Isotopic Precursors Research Articles

Adipose tissue estrogen production and metabolism in premenopausal women

ObjectiveAlthough the ovaries produce the majority of estrogens in women before menopause, estrogen is also synthesized in peripheral tissues such as adipose tissue (AT). The typical female AT distribution, concentrated in subcutaneous and femoro-gluteal regions, is estrogen-mediated, but the significance of estrogen synthesis in AT of premenopausal women is poorly understood. Design and MethodsSerum and subcutaneous and visceral AT homogenates from 28 premenopausal women undergoing non-malignant surgery were analyzed for estrone, estradiol, and serum estrone sulfate (E1S) concentrations with liquid chromatography-tandem mass spectrometry. Isotopic precursors were used to measure enzyme activities of estrone-producing steroid sulfatase and estradiol-producing 17β-hydroxysteroid dehydrogenases (17β-HSD). Messenger RNA (mRNA) expression levels of genes for estrogen-metabolizing enzymes were analyzed using real-time reverse transcription quantitative polymerase chain reaction. ResultsWhile estradiol was the predominant circulating active estrogen, estrone dominated in AT, with a higher concentration in visceral than subcutaneous AT (median, 2657 vs 1459 pmol/kg; P = 0.002). Both AT depots converted circulating E1S to estrone, and estrone to estradiol. Median levels of estrone were five to ten times higher in subcutaneous and visceral AT than in serum (P < 0.001) and the estradiol level in visceral AT was 1.3 times higher than in serum (P < 0.005). The local estrone concentration in visceral AT correlated positively with mRNA expression of estrone-producing enzyme aromatase (r = 0.65, P = 0.003). Waist circumference correlated positively with increased estradiol production in subcutaneous AT (r = 0.60, P = 0.039). ConclusionsPremenopausal AT demonstrated high estrogenic enzyme activity and considerable local estrogen concentrations. This may be a factor promoting female-typical AT distribution in premenopausal women.

Phase and Texture Evolution of Hot-Deformed Sm(Co,Fe,Cu,Zr)z Magnet

Phase and texture evolution of the hot-deformed Sm(Co,Fe,Cu,Zr)z magnet prepared by spark plasma sintering (SPS) have been investigated. The effects of hot deformation temperature and height reduction on the phase structure and magnetic properties of the magnet were studied. The results show that with 90% height reduction at 900 °C, the hot-deformed magnet with TbCu <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">7</sub> structure obtains an obvious c-axis texture. However, with increasing hot deformation temperature, some 1:7H phases decompose into 2:17R, 1:5H, and Zr-rich particle phases in the hot deformation process. The hot-deformed magnet with 85% height reduction possesses a remanence (M <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">r</sub> ) of 6.9 kG, which is higher than that of the isotopic precursor.

Enzymatic Reaction-Related Protein Degradation and Proteinaceous Amino Acid Metabolism during the Black Tea (Camellia sinensis) Manufacturing Process

Amino acids contribute to the nutritional value and quality of black tea. Fermentation is the most important stage of the black tea manufacturing process. In this study, we investigated protein degradation and proteinaceous amino acid metabolism associated with enzymatic reactions during fermentation in the black tea manufacturing process. The results showed that the concentrations of both protein and free amino acids decreased during fermentation. We also confirmed that proteins were broken down into free amino acids by artificially synthesized dipeptide benzyloxycarbonyl glutamyl-tyrosine (Z-Glu-Tyr). Metabolites of the amino acid metabolic pathway increased significantly during fermentation. Furthermore, we confirmed that free amino acids were degraded to volatile compounds in a tracer experiment with the isotope precursor. These results provide information that will help black tea manufacturers improve the quality of black tea.

Comprehensive quali-quantitative profiling of neutral and sialylated O-glycome by mass spectrometry based on oligosaccharide metabolic engineering and isotopic labeling.

Mass spectrometry (MS) analysis combined with stable isotopic labeling is of great importance for quantitatively profiling abnormal sialylated O-glycans associated with disease development, but technically hindered by the poor releasing efficiency of O-glycans from glycoprotein or the labile nature of sialic acid residues at glycans. Herein, we developed an isotopic precursor based metabolic amplification and labeling (IPMAL) technique for relative quantitative profiling of the repertoire O-glycans between normal and tumor cells by ESI-MS. Two groups of cells were incubated with peracetylated benzyl-α-N-acetylgalactosamine (Ac3GalNAc-α-Bnd0) or a heavy labeled peracetylated benzyl-α-N-acetylgalactosamine (Ac3GalNAc-α-Bnd5) precursor respectively to amplify the repertoire of O-glycans as Bnd0/d5-O-glycans which could achieve the quantitative O-glycome analysis by ESI-MS after derivatization. The established method demonstrates desirable feasibility, accuracy (relative error (RE) ≤ 4.20%), reproducibility (coefficient of variation (CV) ≤ 7.61%, n = 3) and good quantitation linearity (R2 > 0.99, n = 3) for five Bn-O-glycans with 2 orders of magnitude. Finally, the method has been successfully applied to quantitative analysis of the repertoire O-glycome changes between normal human liver cell line L02 and human hepatoma cell line SMMC-7721. Moreover, the α-2,3/2,6 sialic acid isomers of Bn-O-glycans from these two cells have been further quantitatively distinguished when involved a sialic acid specific derivatization procedure.

Determining the Turnover of Glycosphingolipid Species by Stable-Isotope Tracer Lipidomics

In this study, we used water-soluble stable mass isotope precursors to measure the turnover of endogenous ceramide (Cer) and glycosphingolipids (GSLs) in HEp-2 cells. Cells incubated in the presence of [13C-U]glucose showed rapid incorporation of hexose residues with an increased mass of 6Da into GSLs. Different turnover rates of GSL classes and their molecular species were observed. Approximately 30% of the glucosylceramide, 50% of the lactosylceramide, and 50% of the globotriaosylceramide species showed a much slower turnover than the rest. This demonstrates the existence of different lipid pools, where a certain fraction of species survived for a long time in the cells. The species with the shortest N-amidated fatty acyl groups (C16:0 and C18:0) showed a more rapid turnover than those with the longest N-amidated fatty acids (C24:0 and C24:1). Experiments with addition of [13C-U]serine were performed to study de novo synthesis of Cer from serine and palmitoyl-CoA. These experiments revealed that de novo synthesis contributes to a minor extent to the total synthesis of new sphingolipids and showed that there is a more rapid formation of the longest Cer species (C24:0 and C24:1) than of the shortest species (C16:0), that is, the opposite as observed for the GSLs in the experiments with [13C-U]glucose. In conclusion, this FLUX lipidomics experimental approach with the addition of [13C-U]glucose to cells allows us to not only study the total turnover but also permit observations of lipid intermediates and metabolic flow of endogenous GSL species at the molecular lipid level.

Deuterium-Labeled Precursor Feeding Reveals a New pABA-Containing Meroterpenoid from the Mango Pathogen Xanthomonas citri pv. mangiferaeindicae.

A new para-aminobenzoic-acid-containing natural product from the mango pathogenic organism Xanthomonas citri pv. mangiferaeindicae is described. By means of stable isotope precursor feeding combined with nontargeted LC-MS/MS, the generated spectra were clustered and visualized in a molecular network. This led to the identification of a new member of the meroterpenoids, termed xanthomonic acid, which is composed of an isoprenylated para-aminobenzoic acid. In vitro cytotoxicity assays demonstrated activity of xanthomonic acid against several human cancer cell lines by induction of autophagy.

Current Bioinformatics Challenges in Proteome Dynamics using Heavy Water-based Metabolic Labeling.

Current Bioinformatics Challenges in Proteome Dynamics using Heavy Water-based Metabolic Labeling.

13C-isotope-based protocol for prenyl lipid metabolic analysis in zebrafish embryos

Metabolism has a decisive role in many fundamental biological processes, including organism development and tissue homeostasis. Here we describe a protocol for fast and reliable (13)C-isotope-based in vivo metabolic profiling. This protocol covers the loading of isotope precursor; extraction, preparation and quantification of the labeled lipid metabolites (e.g., the prenyl lipid CoQ10) by the means of HPLC-MS; and its analysis in zebrafish embryos. This protocol can be applied to different types of experimental settings, including tissue-specific metabolic analyses or dynamic metabolic changes that occur during vertebrate embryogenesis. The protocol takes 5-7 d to complete, requiring minimal equipment and analytical expertise, and it represents a unique alternative to the existing ex vivo (e.g., cell lines) isotope-based metabolic methods. This procedure represents a valuable approach for researchers interested in studying the effect of gene manipulation on lipid metabolism in zebrafish and in understanding the genetic conditions that result in metabolism dysfunction.

Formation of the unusual semivolatile diterpene rhizathalene by the Arabidopsis class I terpene synthase TPS08 in the root stele is involved in defense against belowground herbivory.

Secondary metabolites are major constituents of plant defense against herbivore attack. Relatively little is known about the cell type-specific formation and antiherbivore activities of secondary compounds in roots despite the substantial impact of root herbivory on plant performance and fitness. Here, we describe the constitutive formation of semivolatile diterpenes called rhizathalenes by the class I terpene synthase (TPS) 08 in roots of Arabidopsis thaliana. The primary enzymatic product of TPS08, rhizathalene A, which is produced from the substrate all-trans geranylgeranyl diphosphate, represents a so far unidentified class of tricyclic diterpene carbon skeletons with an unusual tricyclic spiro-hydrindane structure. Protein targeting and administration of stable isotope precursors indicate that rhizathalenes are biosynthesized in root leucoplasts. TPS08 expression is largely localized to the root stele, suggesting a centric and gradual release of its diterpene products into the peripheral root cell layers. We demonstrate that roots of Arabidopsis tps08 mutant plants, grown aeroponically and in potting substrate, are more susceptible to herbivory by the opportunistic root herbivore fungus gnat (Bradysia spp) and suffer substantial removal of peripheral tissue at larval feeding sites. Our work provides evidence for the in vivo role of semivolatile diterpene metabolites as local antifeedants in belowground direct defense against root-feeding insects.

Synthesis of carbon-14, carbon-13 and deuterium labeled forms of thioacetamide and thioacetamide S-oxide.

Thioacetamide (TA) is a model hepatotoxin that undergoes metabolic activation via two successive S-oxidations. The ultimate toxic metabolite thioacetamide S,S-dioxide, or its tautomer acetimidoyl sulfinic acid CH3C(NH)SO2H, then acylates lysine side chains on cellular proteins leading to cellular dysfunction or death. To identify individual target proteins, quantitate the extent of their modification and elucidate the structural details of their modification we required both radio-labeled and stable-labeled forms of TA and its intermediate metabolite thioacetamide S-oxide (TASO). The latter is stable when purified but can be difficult to isolate. Considering currently available isotopic precursors we devised and report here methods for the synthesis and isolation of TA and TASO labeled with C-14, C-13 and/or deuterium. The methods are straightforward, utilize readily available precursors and are amenable to small scale.

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage &lt;em&gt;Caenorhabditis elegans&lt;/em&gt;

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in cellular and mammalian models. Here, we describe detailed methods to perform stable isotopic profiling of intermediary metabolism and metabolic flux in the nematode, Caenorhabditis elegans. Methods are described for profiling whole worm free amino acids, labeled carbon dioxide, labeled organic acids, and labeled amino acids in animals exposed to stable isotopes either from early development on nematode growth media agar plates or beginning as young adults while exposed to various pharmacologic treatments in liquid culture. Free amino acids are quantified by high performance liquid chromatography (HPLC) in whole worm aliquots extracted in 4% perchloric acid. Universally labeled 13C-glucose or 1,6-13C2-glucose is utilized as the stable isotopic precursor whose labeled carbon is traced by mass spectrometry in carbon dioxide (both atmospheric and dissolved) as well as in metabolites indicative of flux through glycolysis, pyruvate metabolism, and the tricarboxylic acid cycle. Representative results are included to demonstrate effects of isotope exposure time, various bacterial clearing protocols, and alternative worm disruption methods in wild-type nematodes, as well as the relative extent of isotopic incorporation in mitochondrial complex III mutant worms (isp-1(qm150)) relative to wild-type worms. Application of stable isotopic profiling in living nematodes provides a novel capacity to investigate at the whole animal level real-time metabolic alterations that are caused by individual genetic disorders and/or pharmacologic therapies.

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage &lt;em&gt;Caenorhabditis elegans&lt;/em&gt;

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in cellular and mammalian models. Here, we describe detailed methods to perform stable isotopic profiling of intermediary metabolism and metabolic flux in the nematode, Caenorhabditis elegans. Methods are described for profiling whole worm free amino acids, labeled carbon dioxide, labeled organic acids, and labeled amino acids in animals exposed to stable isotopes either from early development on nematode growth media agar plates or beginning as young adults while exposed to various pharmacologic treatments in liquid culture. Free amino acids are quantified by high performance liquid chromatography (HPLC) in whole worm aliquots extracted in 4% perchloric acid. Universally labeled 13C-glucose or 1,6-13C2-glucose is utilized as the stable isotopic precursor whose labeled carbon is traced by mass spectrometry in carbon dioxide (both atmospheric and dissolved) as well as in metabolites indicative of flux through glycolysis, pyruvate metabolism, and the tricarboxylic acid cycle. Representative results are included to demonstrate effects of isotope exposure time, various bacterial clearing protocols, and alternative worm disruption methods in wild-type nematodes, as well as the relative extent of isotopic incorporation in mitochondrial complex III mutant worms (isp-1(qm150)) relative to wild-type worms. Application of stable isotopic profiling in living nematodes provides a novel capacity to investigate at the whole animal level real-time metabolic alterations that are caused by individual genetic disorders and/or pharmacologic therapies.

Seeing and Measuring Stable Isotope Tags in Subcellular Domains with Ion Microscopy

Multi-isotope Imaging Mass Spectrometry (MIMS) is the combination of an ion microscope-secondary ion mass spectrometer with tracer methods and intensive quantitative image analysis. MIMS allows one to image the distribution and to measure the accumulation of molecules labelled with stable or radioactive isotopes within subcellular domains in volume smaller than 1 cubic micron. The lateral resolution is better than 30 nm, and the depth resolution is a few atomic layers. Up to seven quantitative atomic mass images (or tags) can be recorded simultaneously. A whole cell can be studied layer-after-layer and rendered in quantitative 3D.MIMS can provide unique information in almost all fields of biomedical research, particularly in two broad areas: 1. metabolic turnover (protein, lipids and sugars in sub-cellular domains using as precursors for example 15N-labeled amino-acids or 13C labeled fatty acids) 2. cell lineage ( tracking individual cells within large populations by labelingDNA, for example with 15N thymidine for the salvage pathway and 18O for the de novo pathway).Because stable isotopes are integral part of living organisms, they are not toxic. This allows one to label DNA with a stable isotope precursors for period of time that could reach years. And, this allows applications of MIMS to human.

Discovery of New Natural Products by Intact‐Cell Mass Spectrometry and LC‐SPE‐NMR: Malbranpyrroles, Novel Polyketides from Thermophilic Fungus Malbranchea sulfurea

Six photosensitive polyketides, malbranpyrroles A-F, were discovered from the thermophilic fungus Malbranchea sulfurea by using intact-cell desorption/ionization on silicon mass (ICD-MS) and LC-SPE-NMR. These two strategies facilitate the searching and structural determination of unstable natural products. The ICD-MS indicated that only brown hyphae of M. sulfurea can produce malbranpyrroles. The biosynthetic pathway of malbranpyrroles was evidenced by 13C isotope precursors and amino acid feeding experiments. The cytotoxicity data revealed that the conformation of the conjugated system in malbranpyrroles does not affect cytotoxic potency against cancer cell lines. In addition, the chlorine atom was shown to be the pharmacophore for cytotoxicity.

Sphingolipidomics: a valuable tool for understanding the roles of sphingolipids in biology and disease

The sphingolipidome is the portion of the lipidome that encompasses all sphingoid bases and their derivatives. Whereas the most studied sphingoid base is sphingosine [(2S,3R,4E)-2-aminooctadecene-1,3-diol], mammals have dozens of structural variants, and hundreds of additional types have been found in other eukaryotic organisms and some bacteria and viruses. Multiplying these figures by the N-acyl-derivatives ("ceramides") and the more than 500 phospho- and glyco- headgroups places the number of discrete molecular species in the tens of thousands or higher. Structure-specific, quantitative information about a growing fraction of the sphingolipidome can now be obtained using various types of chromatography coupled with tandem mass spectrometry, and application of these methods is producing many surprises regarding sphingolipid structure, metabolism, and function. Such large data sets can be difficult to interpret, but the development of tools that display results from genomic and lipidomic studies in a pathway relational, nodal, context can make it easier for investigators to deal with this complexity.

Characterization of essential fatty acid metabolism using the stable isotope tracer technique and gas chromatography/ mass spectrometric assay

To better understand essential fatty acid metabolism, several in vivo and in vitro studies have been performed. Diet controlled rats were orally administrated a mixture of two or more labeled fatty acids: d5‐linoleate, ‐linolenate, ‐dihomo‐gamma‐linolenate (DGLA), and/or 13C‐U‐ linoleate or eicosapentaenoate. GC/MS NCI assay was employed to measure the isotopic precursors, their respective metabolites in rat organs over a time‐course from 4 h up to 25 d. Results and conclusions. 1. Only 2.6% and 6% of linoleate and linolenate, respectively, were elongated and desaturated in rats with about 78% being utilized for beta‐oxidation, excretion, or other loss and 16~18% was stored in tissues, mainly adipose, as the precursor itself. 2. Eicosapentaenoate was a more efficient substrate for docosahexaenoate (DHA) than linolenate; similarly, DGLA is a better substrate for arachidonate and n‐6 docosapentanoate than was linoleate. 3. Dietary DHA is the primary source of brain DHA. 4. Preformed DHA markedly decreased the accumulation of de novo biosynthesized DHA and n‐6 docosapentaenoate in liver, plasma, heart and brain; but no significant differences in rat hepatocytes delta‐5 or ‐6 desaturase activities have been observed. Decreased accumulation of newly‐synthesized DHA by dietary DHA may relate to increased DHA catabolism.This project was funded by the DICBR, NIAAA, NIH.

Biosynthetic studies of Nocathiacin-I

Nocathiacin-I is one of the newest members of thiazolyl peptide class of antibiotics. It is a potent inhibitor of bacterial protein synthesis and showed potent in vitro and in vivo Gram-positive antibacterial activity. Understanding of the biosynthesis of natural products is important for improvement of titer and precursor directed biosynthesis for new compounds. Biosynthesis of nocathiacin-I in Amycolatopsis fastidiosa using stable isotope precursor incorporation is described.

The CDP-ethanolamine Pathway and Phosphatidylserine Decarboxylation Generate Different Phosphatidylethanolamine Molecular Species

In mammalian cells, phosphatidylethanolamine (PtdEtn) is mainly synthesized via the CDP-ethanolamine (Kennedy) pathway and by decarboxylation of phosphatidylserine (PtdSer). However, the extent to which these two pathways contribute to overall PtdEtn synthesis both quantitatively and qualitatively is still not clear. To assess their contributions, PtdEtn species synthesized by the two routes were labeled with pathway-specific stable isotope precursors, d(3)-serine and d(4)-ethanolamine, and analyzed by high performance liquid chromatography-mass spectrometry. The major conclusions from this study are that (i) in both McA-RH7777 and Chinese hamster ovary K1 cells, the CDP-ethanolamine pathway was favored over PtdSer decarboxylation, and (ii) both pathways for PtdEtn synthesis are able to produce all diacyl-PtdEtn species, but most of these species were preferentially made by one pathway. For example, the CDP-ethanolamine pathway preferentially synthesized phospholipids with mono- or di-unsaturated fatty acids on the sn-2 position (e.g. (16:0-18:2)PtdEtn and (18:1-18:2)PtdEtn), whereas PtdSer decarboxylation generated species with mainly polyunsaturated fatty acids on the sn-2 position (e.g. (18:0-20:4)PtdEtn and (18:0-20:5)PtdEtn in McArdle and (18: 0-20:4)PtdEtn and (18:0-22:6)PtdEtn in Chinese hamster ovary K1 cells). (iii) The main PtdEtn species newly synthesized from the Kennedy pathway in the microsomal fraction appeared to equilibrate rapidly between the endoplasmic reticulum and mitochondria. (iv) Newly synthesized PtdEtn species preferably formed in the mitochondria, which is at least in part due to the substrate specificity of the phosphatidylserine decarboxylase, seemed to be retained in this organelle. Our data suggest a potentially essential role of the PtdSer decarboxylation pathway in mitochondrial functioning.

Differential Responses of Male and Female Rats: Composition and Accumulation of Essential Fatty Acids in Plasma

This study explores gender differences in fatty acid composition and metabolism in Long Evans adult rats that were orally administrated a mixture of d5-18:2n-6 and d5-18:3n-3 at dosages of 12 and 60 mg/kg BW, respectively, after over 8 weeks of a controlled diet containing adequate levels of linoleic (LA) and alpha-linolenic (LNA) acids. Caudal vein blood was drawn at 2, 4, 8, 12, 24, 48, 96 and 168 h after administration of the stable isotopes. The unlabeled endogenous fatty acids and stable isotope labeled fatty acids in plasma were quantitated by GC/FID and GC/NCI/MS, respectively. In female rats, a greater percentage of plasma 22:5n-6, 22:6n-3 and a lower percentage of 20:4n-6 was observed relative to males. On a per dosage basis, female rats accumulated more d5-22:4n-6, d5-22:5n-6, d5-18:3n-3, d5-20:5n-3 and d5-22:5n-3 than male rats (P<0.05). In summary, female rats have a tendency to accumulate a higher concentration of 22:5n-6 and 22:6n-3 in the plasma compartment than do the male rats and this is reflected in the in vivo fatty acid metabolism of stable isotopic precursors. This project was funded by the DICBR, NIAAA, NIH.

SphinGOMAP: Lipidomic analysis of [glyco]sphingolipid metabolism

Sphingolipids are among the most structurally complex and diverse components of eukaryotic cells (www.sphingomap.org), and have heretofore been difficult to study because the methods for their analysis have been relatively laborious and insensitive. These technical limitations are disappearing with the development of liquid chromatography-tandem mass spectrometric methods that allow hundreds (to thousands) of compounds to be analyzed quantitatively to provide a “sphingolipidomic” profile of cells. Mass spectrometry can also provide information about pathway dynamics by using stable isotope precursors such as [13C]-acetate or -palmitate, which are incorporated into the fatty acyl-CoA's that are used for the sphingoid base backbone and amide-linked fatty acid. These analyses generate tremendous amounts of data, therefore, new methods of data analysis and visualization are critical. Use of these tools is producing a more comprehensive picture of sphingolipid metabolism and many surprises about the pathways, the partitioning of intermediates, and how diverse factors—from agonists, toxins and pharmaceuticals to development and disease--affect sphingolipid metabolism. The work that will be discussed has been conducted in collaboration with M. Cameron Sullards, May Dongmei Wang and others as well as in conjunction with the LIPID MAPS Consortium and the NCRR Integrated Technology Resource for Biomedical Glycomics.