glycine and their application to the measurement of C-13/C-12 ratios at natural abundance in erythrocyte samples by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). The technique allowed the determination of C-13/C-12 ratios at natural abundance with a precision < 3% and within-day and between-day variabilities both < 4%. The method is able to determine accurately low C-13-enrichments in GSH (0.00241 to 0.00753 Atom Percent Excess) in erythrocyte extracts following incubation with C-13-glycine at low specific enrichment (approx. 1.5 atom %). Excellent agreement was obtained between the calculated GSH fractional synthesis rate (FSR) in human adult blood (approx. 300% day(-1)) using the low-enrichment C-13-glycine/GC/C/IRMS protocol and that using highly enriched C-13-glycine (99 atom %)/GC/MS with the same derivative. The GC/C/IRMS method was shown to be suitable to measure the in vitro GSH FSR (200-660% day(-1)) in human venous and arterial blood from the umbilical cord. This approach provides a good tool for studying the turnover of GSH in vitro in infants, allowing both the use of minimal amounts of tracer and negligible perturbation of endogenous precursor pools