Natural Isotope Abundance in Metabolites: Techniques and Kinetic Isotope Effect Measurement in Plant, Animal, and Human Tissues

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 2H, 18O, 13C, and 15N isotopes in metabolites. Therefore, compound-specific isotopic analysis (CSIA) is crucial to biological and medical applications of stable isotopes. Here, we review methods to implement CSIA for 15N and 13C from plant, animal, and human samples and discuss technical solutions that have been used for the conversion to CO2 and N2 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 δ-value. Therefore, CSIA has an enormous potential for biomedical applications, but its utilization requires precautions for its successful application. © 2017 Elsevier Inc.The authors thank the support of the initiative PLAISIR (Pays de la Loire Association for International Structure on “Isotopomic Research”) funded by the French Regional International Strategy Grant Pays de la Loire, and the Australian Research Council through a Future Fellowship grant (under contract FT140100645)

Cord Blood Glutathione Depletion in Preterm Infants: Correlation with Maternal Cysteine Depletion

Background: Depletion of blood glutathione (GSH), a key antioxidant, is known to occur in preterm infants. Objective: Our aim was to determine: 1) whether GSH depletion is present at the time of birth; and 2) whether it is associated with insufficient availability of cysteine (cys), the limiting GSH precursor, or a decreased capacity to synthesize GSH. Methodology: Sixteen mothers delivering very low birth weight infants (VLBW), and 16 mothers delivering healthy, full term neonates were enrolled. Immediately after birth, erythrocytes from umbilical vein, umbilical artery, and maternal blood were obtained to assess GSH [GSH] and cysteine [cys] concentrations, and the GSH synthesis rate was determined from the incorporation of labeled cysteine into GSH in isolated erythrocytes ex vivo, measured using gas chromatography mass spectrometry. Principal Findings: Compared with mothers delivering at full term, mothers delivering prematurely had markedly lower erythrocyte [GSH] and [cys] and these were significantly depressed in VLBW infants, compared with term neonates. A strong correlation was found between maternal and fetal GSH and cysteine levels. The capacity to synthesize GSH was as high in VLBW as in term infants. Conclusion: The current data demonstrate that: 1) GSH depletion is present at the time of birth in VLBW infants; 2) As VLBW neonates possess a fully active capacity to synthesize glutathione, the depletion may arise from inadequate cysteine availability, potentially due to maternal depletion. Further studies would be needed to determine whether maternal-fetal cysteine transfer is decreased in preterm infants, and, if so, whether cysteine supplementation of mothers at risk of delivering prematurely would strengthen antioxidant defense in preterm neonates

GSTZ1 genotypes correlate with dichloroacetate pharmacokinetics and chronic side effects in multiple myeloma patients in a pilot phase 2 clinical trial

Dichloroacetate (DCA) is an investigational drug targeting the glycolytic hallmark of cancer by inhibiting pyruvate dehydrogenase kinases (PDK). It is metabolized by GSTZ1, which has common polymorphisms altering enzyme or promoter activity. GSTZ1 is also irreversibly inactivated by DCA. In the first clinical trial of DCA in a hematological malignancy, DiCAM (DiChloroAcetate in Myeloma), we have examined the relationship between DCA concentrations, GSTZ1 genotype, side effects, and patient response. DiCAM recruited seven myeloma patients in partial remission. DCA was administered orally for 3 months with a loading dose. Pharmacokinetics were performed on day 1 and 8. Trough and peak concentrations of DCA were measured monthly. GSTZ1 genotypes were correlated with drug concentrations, tolerability, and disease outcomes. One patient responded and two patients showed a partial response after one month of DCA treatment, which included the loading dose. The initial half‐life of DCA was shorter in two patients, correlating with heterozygosity for GSTZ1*A genotype, a high enzyme activity variant. Over 3 months, one patient maintained DCA trough concentrations approximately threefold higher than other patients, which correlated with a low activity promoter genotype (−1002A, rs7160195) for GSTZ1. This patient displayed the strongest response, but also the strongest neuropathy. Overall, serum concentrations of DCA were sufficient to inhibit the constitutive target PDK2, but unlikely to inhibit targets induced in cancer. Promoter GSTZ1 polymorphisms may be important determinants of DCA concentrations and neuropathy during chronic treatment. Novel dosing regimens may be necessary to achieve effective DCA concentrations in most cancer patients while avoiding neuropathy.This work was supported by The Canberra Hospital Private Practice Trust Fund, Cancer Council ACT Project Grant APP1103848, and the Monaro Committee for Cancer Researc

1H-NMR-Based Metabolic Profiling of Maternal and Umbilical Cord Blood Indicates Altered Materno-Foetal Nutrient Exchange in Preterm Infants

Background: Adequate foetal growth is primarily determined by nutrient availability, which is dependent on placental nutrient transport and foetal metabolism. We have used 1H nuclear magnetic resonance (NMR) spectroscopy to probe the metabolic adaptations associated with premature birth. Methodology: The metabolic profile in 1H NMR spectra of plasma taken immediately after birth from umbilical vein, umbilical artery and maternal blood were recorded for mothers delivering very-low-birth-weight (VLBW) or normo-ponderal full-term (FT) neonates. Principal Findings: Clear distinctions between maternal and cord plasma of all samples were observed by principal component analysis (PCA). Levels of amino acids, glucose, and albumin-lysyl in cord plasma exceeded those in maternal plasma, whereas lipoproteins (notably low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) and lipid levels were lower in cord plasma from both VLBW and FT neonates. The metabolic signature of mothers delivering VLBW infants included decreased levels of acetate and increased levels of lipids, pyruvate, glutamine, valine and threonine. Decreased levels of lipoproteins glucose, pyruvate and albumin-lysyl and increased levels of glutamine were characteristic of cord blood (both arterial and venous) from VLBW infants, along with a decrease in levels of several amino acids in arterial cord blood. Conclusion: These results show that, because of its characteristics and simple non-invasive mode of collection, cord plasma is particularly suited for metabolomic analysis even in VLBW infants and provides new insights into the materno-foetal nutrient exchange in preterm infants

Contribution à l'amélioration de la qualité technologique des farines de blé (Triticum aestivum L.) par l'apport foliaire d'azote et de soufre (implication des protéïnes de réserve et du glutathion)

Dans l'objectif d'améliorer la qualité technologique des farines, l'influence des applications foliaires d'azote et de soufre sur les protéines de réserve et sur le glutathion dans le grain de blé tendre, a été étudié. Dans un premier temps, nous avons mis au point une technique de mesure du 34S dans les différentes parties de la plante et les différentes classes protéiques, par analyse élémentaire couplée à la spectrométrie de masse de rapports isotopiques. A partir des mesures de traceurs 34S et 15N, nous avons démontré que le soufre et l'azote apportés par voie foliaire au stade anthèse et post anthèse sont incorporés dans le grain et dans les protéines de réserve. dans un deuxième temps, nous avons caractérisé la teneur et la composition en protéines de réserve et en glutathion à l'aide de différentes techniques analytiques (analyse élémentaire; chromatographie liquide haute performance d'exclusion stérique, en phase inversée et par échanges d'ion.) Nous avons montré que des fertilisations foliaires d'azote et de soufre modifient la synthèse des gluténines et des gliadines, ainsi que des différentes formes du glutathion présentes dans le grain.TOULOUSE-ENSAT-Documentation (315552324) / SudocSudocFranceF

Natural Isotope Abundance in Metabolites: Techniques and Kinetic Isotope Effect Measurement in Plant, Animal, and Human Tissues

International audienceThe 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

S-32/S-34 isotope fractionation in plant sulphur metabolism

Sulphur is a crucial microelement for plant metabolism, involved in response mechanisms to oxidative stress, C-1 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 S-35 in past decades, the use of a natural tracer, allowing the examination of metabolic commitments and mass balance, is desirable. Sulphur stable isotopes (S-32 and S-34) 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 S-32/S-34 isotope fractionations and convenient methods for S-34 analyses. Here, we review documented S-32/S-34 isotope fractionations that may apply to sulphur metabolism, and explain how they should yield disparities amongst sulphur-containing plant compounds

Fast and precise quantitative analysis of metabolic mixtures by 2D H-1 INADEQUATE NMR

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

Absolute quantification of metabolites in breast cancer cell extracts by quantitative 2D 1H INADEQUATE NMR

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 (c) 2012 John Wiley & Sons, Ltd

Reference and normalization methods: Essential tools for the intercomparison of NMR spectra

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