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1.
Ann Nucl Med ; 34(12): 884-891, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33141408

RESUMO

OBJECTIVE: 18F is the most extensively used radioisotope in current clinical practices of PET imaging. This selection is based on the several criteria of pure PET radioisotopes with an optimum half-life, and low positron energy that contributes to a smaller positron range. In addition to 18F, other radioisotopes such as 68Ga and 124I are currently gained much attention with the increase in interest in new PET tracers entering the clinical trials. This study aims to determine the minimal scan time per bed position (Tmin) for the 124I and 68Ga based on the quantitative differences in PET imaging of 68Ga and 124I relative to 18F. METHODS: The European Association of Nuclear Medicine (EANM) procedure guidelines version 2.0 for FDG-PET tumor imaging has adhered for this purpose. A NEMA2012/IEC2008 phantom was filled with tumor to background ratio of 10:1 with the activity concentration of 30 kBq/ml ± 10 and 3 kBq/ml ± 10% for each radioisotope. The phantom was scanned using different acquisition times per bed position (1, 5, 7, 10 and 15 min) to determine the Tmin. The definition of Tmin was performed using an image coefficient of variations (COV) of 15%. RESULTS: Tmin obtained for 18F, 68Ga and 124I were 3.08, 3.24 and 32.93 min, respectively. Quantitative analyses among 18F, 68Ga and 124I images were performed. Signal-to-noise ratio (SNR), contrast recovery coefficients (CRC), and visibility (VH) are the image quality parameters analysed in this study. Generally, 68Ga and 18F gave better image quality as compared to 124I for all the parameters studied. CONCLUSION: We have defined Tmin for 18F, 68Ga and 124I SPECT CT imaging based on NEMA2012/IEC2008 phantom imaging. Despite the long scanning time suggested by Tmin, improvement in the image quality is acquired especially for 124I. In clinical practice, the long acquisition time, nevertheless, may cause patient discomfort and motion artifact.


Assuntos
Elementos Radioativos/química , Marcação por Isótopo/métodos , Neoplasias/diagnóstico por imagem , Tomografia Computadorizada com Tomografia por Emissão de Pósitrons/instrumentação , Tomografia Computadorizada com Tomografia por Emissão de Pósitrons/métodos , Composição de Medicamentos , Radioisótopos de Flúor/química , Radioisótopos de Gálio/química , Humanos , Processamento de Imagem Assistida por Computador/instrumentação , Processamento de Imagem Assistida por Computador/métodos , Radioisótopos do Iodo/química , Imagens de Fantasmas , Doses de Radiação , Traçadores Radioativos , Razão Sinal-Ruído , Fatores de Tempo
2.
PLoS One ; 15(8): e0237612, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32790731

RESUMO

Seventeen glass vessels and twenty glass beads recovered from the excavations at the ancient city of Malindi and the archaeological site of Mambrui in Kenya, east Africa were analysed using electron probe microanalysis (EPMA) and laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). The results show that all of the glass samples are soda-lime-silica glass. They belong to the high alumina -plant ash glass type, characterised by high alumina and relatively low calcium contents, widely distributed in eastern (10th- 16th centuries AD) and southern Africa (13th - 15th centuries AD), Central Asia (9th- 14th centuries AD) and southeast Asia (12th- 13th centuries AD), made with plant ashes and sands. This is an understudied glass type for which previous research has indicated there were three types. When compared with published research on such glasses using Zr, Ti, Ba, Cr, La, Li, Cs, Na2O, MgO and CaO we have identified at least four different compositional groups of v-Na-Al glass: Types A, B, C and D. By comparing the results with contemporary v-Na-Al glass vessels and beads from Central Asia, Africa, and southeast Asia we show that most of the Malindi and Mambrui glass share similar characteristics to the compositions of Mapungubwe Oblate and some of the Madagascar glass beads from southern Africa. They belong to Type A v-Na-Al glass which is characterised by an elevated level of Ti and Ba and a relatively high ratios of Cr/La, relatively low Zr concentrations and low ratios of Zr/Ti. Differences in Zr, Li, MgO and Na2O concentrations in Type A glass indicates that there are subgroups which might derive from different glass workshop(s) specialising in Type A v-Na-Al glass production. Comparison with the chemical compositions of glass from Ghazni, Afghanistan and Termez, Uzbekistan, and by using lead isotope analysis, we suggest v-Na-Al glass was manufactured in Central Asia and possibly worked into vessels and beads there.


Assuntos
Compostos de Cálcio/química , Vidro/química , Óxidos/química , Plantas/química , Dióxido de Silício/química , Hidróxido de Sódio/química , Oligoelementos/análise , Oligoelementos/história , África Oriental , Óxido de Alumínio , Arqueologia , História do Século XV , História do Século XVI , Oceano Índico , Marcação por Isótopo/métodos , Quênia , Espectrometria de Massas
3.
BMC Bioinformatics ; 21(1): 297, 2020 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-32650717

RESUMO

BACKGROUND: Stable isotope tracing has become an invaluable tool for probing the metabolism of biological systems. However, data analysis and visualization from metabolic tracing studies often involve multiple software packages and lack pathway architecture. A deep understanding of the metabolic contexts from such datasets is required for biological interpretation. Currently, there is no single software package that allows researchers to analyze and integrate stable isotope tracing data into annotated or custom-built metabolic networks. RESULTS: We built a standalone web-based software, Escher-Trace, for analyzing tracing data and communicating results. Escher-Trace allows users to upload baseline corrected mass spectrometer (MS) tracing data and correct for natural isotope abundance, generate publication quality graphs of metabolite labeling, and present data in the context of annotated metabolic pathways. Here we provide a detailed walk-through of how to incorporate and visualize 13C metabolic tracing data into the Escher-Trace platform. CONCLUSIONS: Escher-Trace is an open-source software for analysis and interpretation of stable isotope tracing data and is available at https://escher-trace.github.io/ .


Assuntos
Marcação por Isótopo/métodos , Redes e Vias Metabólicas , Software , Gráficos por Computador , Espectrometria de Massas/métodos
4.
PLoS One ; 15(7): e0235752, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32667954

RESUMO

We have limited knowledge of the patterns, causes, and prevalence of elevational migration despite observations of seasonal movements of animals along elevational gradients in montane systems worldwide. While a third of extant Hawaiian landbird species are estimated to be elevational migrants this assumption is based primarily on early naturalist's observations with limited empirical evidence. In this study, we compared stable hydrogen isotopes (δ2H) of metabolically inert (feathers) and active (blood plasma, red blood cells) tissues collected from the same individual to determine if present day populations of Hawaiian honeycreepers undergo elevational movements to track areas of seasonally high flower bloom that constitute significant food resources. We also measured stable carbon isotopes (δ13C) and stable nitrogen isotopes (δ15N) to examine potential changes in diet between time periods. We found that the majority of 'apapane (Himatione sanguinea) and Hawai'i 'amakihi (Chlorodrepanis virens) captured at high elevation, high bloom flowering sites in the fall were not year-round residents at the capture locations, but had molted their feathers at lower elevations presumably in the summer after breeding. δ2H values of feathers for all individuals sampled were higher than blood plasma isotope values after accounting for differences in tissue-specific discrimination. We did not find a difference in the propensity of elevational movement between 'apapane and Hawai'i 'amakihi, even though the 'amakihi is considered more sedentary. However, consistent with a more generalist diet, δ15N values indicated that Hawai'i 'amakihi had a more diverse diet across trophic levels than 'apapane, and a greater reliance on nectar in the fall. We demonstrate that collecting multiple tissue samples, which grow at different rates or time periods, from a single individual can provide insights into elevational movements of Hawaiian honeycreepers over an extended time period.


Assuntos
Migração Animal/fisiologia , Radioisótopos de Carbono/análise , Eritrócitos/metabolismo , Plumas/metabolismo , Marcação por Isótopo/métodos , Radioisótopos de Nitrogênio/análise , Passeriformes/fisiologia , Animais , Hawaii , Dinâmica Populacional
5.
Ann Biol Clin (Paris) ; 78(3): 319-322, 2020 06 01.
Artigo em Francês | MEDLINE | ID: mdl-32540818

RESUMO

The purpose of this work was to compare the measured red-cell volume (RCV) using sodium pertechnétate [RCV-99mTc] compared to the reference technique using sodium radiochromate [RCV-51Cr] and to assess the influence of technetium-99 elution on the RCV-99mTc value. Ten patients had simultaneous measurements of RCV-99mTc and RCV-51Cr. Elution of Tc-99m from red blood cells was 2.9% and led to an average overestimation of RCV-99mTc of 3.7%. The introduction of individual tracer elution rates in the RCV-99mTc calculation corrects this overestimation.


Assuntos
Radioisótopos de Cromo/farmacologia , Volume de Eritrócitos/efeitos dos fármacos , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Marcação por Isótopo/métodos , Tecnécio/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Relação Dose-Resposta a Droga , Contagem de Eritrócitos/métodos , Feminino , Hematócrito/métodos , Humanos , Marcação por Isótopo/efeitos adversos , Masculino , Pessoa de Meia-Idade , Técnica de Diluição de Radioisótopos
6.
PLoS One ; 15(6): e0235114, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32574188

RESUMO

Nursing and weaning periods are poorly understood in cetaceans due to the difficulty of assessing underwater behaviour in the wild. However, the onset and completion of weaning are critical turning points for individual development and survival, with implications for a species' life history including reproductive potential. δ15N and δ13C deposited in odontocete teeth annuli provide a lifetime record of diet, offering an opportunity to investigate variation and trends in fundamental biology. While available reproductive parameters for beaked whales have largely been inferred from single records of stranded or hunted animals and extrapolated across species, here we examine the weaning strategy and nursing duration in northern bottlenose whales (Hyperoodon ampullatus) by measuring stable isotopes deposited in dentine growth layer groups (GLGs). Using a collection of H. ampullatus teeth taken from whales killed during the whaling era (N = 48) and from two stranded specimens, we compared ontogenetic variation of δ15N and δ13C found in annual GLGs across all individuals, by sex and by region. We detected age-based trends in both δ15N and δ13C that are consistent across regions and males and females, and indicate that nursing is prolonged and weaning does not conclude until whales are 3-4 years old, substantially later than previous estimates of 1 year. Incorporating a prolonged period of maternal care into H. ampullatus life history significantly reduces their reproductive potential, with broad implications for models of beaked whale life history, energetics and the species' recovery from whaling.


Assuntos
Dentina/metabolismo , Marcação por Isótopo/métodos , Reprodução/fisiologia , Baleias/fisiologia , Animais , Animais Recém-Nascidos , Isótopos de Carbono/metabolismo , Feminino , Geografia , Islândia , Masculino , Terra Nova e Labrador , Isótopos de Nitrogênio/metabolismo , História Reprodutiva
7.
J Vis Exp ; (159)2020 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-32449746

RESUMO

Many nitrogen fertilizer studies evaluate the overall effect of a treatment on end-of-season measurements such as grain yield or cumulative N losses. A stable isotope approach is necessary to follow and quantify the fate of fertilizer derived N (FDN) through the soil-crop system. The purpose of this paper is to describe a small-plot research design utilizing non-confined 15N enriched microplots for multiple soil and plant sampling events over two growing seasons and provide sample collection, handling, and processing protocols for total 15N analysis. The methods were demonstrated using a replicated study from south-central Minnesota planted to corn (Zea mays L.). Each treatment consisted of six corn rows (76 cm row-spacing) 15.2 m long with a microplot (2.4 m by 3.8 m) embedded at one end. Fertilizer-grade urea was applied at 135 kg N∙ha-1 at planting, while the microplot received urea enriched to 5 atom % 15N. Soil and plant samples were taken several times throughout the growing season, taking care to minimize cross-contamination by using separate tools and physically separating unenriched and enriched samples during all procedures. Soil and plant samples were dried, ground to pass through a 2 mm screen, and then ground to a flour-like consistency using a roller jar mill. Tracer studies require additional planning, sample processing time and manual labor, and incur higher costs for 15N enriched materials and sample analysis than traditional N studies. However, using the mass balance approach, tracer studies with multiple in-season sampling events allow the researcher to estimate FDN distribution through the soil-crop system and estimate unaccounted-for FDN from the system.


Assuntos
Marcação por Isótopo/métodos , Isótopos de Nitrogênio/análise , Solo/química , Zea mays/química , Biomassa , Fertilizantes/análise
8.
Rapid Commun Mass Spectrom ; 34(13): e8814, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32307763

RESUMO

RATIONALE: The electrospray ionization mass spectrometry (ESI-MS) methodology often shows poor ionization reproducibility in the analysis of biological samples. Therefore, normalization of the measured peak intensities is essential. It is believed that quantitative data with high reproducibility can be obtained by adding a constant amount of an internal standard (IS) material labeled with stable isotopes to each sample, thus allowing the correction of the quantitative value of the target compound by that of the IS. We investigated whether the presence or absence of a labeled IS improves the accuracy of these quantitative values. METHODS: Triple quadrupole MS coupled with liquid chromatography was used to analyze fatty acid metabolites in biological samples as target compounds. Two independent systems were used to provide a measure of reproducibility in two different laboratories. RESULTS: Data having poor reproducibility in the raw peak areas were efficiently normalized using the IS, but, crucially, the IS method using stable isotopes was not always necessary. In some cases, the reproducibility was relatively good even without using the IS. In a contaminant matrix, the MS response behavior of the target compound and its stable isotope-labeled material was complicated. Since ion suppression by matrix contaminants was dependent on the concentration of the target compound, the added amounts of the ISs were also important, Furthermore, an equivalent normalization effect was obtained by using a pooled quality control sample as an external standard, thus obviating the need for labeled IS samples, which are often expensive and sometimes not commercially available. CONCLUSIONS: Our results raise the question as to whether the quantitative method using stable-isotope-labeled ISs is always necessary and beneficial. However, the results obtained in this study cannot be generalized because only fatty acid metabolites were examined using ESI-MS and only a highly substituted deuterium-labeled IS was used.


Assuntos
Deutério/química , Ácidos Graxos/análise , Marcação por Isótopo/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Deutério/análise , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Haplorrinos , Humanos , Padrões de Referência , Reprodutibilidade dos Testes
9.
Can J Microbiol ; 66(8): 491-494, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32134703

RESUMO

RNA-based stable isotope probing (RNA-SIP) is used in molecular microbial ecology to link the identity of microorganisms in a complex community with the assimilation of a distinct substrate. The technique is highly dependent on a reliable separation of isotopic-labeled RNA from unlabeled RNA by isopycnic density gradient ultracentrifugation. Here we show that 13C-labeled and unlabeled Escherichia coli RNA can be sufficiently separated by isopycnic ultracentrifugation even in the absence of formamide. However, a slightly lower starting density is needed to obtain a distribution pattern similar to that obtained when formamide was used. Hence, the commonly used addition of formamide to the centrifugation solution might not be needed to separate 13C-labeled RNA from unlabeled RNA, but this must be verified for more complex environmental mixtures of RNA. Clearly, an omission of formamide would increase the safety of RNA-SIP analyses.


Assuntos
Escherichia coli/genética , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , Isótopos de Carbono/química , Centrifugação com Gradiente de Concentração/métodos , Escherichia coli/química , Formamidas/química , Marcação por Isótopo/métodos , RNA Bacteriano/química , Ultracentrifugação/métodos
10.
Nat Commun ; 11(1): 1454, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32193396

RESUMO

Enzymes dependent on nicotinamide cofactors are important components of the expanding range of asymmetric synthetic techniques. New challenges in asymmetric catalysis are arising in the field of deuterium labelling, where compounds bearing deuterium (2H) atoms at chiral centres are becoming increasingly desirable targets for pharmaceutical and analytical chemists. However, utilisation of NADH-dependent enzymes for 2H-labelling is not straightforward, owing to difficulties in supplying a suitably isotopically-labelled cofactor ([4-2H]-NADH). Here we report on a strategy that combines a clean reductant (H2) with a cheap source of 2H-atoms (2H2O) to generate and recycle [4-2H]-NADH. By coupling [4-2H]-NADH-recycling to an array of C=O, C=N, and C=C bond reductases, we demonstrate asymmetric deuteration across a range of organic molecules under ambient conditions with near-perfect chemo-, stereo- and isotopic selectivity. We demonstrate the synthetic utility of the system by applying it in the isolation of the heavy drug (1S,3'R)-[2',2',3'-2H3]-solifenacin fumarate on a preparative scale.


Assuntos
Biocatálise , Técnicas de Química Sintética/métodos , Deutério/química , Marcação por Isótopo/métodos , Oxirredutases/química , Óxido de Deutério/química , Estrutura Molecular , Niacinamida/química , Succinato de Solifenacina/química , Estereoisomerismo
11.
Nat Chem Biol ; 16(6): 630-634, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32203414

RESUMO

The proposal that N6-methyl-deoxyadenosine (m6dA) acts as an epigenetic mark in mammals remains controversial. Using isotopic labeling coupled to ultrasensitive mass spectrometry, we confirm the presence of low-level m6dA in mammalian DNA. However, the bulk of genomic m6dA originates from ribo-N6-methyladenosine, which is processed via the nucleotide-salvage pathway and misincorporated by DNA polymerases. Our results argue against m6dA acting as a heritable, epigenetic DNA mark in mammalian cells.


Assuntos
DNA/química , DNA/metabolismo , Desoxiadenosinas/análise , Genômica , Marcação por Isótopo/métodos , Aminoácidos/química , Animais , Linhagem Celular , Metilação de DNA , DNA Polimerase Dirigida por DNA/metabolismo , Genoma , Humanos , Espectrometria de Massas , Metiltransferases/metabolismo , Camundongos
12.
Sci Rep ; 10(1): 5317, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32210336

RESUMO

The kidney is comprised of highly complex structures that rely on self-maintenance for their functions, and tissue repair and regeneration in renal diseases. We devised a proteomics assay to measure the turnover of individual proteins in mouse kidney. Mice were metabolically labeled with a specially formulated chow containing nitrogen-15 (15N) with the absence of normal 14N atoms. Newly synthesized proteins with 15N contents were distinguished from their 14N counterparts by mass spectrometry. In total, we identified over 4,000 proteins from the renal cortex with a majority of them contained only 15N. About 100 proteins had both 14N- and 15N-contents. Notably, the long-lived proteins that had large 14N/15N ratios were mostly matrix proteins. These included proteins such as type IV and type VI collagen, laminin, nidogen and perlecan/HSPG2 that constitute the axial core of the glomerular basement membrane (GBM). In contrast, the surface lamina rara proteins such as agrin and integrin had much shorter longevity, suggesting their faster regeneration cycle. The data illustrated matrix proteins that constitute the basement membranes in the renal cortex are constantly renewed in an ordered fashion. In perspective, the global profile of protein turnover is usefully in understanding the protein-basis of GBM maintenance and repair.


Assuntos
Membrana Basal/metabolismo , Isótopos de Nitrogênio/metabolismo , Proteômica/métodos , Animais , Membrana Basal/diagnóstico por imagem , Membrana Basal/fisiologia , Proteínas da Matriz Extracelular/metabolismo , Feminino , Membrana Basal Glomerular/metabolismo , Marcação por Isótopo/métodos , Rim/metabolismo , Nefropatias/metabolismo , Laminina/metabolismo , Masculino , Espectrometria de Massas/métodos , Camundongos , Camundongos Endogâmicos C57BL , Estabilidade Proteica
13.
Int J Nanomedicine ; 15: 31-47, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32021163

RESUMO

Purpose: Using our chelate-free, heat-induced radiolabeling (HIR) method, we show that a wide range of metals, including those with radioactive isotopologues used for diagnostic imaging and radionuclide therapy, bind to the Feraheme (FH) nanoparticle (NP), a drug approved for the treatment of iron anemia. Material and methods: FH NPs were heated (120°C) with nonradioactive metals, the resulting metal-FH NPs were characterized by inductively coupled plasma mass spectrometry (ICP-MS), dynamic light scattering (DLS), and r1 and r2 relaxivities obtained by nuclear magnetic relaxation spectrometry (NMRS). In addition, the HIR method was performed with [90Y]Y3+, [177Lu]Lu3+, and [64Cu]Cu2+, the latter with an HIR technique optimized for this isotope. Optimization included modifying reaction time, temperature, and vortex technique. Radiochemical yield (RCY) and purity (RCP) were measured using size exclusion chromatography (SEC) and thin-layer chromatography (TLC). Results: With ICP-MS, metals incorporated into FH at high efficiency were bismuth, indium, yttrium, lutetium, samarium, terbium and europium (>75% @ 120 oC). Incorporation occurred with a small (less than 20%) but statistically significant increases in size and the r2 relaxivity. An improved HIR technique (faster heating rate and improved vortexing) was developed specifically for copper and used with the HIR technique and [64Cu]Cu2+. Using SEC and TLC analyses with [90Y]Y3+, [177Lu]Lu3+ and [64Cu]Cu2+, RCYs were greater than 85% and RCPs were greater than 95% in all cases. Conclusion: The chelate-free HIR technique for binding metals to FH NPs has been extended to a range of metals with radioisotopes used in therapeutic and diagnostic applications. Cations with f-orbital electrons, more empty d-orbitals, larger radii, and higher positive charges achieved higher values of RCY and RCP in the HIR reaction. The ability to use a simple heating step to bind a wide range of metals to the FH NP, a widely available approved drug, may allow this NP to become a platform for obtaining radiolabeled nanoparticles in many settings.


Assuntos
Óxido Ferroso-Férrico/química , Marcação por Isótopo/métodos , Nanopartículas/química , Radioisótopos/química , Quelantes/química , Cromatografia em Gel , Radioisótopos de Cobre/química , Difusão Dinâmica da Luz , Lutécio/química , Espectroscopia de Ressonância Magnética , Compostos Radiofarmacêuticos/química
14.
J Am Soc Mass Spectrom ; 31(2): 183-189, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-32031397

RESUMO

Previous studies have shown the benefits of the amine-reactive, CID-MS/MS-cleavable cross-linker disuccinimidyl dibutyric urea (DSBU) for structural proteomics studies via cross-linking/MS (XL-MS). To further facilitate the automation of XL-MS experiments, we synthesized a deuterated (D12) version of the DSBU cross-linker combining the advantages of MS-cleavable linkers and isotope labeling. The rationale of conducting XL-MS with a mixture of unlabeled and stable isotope-labeled DSBU is to obtain characteristic mass differences at the MS level indicating cross-linked species. These cross-linked species can then be selected for fragmentation by collisional activation. At the MS/MS level, the characteristic 26-u doublets arising from cleavage of the central urea group in DSBU confirm the amino acid sequences of cross-linked peptides as well as the exact cross-linking sites. D12-labeled DSBU was tested on three systems with increasing complexity: (i) bovine serum albumin as purified protein, (ii) Escherichia coli ribosome as large, multimeric protein assembly, and (iii) Drosophila embryo extract as complete proteome. We demonstrate the benefits arising from the use of isotope-labeled DSBU for an automated assignment of cross-linked products. Combining isotope labeling and MS cleavability in one cross-linker resulted in higher cross-link identification numbers especially for highly complex protein mixtures.


Assuntos
Reagentes para Ligações Cruzadas/química , Proteínas/química , Succinimidas/química , Espectrometria de Massas em Tandem/métodos , Ureia/química , Deutério/química , Marcação por Isótopo/métodos , Conformação Proteica , Proteínas/análise
15.
Artigo em Inglês | MEDLINE | ID: mdl-32045699

RESUMO

Untargeted lipidomics is a powerful tool to discover new biomarkers and to understand the physiology and pathology of lipids. The use of stable isotopes as tracers to investigate the kinetics of lipids is another tool able to supply dynamic information on lipid synthesis and catabolism. Coupling the two methodology is then very appealing in the study of lipid metabolism. The main issue to face is to perform thousands of calculations in order to obtain kinetic parameters starting from the MS raw data. An automated computerized routine able to do accomplish such task is presented in this paper. We analyzed the lipid kinetics of palmitic acid (PA) in hepatoma liver cells cultured in vitro in which insulin resistance has been induced by high glucose supplementation. The deuterated palmitate tracer (d5PA) was administered as a bolus and the cells were harvested daily for 48 h. 5dPA was incorporated into 326 monoisotopic compounds and in 84 of their [M + 1] isotopologues detected by high resolution orbitrap MS. The differences between the kinetics curves showed that at least four long chain triglycerides (TG) species incorporated more PA in glucose treated cells, while phosphocholines, sphingomyelins, mono- and di-glycerides and ceramides (Cer) incorporated less tracer under glucose treatment. Nevertheless, Cer amount was increased by glucose treatment. In conclusion we developed an automated powerful algorithm able to model simultaneously hundreds of kinetic curves obtained in a cell culture spiked with a stable isotope tracer, and to analyze the difference between the two different cell models.


Assuntos
Hepatócitos/metabolismo , Metabolismo dos Lipídeos , Lipidômica/métodos , Esfingolipídeos/metabolismo , Algoritmos , Meios de Cultura/metabolismo , Deutério , Ácidos Graxos não Esterificados/análise , Ácidos Graxos não Esterificados/metabolismo , Cromatografia Gasosa-Espectrometria de Massas/métodos , Glucose/metabolismo , Células Hep G2 , Humanos , Resistência à Insulina , Marcação por Isótopo/métodos , Cinética , Ácido Palmítico/análise , Ácido Palmítico/metabolismo , Software , Esfingolipídeos/análise , Fluxo de Trabalho
16.
Anal Chim Acta ; 1104: 87-94, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32106961

RESUMO

Mass spectrometry analysis coupled with stable isotope labeling is a powerful strategy for comparative analysis of N-glycans for glycan biomarker discovery. Here, a novel method combining dual isotopic labeling and fluorous solid-phase extraction was developed, enabling selective enrichment, simultaneous quantitation and recognition of neutral/sialylated glycans from serum samples by mass spectrometry. The isotopic label of fluorous compound on the reducing end of glycans acts as an enrichment tag and provides mass difference between neutral glycans from different samples, while the isotopic label on the non-reducing end of glycans protects the sialic acid residue and provides additional mass difference for sialylated glycans. Therefore, the neutral/sialylated glycans could be simultaneously enriched through the fluorous solid-phase extraction (FSPE) and quantified by mass spectrometry. This method provided a good linearity (R2 > 0.99) and high reproducibility (CV < 20%) within 2 orders of magnitude in the dynamic range. Finally, this strategy was successfully applied to investigate the N-glycome alteration in serum associated with gastric cancer (GC). Bisecting GlcNAc and triantennary glycan compositions were found to be significantly changed between GC cases (n = 50) and healthy control, indicating the great potential to be novel biomarkers for GC early diagnosis.


Assuntos
Biomarcadores Tumorais/sangue , Marcação por Isótopo/métodos , Polissacarídeos/sangue , Extração em Fase Sólida/métodos , Neoplasias Gástricas/sangue , Glicosilação , Humanos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
17.
Anal Bioanal Chem ; 412(12): 2841-2849, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32078005

RESUMO

Free fatty acid (FFA) and acylcarnitine (AcCar) are key elements of energy metabolism. Dysregulated levels of FFA and AcCar are associated with genetic defects and other metabolic disorders. Due to differences in the physicochemical properties of these two classes of compounds, it is challenging to quantify FFA and AcCar in human plasma using a single method. In this work, we developed a chemical isotope labeling (CIL)-based liquid chromatography-multiple reaction monitoring (LC-MRM) method to simultaneously quantify FFA and AcCar. Dansylhydrazine (DnsHz) was used to label the carboxylic acid moiety on FFA and AcCar. This resulted in the formation of a permanently charged ammonium ion for facile ionization in positive ionization mode and higher hydrophobicity for enhanced retention of short-chain analogs on reversed-phase LC columns and enabled absolute quantification by using heavy labeled DnsHz analogs as internal standards. Labeling conditions including the concentration and freshness of cross-linker, reaction time, and temperature were optimized. This method can successfully quantify all short-, medium- and long-chain FFAs and AcCars with greatly enhanced sensitivity. Using this method, 25 FFAs and 13 AcCars can be absolutely quantified and validated in human plasma samples within 12 min. Simultaneous quantification of FFA and AcCar enabled by this CIL-based LC-MRM method facilitates the investigation of fatty acid metabolism and has potential in clinical applications.


Assuntos
Isótopos de Carbono/análise , Carnitina/análogos & derivados , Cromatografia Líquida/métodos , Compostos de Dansil/química , Hidrazinas/química , Marcação por Isótopo/métodos , Espectrometria de Massas em Tandem/métodos , Carnitina/sangue , Humanos
18.
BMC Bioinformatics ; 21(1): 37, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-32000676

RESUMO

BACKGROUND: DNA-stable isotope probing (DNA-SIP) links microorganisms to their in-situ function in diverse environmental samples. Combining DNA-SIP and metagenomics (metagenomic-SIP) allows us to link genomes from complex communities to their specific functions and improves the assembly and binning of these targeted genomes. However, empirical development of metagenomic-SIP methods is hindered by the complexity and cost of these studies. We developed a toolkit, 'MetaSIPSim,' to simulate sequencing read libraries for metagenomic-SIP experiments. MetaSIPSim is intended to generate datasets for method development and testing. To this end, we used MetaSIPSim generated data to demonstrate the advantages of metagenomic-SIP over a conventional shotgun metagenomic sequencing experiment. RESULTS: Through simulation we show that metagenomic-SIP improves the assembly and binning of isotopically labeled genomes relative to a conventional metagenomic approach. Improvements were dependent on experimental parameters and on sequencing depth. Community level G + C content impacted the assembly of labeled genomes and subsequent binning, where high community G + C generally reduced the benefits of metagenomic-SIP. Furthermore, when a high proportion of the community is isotopically labeled, the benefits of metagenomic-SIP decline. Finally, the choice of gradient fractions to sequence greatly influences method performance. CONCLUSIONS: Metagenomic-SIP is a valuable method for recovering isotopically labeled genomes from complex communities. We show that metagenomic-SIP performance depends on optimization of experimental parameters. MetaSIPSim allows for simulation of metagenomic-SIP datasets which facilitates the optimization and development of metagenomic-SIP experiments and analytical approaches for dealing with these data.


Assuntos
DNA/química , DNA/genética , Marcação por Isótopo/métodos , Metagenômica/métodos , Composição de Bases , Bases de Dados Genéticas , Biblioteca Gênica , Isótopos/análise , Metagenoma
19.
Methods Mol Biol ; 2102: 291-302, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31989562

RESUMO

32P-Postlabeling analysis is an ultra-sensitive method for the detection of DNA adducts, such as those formed directly by the covalent binding of carcinogens and mutagens to bases in DNA, and other DNA lesions resulting from modification of bases by endogenous or exogenous agents (e.g., oxidative damage). The procedure involves four main steps: enzymatic digestion of DNA sample; enrichment of the adducts; radiolabeling of the adducts by T4 kinase-catalyzed transference of 32P-orthophosphate from [γ-32P]ATP; chromatographic separation of labeled adducts, and detection and quantification by means of their radioactive decay. Using 10 µg of DNA or less, it is capable of detecting adduct levels as low as 1 adduct in 109-1010 normal nucleotides. It is applicable to a wide range of investigations, including monitoring human exposure to environmental or occupational carcinogens, determining whether a chemical has genotoxic properties, analysis of the genotoxicity of complex mixtures, elucidation of the pathways of activation of carcinogens, and monitoring DNA repair.


Assuntos
Adutos de DNA/análise , Adutos de DNA/química , Marcação por Isótopo/métodos , Animais , Carcinógenos/química , Carcinógenos/toxicidade , Cromatografia Líquida de Alta Pressão/métodos , Adutos de DNA/genética , Dano ao DNA/efeitos dos fármacos , Proteínas Fúngicas , Humanos , Mutagênicos/química , Mutagênicos/toxicidade , Estresse Oxidativo/genética , Radioisótopos de Fósforo , Fosfotransferases , Endonucleases Específicas para DNA e RNA de Cadeia Simples , Fluxo de Trabalho
20.
Methods Mol Biol ; 2088: 1-16, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31893367

RESUMO

The accurate and precise analysis of isotopologue and tandem mass isotopologue ratios in heavy stable isotope labeling experiments is a critical part of assessing absolute intracellular metabolic fluxes. Resulting from feeding the organism of interest with a specifically isotope-labeled substrate, the principal characteristics of these labeling experiments are the metabolites' non-naturally distributed isotopologue patterns. For the purpose of inferring metabolic rates by maximizing the fit between a priori simulated and experimentally obtained labeling patterns, 13C is the preferred stable isotope of use.The analysis of the obtained labeling patterns can be approached by different mass spectrometric approaches. Gas chromatography (GC) features broad metabolite coverage and excellent separation efficiency of biologically relevant isomers. These advantages compensate for laborious derivatization steps and the resulting need for interference correction for natural abundant isotopes.Here, we describe a workflow based on GC-high resolution mass spectrometry with chemical ionization for the analysis of carbon-isotopologue distributions and some positional labeling information of primary metabolites. To study the associated measurement uncertainty of the resulting 13C labeling patterns, guidance to uncertainty estimation according to the EURACHEM guidelines with Monte-Carlo simulation is provided.


Assuntos
Isótopos de Carbono/química , Cromatografia Gasosa-Espectrometria de Massas/métodos , Espectrometria de Massas em Tandem/métodos , Marcação por Isótopo/métodos , Metabolômica/métodos , Método de Monte Carlo , Pichia/metabolismo , Incerteza
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