Dissertations / Theses on the topic 'Stable isotope dilution assay'
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Brown, Rachel Christine, and rcbrown@adam com au. "gamma-Lactones in wine: Synthesis, quantification and sensory studies." Flinders University. School of Chemistry, Physics and Earth Sciences, 2007. http://catalogue.flinders.edu.au./local/adt/public/adt-SFU20080226.234630.
Full textAbstract:
gamma-Lactones are found in a wide variety of food and beverage products, in particular grapes and wine. This thesis details the work completed on some gamma-lactones in wine: their synthetic preparation, development of quantification methodologies and sensory studies.
Chapter 1 outlines the history of the Australian wine industry from the arrival of the first vines on the First Fleet in 1788 with Captain Arthur Philip. This chapter provides: an overview of Australias position in the world of grape and wine production; an analysis of the export arm of the industry; and a look at the different wine producing regions around the country. The latter part of the chapter focuses on the different volatile compounds found in wine.
Part A:
Chapter 2 provides an overview on the history of barrel manufacture and the use of oak wood in cooperage, with an emphasis on oaks well known ability to impart desirable characteristics to wine through the extraction of volatile aroma compounds. This chapter provides a summary of these odorants with a particular emphasis on the oak lactones. Previous sensory studies and synthetic work are discussed. Of great importance to this work are the recent advancements in 1,2-dioxine chemistry, highlighted in this chapter.
Chapter 3 details the synthetic work completed for the preparation of all four possible oak lactone stereoisomers. A suitably substituted racemic 1,2-dioxine featured as the common intermediate and enabled preparation of the gamma-lactone moiety upon reaction with a chiral malonate diester and separation of the diastereomers by column chromatography. A key step involved the decarboxylation of the ester cleaved gamma-lactone diastereomers, which could be directed to give either the cis- or trans-products. Standard chemical transformations were then utilised to produce the desired stereoisomers of oak lactone.
Chapter 4 describes the results from the sensory studies that were completed on the synthetic oak lactone samples. Odour detection thresholds were measured in both a white and a red wine. The thresholds in the former medium were calculated to be 24 ug/L, 172 ug/L, 132 ug/L and 305 ug/L, while in the latter medium the thresholds were calculated to be 57 ug/L, 380 ug/L, 175 ug/L and 285 ug/L, for (4S,5S)-cis-, (4S,5R)-trans-, (4R,5R)-cis- and (4R,5S)-trans-oak lactone, respectively. Difference testings were completed on the pairs of enantiomers and also on mixtures of the nature-identical isomers: between the cis-enantiomers a significant difference was found at the 99% confidence level, while between the trans-enantiomers and also the mixtures of cis- and trans-isomers little difference was observed.
Chapter 5 contains the experimental procedures for Part A.
Part B:
Chapter 6 discusses the sensory properties of some gamma- and delta-lactones, with the focus on a series of five-alkyl substituted gamma-lactones: gamma-octalactone, gamma-nonalactone, gamma-decalactone and gamma-dodecalactone. Topics covered in this chapter include chirality, biosynthetic pathways and quantification results in wine from previous studies for these gamma-lactones.
Chapter 7 concerns the method development for the quantification of gamma-lactones in wine using a stable isotope dilution assay (SIDA). Deuterated analogues were prepared from commercially available racemic gamma-lactones for use as internal standards. Initially a head space solid-phase microextraction (HS SPME) method was developed using d5-standards; however, analysis of bottled wine samples revealed the presence of co-eluting compounds that contained several of the selected ions. Thus an alternative method was developed using d7-standards, with a specific focus on sample clean-up, via solid-phase extraction (SPE). Using this procedure, 44 white and 120 red wines were analysed for their gamma-lactone content. The lactones were found to be significantly more common in the red wines, with gamma-nonalactone the most abundant lactone in this series.
Chapter 8 deals with the extension of the SIDA method, as developed in Chapter 7, for use with a chiral gas chromatography column. Optically pure standards were prepared, from either L- or D-glutamic acid, and used to determine the order of elution of the enantiomers. A method was developed for the quantification of the individual enantiomers of gamma-octalactone, gamma-nonalactone, gamma-decalactone and gamma-dodecalactone. The enantiomeric distribution of gamma-nonalactone was investigated in 34 red wines; the (R)-stereoisomer was found to be dominant with an average of 59%, although there were wines analysed that did contain the (S)-stereoisomer in greater amounts.
Chapter 9 describes the results from the sensory studies that were completed on the individual enantiomers of the gamma-lactones. Odour detection thresholds were measured in a red wine. The thresholds were calculated to be 238 ug/L, 285 ug/L, 34 ug/L and 8 ug/L for the (R)-enantiomers, while the thresholds were calculated to be 135 ug/L, 91 ug/L, 47 ug/L and 39 ug/L for the (S)-enantiomers, of gamma-octalactone, gamma-nonalactone, gamma-decalactone and gamma-dodecalactone, respectively.
Chapter 10 contains the experimental procedures for Part B.
Chapter 11 contains the appendices, followed by the references in Chapter 12.
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Zech, Julie [Verfasser], Leif-Alexander [Akademischer Betreuer] Garbe, Hajo [Gutachter] Haase, Juri [Gutachter] Rappsilber, and Sascha [Gutachter] Rohn. "Analysis of bisphenols and bisphenol A diglycidyl ethers by stable isotope dilution assay liquid chromatography-tandem mass spectrometry / Julie Zech ; Gutachter: Hajo Haase, Juri Rappsilber, Sascha Rohn ; Betreuer: Leif-Alexander Garbe." Berlin : Technische Universität Berlin, 2016. http://d-nb.info/1156011728/34.
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Hu, Ling [Verfasser], Michael [Akademischer Betreuer] Rychlik, and Peter [Akademischer Betreuer] Köhler. "Development and Application of Stable Isotope Dilution Assays for the Fusarium Mycotoxins Enniatins and Beauvericin / Ling Hu. Gutachter: Peter Köhler ; Michael Rychlik. Betreuer: Michael Rychlik." München : Universitätsbibliothek der TU München, 2014. http://d-nb.info/1047185504/34.
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Marzouk, Ezzat Rashad El-Said. "Using multi-element stable isotope dilution to quantify metal reactivity in soil." Thesis, University of Nottingham, 2012. http://eprints.nottingham.ac.uk/28914/.
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Determining the total concentration of elements in soils seldom provides sufficient insight into trace metal bioavailability. However, measurement of ‘isotopically exchangeable’ metal can provide a better evaluation of metal reactivity and potential toxicity. Traditionally this requires the use of problematic radio-isotopes (e.g. 109Cd (γ)). Fortunately, increasing access to Inductively Coupled Plasma Mass Spectrometry (ICP-MS) in recent years has led to greater use of enriched stable isotopes of trace metals. The lability of heavy metals has been determined through a variety of approaches, including single and sequential extraction or predicted by geochemical models. In the present work, multi-element stable isotopes methods were developed for simultaneously determination of the labile pool of Fe, Zn, Cd and Pb using isotopic exchange principles. This included experimental and instrumental development for an accurate and precise determination of labile metal pool in soils. This approach was then validated by quantifying Zn, Cd and Pb in contaminated soils (Derbyshire; n = 8 and Weardale catchment; n = 246) and comparing the outcome results with common traditional extraction procedures. The variation of metal lability with soil characteristics was used to predict metal lability from the simple soil measurements using a multiple regression approach. In addition, E-values of Fe, Zn, Cd and Pb was used as input to WHAM(VI) (Windermere Humic-Aqueous Model) to predict metal solubility, emphasising in the role of Fe under reducing conditions in this regard. The results showed that isotopic dilution is a robust mechanistic method for assessing the ‘reactive’ pool of multiple trace metals over a wide range of soil characteristics. The results showed a very wide range of metal reactivities (almost 1%-100%) for Zn, Cd and Pb that were consistent over a range of spike concentrations. Sub-micron forms of non labile metal are perhaps most likely to occur in suspension either strongly bonded to humic/fulvic acids or occluded within CaCO3 particles. It appears that E values have no consistent correspondence to any chemical extraction procedure. Nevertheless, the use of 0.43 M HNO3 to extract labile metal in organic soils at pH < 6 appears justifiable - especially where humus is likely to be the principal adsorption surface. It is also important to acknowledge that extractions are not necessarily intended to estimate the entire reactive fraction. Thus, DTPA has been successfully applied as an empirical prediction of plant uptake but its extraction capacity is particularly limited in calcareous systems where it substantially underestimates the isotopically exchangeable metal pool. Speciation calculations showed that prediction of metal solubility was much better when the isotopically reactive metal pools were used as input to WHAM(VI). The soil samples that fitted best had pH values less than 4.0 and high organic matter contents reflecting the strength of the humic binding component of WHAM(VI) particularly in the case of Zn. The changes in metal solubility and lability under reducing conditions were mainly affected by pH. Moreover, the measurement of Fe2+ in the solution phase was considerably lower than that of the isotopically labile Fe2+ which calls into question the dependence on soluble Fe2+ to predict reductive dissolution of Fe-oxides. In addition, under reducing conditions the variables input of Fe to WHAM(VI) showed greatest effects on predicting metal solubility. It was found that Zn and Cd were affected only by Fe2+ competition for adsorption sites while predicted Pb solubility was more affected by loss of oxides than competition processes. The fractionation results, output from WHAM(VI), showed that a significant proportion of Pb was associated with Mn-oxides. Therefore, the calculation of loss of the adsorption site of Mn-oxides depending on Mn2+ measured in the solution phase did not improve the predicted Pb solubility where the model underestimate the adsorbed labile Mn as inference from Fe results.
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Lambertsson, Lars. "Mercury species transformations in marine and biological systems studied by isotope dilution mass spectrometry and stable isotope tracers." Doctoral thesis, Umeå : Univ, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-467.
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Kelly, Robert Noel. "Towards the absolute quantification of protein isoforms through the use of stable-isotope dilution mass spectrometry." Thesis, University of Glasgow, 2013. http://theses.gla.ac.uk/4401/.
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While the existence of protein was first described by Berzelius and Mulder back in 1838 and a single empirical formula noted (C400H620N100O120P1S1) (Vickery, 1950, Brand, 1946), early protein-based research was limited to the analysis of proteins which could be easily purified in large quantities, such as those obtained from blood, egg whites and those obtainable from slaughterhouses, such as digestive and metabolic enzymes (Chapman, 2005). Indeed, despite the development of recombinant deoxyribonucleic acid technologies in the 1970s (enabling protein expression) and the increasing sensitivity of techniques which enable the identification and sequencing of proteins separated by gel electrophoresis (Patterson and Aebersold, 2003), it was not until the late 1980s, with the description of soft biomolecule ionisation that large scale proteomic analyses were undertaken, based upon the use of mass spectrometry (Guerrera and Kleiner, 2005). While early mass spectrometry-based proteomic analyses focussed on the systematic identification of a great number of proteins within a single organism, the field of proteomics is now becoming increasingly quantitative (Baak et al., 2005), enabling the relative comparison of protein expression patterns between phenotypes, but also the targeted absolute quantification of specific proteins. During this project, a stable isotopically labelled internal standard based absolute quantitative technique, first described by Gerber and co-workers in 2003 (S. A. Gerber et al., 2003), was applied to the absolute quantification of three families of multiple protein isoforms. This area of research is of particular scientific interest as it is thought that up to 95% of human multi-exon genes may be subject to alternative splicing, making alternative splicing the rule, not the exception (Pan et al., 2008a). Indeed alternative splicing has also been implicated as both a cause and a consequence of disease. This technique should therefore enable both the confirmation of disease, based upon the identification of a set of phenotype specific protein biomarkers, but also the mapping of a disease’s progression (Venables, 2004). During this study, stable isotopically labelled internal standard peptides were selected for the absolute quantification of 11 confirmed protein isoforms, and two predicted protein isoforms. In addition, a separate MRM based LC-MS acquisition method was developed for the absolute quantification of each of the three families of protein isoforms (A-Raf, PDE4B and SERCA2) within a single analysis, and finally, these acquisition methods were applied to the absolute quantification of a range of immunoprecipitated, exogenously expressed protein isoforms. This project was, however, hindered by the sensitivity of the mass spectrometers available for use, preventing these acquisition methods from being applied to the absolute quantification of the endogenous levels of protein expression. While beyond the scope of this project, the further development of this quantitative technique should enable future researchers to: (i) Quantify each endogenously expressed protein isoform within a family of multiple protein isoforms. (ii) Assess any changes in the expression of each isoform in a range of cellular states, and (iii) Assess how a targeted drug treatment may affect the expression ratio of these protein isoforms.
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Lindner, Johanna Magdalena [Verfasser], and Michael [Akademischer Betreuer] Vogeser. "Novel approaches to corticosteroid profiling by stable isotope dilution tandem mass spectrometry / Johanna Magdalena Lindner ; Betreuer: Michael Vogeser." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2018. http://d-nb.info/1174142774/34.
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Vaiglova, Petra. "Neolithic agricultural management in the Eastern Mediterranean : new insight from a multi-isotope approach." Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:c8824136-da35-43b2-a700-f458d0cc2fdf.
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The work presented in this dissertation explores the nature of agro-pastoral strategies developed by Neolithic farmers as a way to understand how early food production was inter-twined with environmental and socio-economic opportunities and constraints. Towards this end, a multi-isotope approach is used to address questions of scale and intensity of crop cultivation and animal management at the archaeological sites of Kouphovouno, southern Greece, Makriyalos, northern Greece, and Çatalhöyük, south-central Turkey. Measurements of stable carbon, nitrogen, oxygen and strontium isotope values of carbonized plant remains, human and animal bone collagen and animal tooth enamel are used to examine the similarities and differences in the types of treatments that individual species of plants and animals received during the agricultural cycle at the distinct locations. The results show that farmers at the three sites developed variable methods for exploiting the arable and pastoral landscape and catering to their economic and culinary needs. The discussion considers the implications of these findings to our understanding of the complexity and adaptability of early farming systems.
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Schuster, Carina [Verfasser], and Michael [Akademischer Betreuer] Vogeser. "Investigation and development of stable isotope dilution mass spectrometry methods for therapeutic drug monitoring of anti-infective drugs used in the critically ill / Carina Schuster ; Betreuer: Michael Vogeser." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2020. http://d-nb.info/1212362896/34.
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Haynes, Christopher Allen. "Development of an assay for fatty acyl-CoAs using liquid chromatography-electrospray ionization-tandem mass spectrometry and its application to the stable isotope labeling and quantitation of sphingolipid metabolism." Diss., Georgia Institute of Technology, 2009. http://hdl.handle.net/1853/37171.
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Fatty acyl-Coenzyme As are metabolites of lipid anabolism and catabolism. A method was developed for their quantitation in extracts of cultured mammalian cells using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Palmitoyl-CoA (C16:0-CoA) is utilized for de novo sphingolipid biosynthesis catalyzed by serine palmitoyltransferase (SPT), which condenses palmitoyl-CoA and serine to form 3-ketosphinganine. After reduction to form sphinganine (Sa), dihydroceramide synthase (CerS) can N-acylate the Sa using a second fatty acyl-CoA molecule, forming dihydroceramide (DHCer). The CerS enzyme family utilizes different acyl chain lengths of fatty acyl-CoAs in an isoform-specific manner, resulting in DHCer with N-acyl chains ranging from C16 to C26 [and even longer] in mammalian tissues. DHCer is trans-4,5-desaturated to yield ceramide, which is further metabolized by the addition of moieties at the 1-O-position, forming sphingomyelin (SM) and ceramide monohexose (CMH).
The rates of fatty acyl-CoA and sphingolipid biosynthesis were determined using stable isotope-labeling and LC-ESI-MS/MS analysis of the analyte isotopologues and isotopomers. Isotopic labeling of palmitoyl-CoA with [U-13C]-palmitate in HEK293 and RAW264.7 cells was robust and rapid (~ 60% labeling of the metabolite pool in 3 hr). Isotopic labeling of sphingolipids indicated utilization of [M + 16]-palmitoyl-CoA by SPT and CerS isoforms in both cell types. Metabolic flux modeling was applied to the data for [U-13C]-palmitate activation to [M + 16]-palmitoyl-CoA and its subsequent utilization in de novo sphingolipid biosynthesis, and this analysis indicated rapid turn-over rates for palmitoyl-CoA and ceramide in both cell types.
Palmitate treatment of cultured cells alters their metabolic status and gene expression, therefore labeling of palmitoyl-CoA by treatment with [1-13C]-acetate was employed. A distribution of mass-shifted palmitoyl-CoA species (isotopologues) is observed based on the number of incorporations of [1-13C]-acetate during de novo biosynthesis, requiring computational analysis to derive two parameters: the isotopic enrichment of the precursor pool, and the fraction of palmitoyl-CoA that was biosynthesized during the experiment. Previous reports by others describe mass isotopomer distribution analysis (MIDA) and isotopomer spectral analysis (ISA) for this purpose, and both calculation approaches indicated concurrent results.
In summary, the quantitation of fatty acyl-CoAs and their isotopic enrichment during stable isotope-labeling studies of lipid metabolism can provide data that significantly change the interpretation of analyte quantitation in these experiments, as demonstrated here for investigations of de novo sphingolipid biosynthesis.
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Lee, Sungeun. "Virus-host interactions across a soil pH gradient at the community and individual scale." Thesis, Lyon, 2020. http://www.theses.fr/2020LYSEC020.
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Les virus du sol sont capables d'influencer la structure de la communauté microbienne et le fonctionnement de l'écosystème en affectant l'abondance des cellules hôtes par lyse et par leurs caractéristiques à transférer des gènes entre les hôtes. Bien que notre compréhension sur la diversité et la fonction virales s’améliore, la connaissance des interactions hôte-virus dans le sol reste limitée. Pour mieux comprendre les interactions hôte-virus, un gradient du sol à long terme manipulé par le pH dans lequel la communauté microbienne change à travers, a été étudié. Les principaux objectifs de cette thèse ont consisté à (1) déterminer l'influence de la structure de la communauté microbienne et du pH du soil sur les virus par séquençage d’ADN haut-débit (Chapitre II), (2) déterminer l’infectivité des populations virales à partir de niches de sol co-localisées et non co-localisées avec son hôte grâce à une approche de l’essai de plaque combinée à un séquençage hybride (Chapitre III), (3) identifier les populations virales infectant des groupes fonctionnels microbiens spécifiques du sol, en particulier les méthanotrophes (Chapitre IV) et les nitrifiants (Chapitre V ), à l’aide d’une sonde isotopique stable à l'ADN. Nos premiers résultats ont montré que la structure de la communauté virale change selon le pH du sol, ce qui souligne que la communauté virale est étroitement liée aux populations hôtes. L’analyse de CRISPR systèmes a révélé des interactions virus-hôte dynamiques, avec le nombre et la taille des CRISPR systèmes distincts selon le soil à pH contrasté. L’analyse taxonomique de cette CRISPR systèmes suggère que les virus jouent un rôle essentiel dans la composition et de la fonction de la communauté procaryote du sol. Les processus co-évolutionnaires entre l'hôte (le système de restriction-modification et le CRISPR-Cas système) et les populations virales co-localisées (la mutation d’une séquence espaceur « spacer » et la méthyltransférase codée par le virus) fournissent des preuves de l'adaptation locale et que les interactions virus-hôte jouent un rôle important dans la susceptibilité d'un hôte à l'infection et par conséquent la régulation des populations bactériennes du sol. L'ADN-SIP-métagénomique ciblant des groupes fonctionnels microbiens spécifiques a permis l’analyse des populations hôte-virus individuelles. Le suivi du flux de carbone à travers les populations procaryotes et virales a révélé des interactions actives entre les virus et les hôtes méthanotrophes et nitrifiants, et les préférences de niches de pH du sol. Notre étude a montré une preuve de transfert horizontal de gènes et des gènes métaboliques auxiliaires codés par le virus, indiquant que les virus contribuent de manière significative aux cycles biogéochimiques dans le sol, tels que le carbone (les gènes qui codent pour les familles GH, peptidases et la sous-unité C de méthane monooxygénase particulaire), et l'azote (les gènes qui codent pour la nitrogénase et le cytochrome cd1-nitrite réductase). Dans l’ensemble, ces résultats ont montré que les virus du sol sont des régulateurs importants des communautés microbiennes par la lyse spécifique de l’hôte et des interactions dynamiques virus-hôteSoil viruses have potential to influence microbial community structure and subsequent ecosystem functioning by directly affecting the abundance of host cells by lysis and through their ability to transfer genes between hosts. Although our understanding of soil viral diversity and functioning has increased, the role of viruses and their interactions with prokaryotes in soil is limited. To gain a better understanding of virus-host interactions in soil, a long-term pH-manipulated soil gradient, which microbial community structure changes across, was investigated. The main objectives of this thesis were to 1) determine the influence of microbial community structure and soil pH on viruses using metagenomics and viromics (Chapter II), 2) determine the infectivity of soil viral populations from co-localized and foreign pH soil niches using a plaque assay approach combined with hybrid metagenomics sequencing (Chapter III) and 3) identify virus populations infecting specific soil microbial functional groups, specifically methanotrophs (Chapter IV) and nitrifiers (Chapter V), using DNA stable isotope probing combined with metagenomic deep sequencing. Viral community structure was found to change with soil pH, demonstrating that viral communities are tightly linked to host populations, but also may have narrow host ranges. Analysis of clustered regularly interspaced short palindromic repeats (CRISPR) arrays revealed dynamic virus-host interactions, with the number and size of CRISPR arrays distinct across contrasting pH soil. Profiling of the host-virus linkages between soil pH, suggests that viruses play a critical role in shaping the composition and function of the soil prokaryotic community. Surprisingly, greater infectivity of a host bacterium by virus populations was found when viruses and host bacterium were not co-localized in the same pH soil. Coevolutionary processes between the host and virus populations, such as restriction modification/virus-encoded methyltransferase and CRISPR-Cas system/spacer mutation, provide evidence for local adaptation, and that virus-bacterial host interactions play an integral part in the susceptibility of a host to infection and consequently in the regulation of soil bacterial populations. Targeting specific microbial functional groups via stable isotope probing allowed analysis of individual host-virus populations. Tracking carbon flow through prokaryotic and viral populations revealed active interactions between viruses and methanotroph and nitrifier hosts, and soil pH niche preferences. Evidence of horizontal gene transfer and virus-encoded auxiliary metabolic genes, such as glycoside hydrolase families, peptidases, particulate methane monooxygenase subunit C (pmoC), nitrogenase (nifH) and cytochrome cd1-nitrite reductase, supports that viruses are significant contributors to host functioning and carbon and nitrogen cycling in soil. Overall, this work demonstrated that soil viruses are important regulators of microbial communities through specific host lysis and dynamic virus-host interactions
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Lin, Chao Ruei, and 林朝瑞. "Quantitative Assay for Ethylpurine Adducts in Human Salivary DNA and in Urine by Stable Isotope Dilution nanoLC-Nanospray Ionization Tandem Mass Spectrometry." Thesis, 2013. http://ndltd.ncl.edu.tw/handle/19302019482206000754.
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碩士國立中正大學
化學暨生物化學研究所
101
Cigarette smoke contains many alkylating agents which damage DNA producing DNA adducts, such as N3-ethyladenine (3-EtA) and N7-ethylguanine (7-EtG). Cells of the oral cavity have been employed to measure DNA adducts that modified by tobacco carcinogen phenylimidazo[4,5-b]pyridine (PhIP) following exposure to tobacco smoke and levels of dG-C8-PhIP in smokers were higher than in nonsmoker. In this study, a highly specific and sensitive assay based on stable isotope dilution nanoLC-nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) was used to measure 3-EtA and 7-EtG in human salivary DNA and urine. Neutral thermal hydrolysis was used to release 3-EtA and 7-EtG from DNA, while the supernatant of urine containing adducts was obtained after centrifugation. These adducts from the supernatant of DNA hydrolysate and urine were enriched by a reversed-phase solid-phase extraction column before nanoLC-NSI/MS/MS analysis. The on-column detection limits (S/N ≥ 3) of 3-EtA and 7-EtG were 92 and 56 amol, respectively. The quantification limits of 3-EtA and 7-EtG were 310 and 280 amol, respectively. In human salivary DNA, levels of 3-EtA and 7-EtG in 14 smokers were 12.5 ± 7.2 and 14.0 ± 8.5 in 108 normal nucleotides, respectively. In 15 nonsmokers, levels of 3-EtA and 7-EtG were 9.7 ± 5.3 and 3.8 ± 2.8 in 108 normal nucleotides, respectively. The level of 7-EtG was statistically significantly higher in smokers than in nonsmokers (p < 0.05). In human urinary samples, adduct concentrations of 3-EtA and 7-EtG in 20 smokers were 66.5 ± 28.6 and 18.5 ± 14.2 pg/mL urine, respectively. In 20 nonsmokers, adduct concentrations of 3-EtA and 7-EtG were 3.5 ± 3.8 and 2.4 ± 3.0 pg/mL urine, respectively. The concentrations of 3-EtA and 7-EtG were statistically significantly higher in smokers than in nonsmokers (p < 0.05). This highly specific and sensitive assay based on stable isotope dilution nanoLC-NSI/MS/MS assay should be clinically useful in assessing the possibility of measuring ethylpurines in human salivary DNA and in urine as risk biomarkers for smoking-related cancers.
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Liu, Yen Fu, and 劉彥甫. "Quantitative Assay for Ethylpurine Adducts in Human White Blood Cells DNA and Human Urine by Stable Isotope Dilution Capillary LC-Nanospray Ionization Tandem Mass Spectrometry." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/26809395868158430724.
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碩士國立中正大學
化學暨生物化學研究所
100
Cigarette smoke contains many alkylating agents which damage DNA producing DNA adducts, such as 3-ethyladenine (3-EtA) and 7-ethylguanine (7-EtG). To quantify ethylated adducts on DNA, I used neutral thermal hydrolysis to release 3-EtA and 7-EtG from DNA. The adducts were purified by a reversed-phase solid-phase extraction column and analyzed by stable isotope dilution capillary liquid chromatography nanospray ionization tandem mass spectrometry (capLC-NSI/MS/MS) under the highly selected reaction monitoring (H-SRM) mode. The amount of ethylated DNA adducts represents the steady-state levels of DNA adducts resulting from the ethylating agent after repair in vivo. The detection limit of 3-EtA and 7-EtG was 30.7 and 55.9 amol, respectively. Levels of 3-EtA and 7-EtG are 16.0 ± 7.8 and 9.7 ± 8.3 in 108 normal nucleotides, respectively, in white blood cells (WBC) DNA of 20 smokers, which are statistically significantly higher than those in 20 nonsmokers with 5.4 ± 2.6 3-EtA and 0.3 ± 0.8 7-EtG in 108 normal nucleotides (p < 0.0001). These DNA adducts are repaired in vivo and then released in human urine. The urine samples were purified by a mixed-mode cation exchange followed by a reversed-phase solid-phase extraction column and analyzed by capLC-NSI/MS/MS under the H-SRM. The urinary concentration of 3-EtA and 7-EtG are 120.9 ± 33.5 pg/mL and 61.0 ± 8.5 pg/mL in 5 smokers. The highly sensitive and specific stable isotope dilution nanoLC-NSI/MS/MS assay should be clinically useful in assessing the possibility of measuring ethylpurines in human WBC DNA and in urine as risk biomarkers for smoking-related cancers.
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Nxumalo, Wonder Praise-God. "Improved sample preparation ensures accurate quantification of multiple mycotoxins in maize by Liquid Chromatography-stable Isotope Dilution Assay-Tandem Mass Spectrometry (LC-SIDA-MS/MS)." Diss., 2016. http://hdl.handle.net/2263/57278.
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The objective of this study was to develop and validate an improved sample preparation technique for accurate quantification of aflatoxin B1 (AFB1), AFB2, AFG1, AFG2, deoxynivalenol (DON), fumonisin B1 (FB1), FB2, Ochratoxin A (OTA), zearalenone (ZEN), HT-2 toxin and T-2 toxin in maize using liquid chromatography-isotope dilution mass spectrometry.
Mycotoxin contamination in agricultural commodities poses a threat to human health. Contamination of food is recognised as a source of food borne illness by the World Health Organisation (WHO). The toxicity of mycotoxins has been evaluated by the Joint Food and Agricultural Organisation (FAO)/WHO Expert Committee on Food Additives (JECFA) and the maximum levels (MLs) for the agricultural important mycotoxins have been established. Agricultural commodities need to be tested to ensure food safety prior to human consumption; this requires accurate analytical methods for identification and quantification of these mycotoxins at the regulatory levels.
Analytical methods based on liquid chromatography coupled to mass spectrometry have been developed for identification and quantification of mycotoxins. However, MS based analysis is affected by matrix effects that results from ionisation inefficiency of the target analyte due to co-eluting matrix components. Therefore, there is a need for improved sample preparation methods which can minimise, or possibly eliminate, matrix components prior to mass spectrometric analysis. Dilute-and-shoot , Quick, Easy, Cheap, Efficient, Rugged and Safe (QuEChERS) and solid phase extraction (SPE) techniques were evaluated for matrix removal efficiency in multi mycotoxin determination in maize. Isotopically labelled mycotoxin standards were used to compensate for variations during the analysis. Spiked blank maize samples and matrix reference materials were used to evaluate the performance of each sample preparation technique.
Dilute-and-shoot technique was used as a first approach to estimate expected matrix effects and to verify whether isotopically labelled internal standards can compensate for matrix effects during the analysis. All the analytes were affected by the presence of matrix effects, signal suppression/enhancement (SSE) ranged between 88% - 194%. When %REC > 130% it was deemed enhanced. The QuEChERS method was ineffective in isolating mycotoxins from the matrix. Results from dilute-and-shoot and QuEChERS highlighted the need of a selective clean-up step to reduce matrix effects. Different SPE columns with different sorbents were evaluated for matrix removal efficiency and analyte retention performances. Columns with analyte(s) selective sorbents were effective in improving recoveries for those specific analytes. Also, minimum matrix effects were observed from these columns. However, for multi mycotoxin determination, an ideal clean-up step should yield good recoveries for all the mycotoxins with varying physicochemical properties. Hydrophilic-lipophilic balanced (HLB) SPE column gave good recoveries for most analytes despite relatively high matrix effects with respect to selective sorbents. A clean-up method based on HLB clean-up was optimised to improve matrix removal efficiency.
An accurate, precise and robust method for the determination of multiple mycotoxins in maize was developed and validated. This method is based on ultrasonic extraction, economical HLB SPE clean-up and ultra-high performance liquid chromatography-stable isotope dilution assay-tandem mass spectrometry (UHPLC-SIDA-MS/MS). Sample extraction based on two extraction steps using acidified methanol/water mixture and HLB SPE clean-up resulted in good analyte recoveries 57% ? %REC ? 142% for most analytes. Fast polarity switching mode was used to determine all the analytes in one chromatographic run without compromising chromatographic resolution. Method performance results indicate that the method can be used to detect and quantify mycotoxins at the regulated levels.
Keywords: Mycotoxins, maize, stable isotope dilution assay, ultra-high performance liquid chromatography, tandem mass spectrometry.Dissertation (MSc)--University of Pretoria, 2016.
tm2016
Chemistry
MSc
Unrestricted
15
Huang, Yi-chiuan, and 黃奕銓. "1. An improved assay for simultaneous analysis of 3-nitrotyrosine and 3-bromotyrosine in human urine by stable isotope dilution capillary liquid chromatography nanospray ionization tandem mass spectrometry2. Analysis of Thiol-Containing Amino Acids in Hu." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/75274478314817320035.
Full textAbstract:
碩士國立中正大學
化學所
98
Post-translational modification of proteins could affect protein functions. Nitration of protein tyrosine (Tyr), forming 3-nitrotyrosine (3NT), is implicated in cardiovascular diseases, cancer, and inflammatory diseases. At physiological concentrations of bromide, hypobromous acid can be a major oxidant produced by eosinophil peroxidase, leading to bromination of Tyr forming 3-bromotyrosine (3BT), which is considered a biomarker of cancer, allergic inflammatory disorders, asthma and other respiratory diseases. In this study, we have developed an isotope dilution capillary liquid chromatography nanospray ionization tandem mass spectrometry (capLC-NSI/MS/MS) assay for simultaneous analysis of protein-bound 3NT and 3BT in human urine. The detection limits were 0.2 pg (881 amol) for 3NT (S/N = 8) and 0.5 pg (1.92 fmol) for 3BT (S/N = 11) injected on-column, which were 50- and 10-fold lower for 3NT and 3BT, respectively, compared to the previous report ( Toxicol. Lett. 181, 31–39). Urinary protein was hydrolyzed by acid and purified by only one reversed phase solid-phase extraction (SPE) column to enrich Tyr, 3NT and 3BT. The fraction containing enriched 3NT and 3BT was analyzed by capLC-NSI/MS/MS. The high content Tyr in urinary proteins was quantified by high-performance liquid chromatography with fluorescence detection. Both 3NT and 3BT were detected and quantified in 0.1 mL of human urine samples. The use of a single SPE column in sample preparation simplifies the assay procedures. The high sensitivity and specificity of this capLC-NSI/MS/MS method render it a valuable tool in measurement of 3NT and 3BT in the human urinary protein as promising noninvasive biomarkers for protein tyrosine nitration and bromination in vivo.
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Stiles, Kyra. "Quantification Of Gross Nitrogen Transformation Rates Within A Conventional Potato Rotation Using Stable Isotopes." 2012. http://hdl.handle.net/10222/15821.
Full textAbstract:
This study used the isotope pool dilution method to estimate gross rates of mineralization, nitrification, NH4+ and NO3- consumption, and denitrification emissions over two growing seasons within a conventional barley-red clover-potato crop rotation on Prince Edward Island. Gross rates within the 2010 season were, in most cases, not significant across crop species or sampling date. In comparison, gross nitrification, NH4+ consumption, and NO3- consumption rates in 2011 were greatest within the potato crop following planting and hilling. However, rates were highly variable within both seasons. Error analysis indicated that variation in soil mineral nitrogen concentrations between duplicate cores was the greatest source of error. The use of the isotope pool dilution method to estimate gross nitrogen transformation rates using intact cores was not viable within this production system due to high and variable soil mineral nitrogen concentrations, particularly following fertilizer application.
17
Wang, Hsueh-Chun, and 王學鈞. "Analysis of Multiple DNA Adducts in Cancer Patients by Stable Isotope Dilution Nanoflow UPLC-Nanospray Ionization Tandem Mass Spectrometry." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/989um9.
Full textAbstract:
碩士國立中正大學
化學暨生物化學研究所
102
We used stable isotope dilution method by nanoflow ultra performance liquid chromatography-nanospray ionization tandem mass spectrometry (nanoUPLC-NSI/MS/MS) under the highly-selective reaction monitoring (H-SRM) mode to analyze multiple DNA adducts in cancer patients DNA to examine the role of these adducts in cancer formation and their potential as the cancer biomarkers. We focus on four groups of DNA adducts, namely exocyclic adducts, oxidized adduct 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxo-dG), ethylated adducts, and halogenated adducts. Exocyclic adducts include three lipid peroxidation-related etheno adducts 1,N6-etheno-2'-deoxyadenosine (εdAdo), 3,N4-etheno-2'-deoxycytidine (εdCyd), 1,N2-etheno-2'-deoxyguanosine (1,N2-εdGuo) and two 1,N2-propano-2'-deoxyguanosine derived from acrolein (AdG) and crotonaldehyde (CdG). Ethylated adducts include, O2-, O4-, N3-ethylthymidine (edT), and N7-ethylguanine (7-EtGua). The halogenated adducts are 5-chloro-2'-deoxycytidine (5-Cl-dC) and 5-bromo-2'-deoxycytidine (5-Br-dC). These DNA adducts are associated with atherosclerosis, inflammation, oxidative-stress-induced DNA damage, environmental and chemical exposed. In the first part of the study, we quantified the levels of the five exocyclic DNA adducts and 7-EtGua in salivary DNA of five 4th stage oral cancer patients and five healthy donors. In the second part, we analyzed the twelve adducts in DNA from esophageal, gastric, and colorectal cancer patients’ tumor and normal tissue. Only 25 micrograms of DNA was use for each analysis, this highly sensitive, specific, and accurate nanoUPLC-NSI/MS/MS assay might be clinically feasible for simultaneous quantification of these adducts as potential biomarkers of cancer diagnosis and to investigate the role of these adducts in cancer etiology.
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Chen, Sheng-Hong, and 陳勝宏. "Simultaneous Quantification of Five DNA Adducts and Cotinine in Human Urine by Stable Isotope Dilution Nanoflow Liquid Chromatography Nanospray Ionization Tandem Mass Spectrometry." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/46c74b.
Full textAbstract:
碩士國立中正大學
化學暨生物化學研究所
102
Exposure to environmental exogenous chemicals and endogenous reactive species in human can lead to the formation of structurally modified DNA bases (DNA adducts), which play a key role on multi-stage carcinogenesis process, and are the initial step of carcinogenesis. If DNA adducts have not been repaired by the body, these modified DNA bases could mispair by base to base, and lead to mutation during DNA replication, and develop into cancer eventually. These promutagenic DNA adducts are excised by base excision repair and nucleotide excision repair mechanisms and they are excreted into urine or blood as adducted bases and the nucleosides. In this report, we have developed an assay of high sensitivity and high specificity for simultaneous quantification of the five DNA adducts and cotinine by isotope dilution nanoflow liquid chromatography-nanospray ionization-tandem mass spectrometry (nanoLC-NSI/MS/MS)in human urine. Sample purification in urine before analysis by MS only requires a Strata-X reversed phase solid phase extraction column. The urine concentration of εAde、εCyt、1,N2-εGua、7-EtG、8-OH-dG and COT in 5 smokers are 171.2 ± 77.1 pg/mL、285.8 ± 193.5 pg/mL、104.0 ± 61.2 pg/mL、110.1 ± 64.6 pg/mL、3.38 ± 1.14 ng/mL and 784.8 ± 536.3 ng/mL, respectively；those in 8 nonsmokers are 85.2 ± 144.1 pg/mL、202.3 ± 247.3 pg/mL、31.5 ± 31.6 pg/mL、56.6 ± 68.5 pg/mL、2.31 ± 2.35 ng/mL and 0.47 ± 0.43 ng/mL, respectively. This highly sensitive and specific assay based on stable isotope dilution nanoLC-NSI/MS/MS assay provides a useful assay in measuring etheno、ethylpurine and oxidative adducts as noninvasive biomarkers in cancer risk ssessment.
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Chen, Yin-Jung, and 陳胤融. "Simultaneous Quantification of Etheno Adducts and 8-Hydroxy-2’-deoxyguanosine in Human Urine by Stable Isotope Dilution Capillary Liquid Chromatography/Nanospray Ionization-Tandem Mass Spectrometry." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/67776576736619703600.
Full textAbstract:
碩士國立中正大學
化學暨生物化學研究所
99
The promutagenic etheno DNA adducts, including 1,N6-ethenoadensine (εAde), 3,N4-ethenocytosine (εCyt), and 1,N2-ethenoguansine (1,N2-εGua), are derived from exogenous as well as endogenous sources, such as lipid peroxidation. And oxidation DNA adduct, 8-hydroxy-2’-deoxyguanosine (8OHdG) is derived from oxidation stress. The levels of etheno DNA adducts are elevated in cancer-prone tissues, and increasing to high oxidation stress. In this study, we developed a highly specific and sensitive method for simultaneous quantification of etheno DNA adducts: εAde, εCyt , 1,N2-εGua, and 8OHdG in human urine by stable isotope dilution capillary LC-nanospray ionization tandem mass spectrometry (capillary LC-NSI/MS/MS) under the highly selective reaction monitoring (H-SRM) mode. A C18-OH solid phase extraction column was used to enrich the εAde and 8OHdG, which were analyzed by capillary LC-NSI/MS/MS on a triple quadrupole instrument. Also tried is the mixed-mode cation exchange cartridge , that to enrich εCyt and 1,N2-εGua. The detection limit of εAde, εCyt and 1,N2-εGua injected on-column was 0.63, 0.74 and 2.27 fmol, respectively. With a minimum amount of urine (10 μl), this capillary LC-NSI/MS/MS method provides a useful assay in measuring etheno adducts and oxidative adduct as noninvasive biomarkers in cancer risk assessment.
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Lin, Guan-Jih, and 林冠誌. "Stable Isotope Dilution LC-Nanospray Ionization Tandem Mass Spectrometric Analyses for Simultaneous Quantification of (1) Three Etheno DNA Adducts in Human Blood and (2) Ethylpurine Adducts in Human Urine." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/67813177758754924904.
Full textAbstract:
碩士國立中正大學
化學所
97
(1)The promutagenic etheno DNA adducts are derived from exogenous as well as endogenous sources, such as lipid peroxidation. The levels of etheno DNA adducts are elevated in cancer-prone tissues. In this study, we developed a highly specific and sensitive method for simultaneous quantification of etheno DNA adducts: 1,N6-etheno-2´-deoxyadenosine (εdAdo), 3,N4-etheno-2´-deoxycytidine (εdCyd), and 1,N2-etheno-2´-deoxyguanosine (εdGuo) in human blood by stable isotope dilution nanoflow LC-nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) under the highly selective reaction monitoring (H-SRM) mode. We extracted human white blood cell (WBC) DNA from whole blood and digested DNA into nucleosides by enzyme hydrolysis. A C18 solid phase extraction column was used to enrich the etheno DNA adducts, which were analyzed by nanoLC-NSI/MS/MS on a triple quadrupole instrument. The detection limit of εdAdo, εdCyd and εdGuo injected on-column was 1.8, 20 and 86 amol, respectively. The levels of εdAdo, εdCyd and εdGuo in 7 human WBC DNA samples were 4.6 ± 1.5, 6.1 ± 2.7, and 4.9 ± 4.1 (mean ± S.D.) adducts per 107 parent nucleosides, respectively. With minimum amount of DNA (6 μg), this nanoLC-NSI/MS/MS method provides a useful assay in measuring etheno DNA adducts as noninvasive biomarkers in cancer risk assessment.(2)Tobacco smoke contains over 4,000 compounds, which includes alkylating agents. According to the literature, exposure to tobacco smoke results in increased levels of 3-ethyladenine (3-EtA) and 7-ethylguanine (7-EtG) and their levels in smokers’ urine are higher than those in non-smokers’. In this study, we attempted to analyze 3-EtA and 7-EtG in human urine simultaneously. Urine was centrifuged and the adducts were enriched by a C18-OH SPE column. The fractions containing 3-EtA and 7-EtG were analyzed by capillary liquid chromatography nanospray ionization tandem mass spectrometry (capLC-NSI/MS/MS) under the highly selected monitoring mode. Because urine contains many impurities, we use on-line sample purification with a trap column before MS analysis. The detection limit of 3-EtA and 7-EtG injected on-column was 0.3 and 0.55 fmol, respectively. In order to remove the interference in the urine samples, so we tried to use different pretreatment procedure such as NH2 SPE column or SCX SPE column. The NH2 SPE column did not remove the interference in the urine samples. Because 3-EtA and 7-EtG retained in SCX SPE column, it’s possible to use SCX SPE column for adduct enrichment and sample clean-up. We hoped this assay can be useful in evaluation of urinary 3-EtA and 7-EtG as non-invasive biomarkers for measuring DNA damage following exposure to ethylating carcinogens.
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Dang, Khanh B. "Detection and quantification of staphylococcus aureus enterotoxin B in food product using isotopic dilution techniques and mass spectrometry." Thèse, 2012. http://hdl.handle.net/1866/9019.
Full textAbstract:
L’entérotoxine B staphylococcique (SEB) est une toxine entérique hautement résistante à la chaleur et est responsable de plus de 50 % des cas d’intoxication d’origine alimentaire par une entérotoxine. L’objectif principal de ce projet de maîtrise est de développer et valider une méthode basée sur des nouvelles stratégies analytiques permettant la détection et la quantification de SEB dans les matrices alimentaires. Une carte de peptides tryptiques a été produite et 3 peptides tryptiques spécifiques ont été sélectionnés pour servir de peptides témoins à partir des 9 fragments protéolytiques identifiés (couverture de 35 % de la séquence). L’anhydride acétique et la forme deutérée furent utilisés afin de synthétiser des peptides standards marqués avec un isotope léger et lourd. La combinaison de mélanges des deux isotopes à des concentrations molaires différentes fut utilisée afin d’établir la linéarité et les résultats ont démontré que les mesures faites par dilution isotopique combinée au CL-SM/SM respectaient les critères généralement reconnus d’épreuves biologiques avec des valeurs de pente près de 1, des valeurs de R2 supérieure à 0,98 et des coefficients de variation (CV%) inférieurs à 8 %. La précision et l’exactitude de la méthode ont été évaluées à l’aide d’échantillons d’homogénat de viande de poulet dans lesquels SEB a été introduite. SEB a été enrichie à 0,2, 1 et 2 pmol/g. Les résultats analytiques révèlent que la méthode procure une plage d’exactitude de 84,9 à 91,1 %. Dans l’ensemble, les résultats présentés dans ce mémoire démontrent que les méthodes protéomiques peuvent être utilisées efficacement pour détecter et quantifier SEB dans les matrices alimentaires.
Mots clés : spectrométrie de masse; marquage isotopique; protéomique quantitative; entérotoxinesStaphylococcal enterotoxin B is a highly heat-resistant enteric toxin and it is responsible for over 50% of enterotoxin food poisoning. It represents a particular challenge during food processing since, even if the bacteria have been destroyed, the biological activity of the toxin remains unchanged. The objective of this study was to develop and validate a new method based on a novel proteomic strategy to detect and quantify SEB in food matrices. Tryptic peptide map was generated and 3 specific tryptic peptides were selected and used as surrogate peptides from 9 identified proteolytic fragments (sequence coverage of 35%). Peptides were label with light and heavy form of acetic anhydride to create an isobaric tag that will allow quantification. The linearity was tested using mixtures of different molar ratios and the results showed that measurements by LC-MS/MS were within generally accepted criteria for bioassays with slope values near to 1, values of R2 above 0.98 and less than 8% coefficient of variation (%CV). The precision and accuracy of the method were assessed using chicken meat homogenate samples spiked with SEB at 0.2, 1 and 2 pmol/g. The results indicated that the method can provide accuracy within 84.9 – 91.1% range. Overall, the results presented in this thesis show that proteomics-based methods can be effectively used to detect, confirm and quantify SEB in food matrices. Keywords: mass spectrometry; stable isotope labeling; quantitative proteomics; enterotoxins
22
Lee, Jing Rong, and 李京融. "Stable Isotope Dilution Nanoflow Liquid Chromatography /Nanospray Ionization Tandem Mass Spectrometry Methods for Analyses of Three Ethyl-Thymidine Adducts in Human Saliva DNA and 3-Hydroxypropyl¬mercapturic Acid and 3-Hydroxy-1-methylpropylmercapturic A." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/61639496714013355586.
Full textAbstract:
碩士國立中正大學
化學暨生物化學研究所
100
Studies showed that levels of certain ethylated DNA adducts in certain tissues and urine are higher in smokers than in nonsmokers. Because cigarette smoking is a major risk factor of various cancers, DNA ethylation might play an important role in cigarette smoke-induced cancer formation. Among the ethylated DNA adducts, O2-ethylthymidine (O2-edT) and O4-ethylthymidine (O4-edT) are poorly repaired and are accumulated in the body. In addition, O4-edT possesses promutagenic properties. A highly sensitive, accurate, and quantitative assay has been developed based on isotope dilution nanoflow liquid chromatography¬−nanospray ionization tandem mass spectrometry (nanoLC¬−NSI/MS/MS) for simultaneous detection and quantification of O2-edT, N3-edT (N3-ethylthymidine), and O4-edT adducts in human leukocyte DNA by this laboratory. In this study, we used this method for detection and quantification of O2-edT, N3-edT, and O4-edT adducts in human salivary DNA. Saliva is an easily accessible and available biological fluid and a potential target of noninvasive biomarkers. Under the highly selected reaction monitoring (H-SRM) mode, salivary samples from 11 smokers and 10 nonsmokers were analyzed. Starting with 50 μg of DNA isolated from about 3.5 mL of saliva, levels of O2-edT, N3-edT, and O4-edT in 11 smokers’ saliva DNA were 5.0 ± 5.9, 5.8 ± 9.3, 5.8 ± 10.0 in 108 normal nucleotides, respectively, while those in 4 nonsmokers were non-detectable. Clearly, more salivary DNA samples are needed to evaluate the effect of smoking on levels of these 3 edT adducts in salivary DNA, which might be potential biomarkers for exposure to ethylating agents and for cancer risk assessment. Cigarette smoking is a major source of human exposure to acrolein and crotonaldehyde, a widespread environmental pollutant and toxicant that is also formed endogenously through metabolism of lipid peroxidation. Cigarette smoke was found to contain about 18―140 μg acrolein/cigarette and 72―228 μg crotonaldehyde/ cigarette. The reactivity of acrolein and crotonaldehyde is attributed to the electrophilic center of their β-carbon with the electron-rich group of biomolecules, such as thiols and the amino groups, forming the Michael addition product. These unsaturated aldehydes conjugate with glutathione, following reduction and normal metabolic processes, and generate two major metabolites：3-hydroxypropylmercapturic acid (3-HPMA) and 3-hydroxy-1- methylpropylmercapturic acid (HMPMA), respectively. LC-MS/MS methods for the determination of 3-HPMA and HMPMA in human urine have been developed. Studies show that 3-HPMA and HMPMA are higher in the urine of smokers than nonsmokers. We attempt to develop a highly sensitive and quantitative assay for simultaneous detection and quantification of 3-HPMA and HMPMA by isotope dilution nanoflow liquid chromatography nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) under the highly selected reaction monitoring mode (H-SRM). Using this assay with increased sensitivity, we try to find the relationship between these two metabolites and diseases
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Wu, Chia-yen, and 吳佳燕. "1.Simultaneous Quantification of Etheno Adducts in human urine and DNA samples by Stable Isotope Dilution Capillary Liquid Chromatography/Nanospray Ionization-Tandem Mass Spectrometry 2.Post-translational modifications on human hemoglobin by glyoxal and." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/39716950631837908475.
Full textAbstract:
碩士國立中正大學
化學所
96
Etheno DNA adducts are promutagenic DNA lesions derived from exogenous as well as endogenous sources. The levels of etheno adducts in tissue DNA are elevated in cancer-prone tissues, and the urinary excretion of etheno adducts is associated with oxidative stress. In this report, we have developed an assay for the accurate quantification of etheno DNA adducts by isotope dilution capillary liquid chromatography/nanospray ionization-tandem mass spectrometry (capillary LC-NSI/MS/MS) in human urine and tissue samples. These promutagenic etheno DNA adducts are excised by base excision repair and possibly the nucleotide excision repair mechanisms and they are excreted into urine as adducted bases and the nucleosides. The etheno DNA adducts investigated in this study include 1,N6-ethenoadenosine(eAde),3,N4-ethenocytidine(eCyt),1,N6-etheno-2?-deoxyadenosine(edAdo),3,N4-etheno-2?-deoxycytosine(edCyt),and1,N2-etheno-2?-deoxyguanosine(edGuo). Sample purification before analysis by MS only requires a reversed phase solid phase extraction column. The detection limit (LOD) of eAde, eCyt, edAdo, edCyt and edGuo injected on-column using this capillary LC-NSI/MS/MS is 300 , 120, 1.8, 20, and 86 amol, respectively. Levels of eAde, eCyt and edAdo in 12 human urine are 88 ± 48,109 ± 96 and 4.4 ± 3.0 pg/mL. The origin of DNA are calf thymus, human placental and human white blood cell DNA. Levels of these etheno adducts in calf thymus DNA and human placental DNA were compared as nucleosides using six different enzyme hydrolysis procedures. In human placental DNA, the levels of edAdo, edCyd and edGuo was 6.45 ± 0.46, 27.8 ± 0.27 and 7.76 ± 0.64 adducts per 107 parent nucleoside, respectively. But in some calf thymus and WBC DNA samples, the adduct levels were below the detection limits. Larger quantities of DNA is needed for simultaneous quantification of low levels of these three etheno adducts. The non-enzymatic conjugated addition product of glucose or aldehyde derivatives and glycation reaction of protein are the main cause of vascular complications of diabetes. When the concentration of glucose remains at high level in the body, the amounts of ?dicarbonyl compounds, such as glyoxal and methylglyoxal, also increase. Glyoxal is derived from glucose and amino acid oxidation as well as lipid peroxidation; whereas methylglyoxal is mainly from self-decomposition of glyceraldehyde-3-phospate (G-3-P), a degradation intermediate product. This research makes use of liquid chromatography nanospray ionization tandem mass spectrometry to investigate sites of reaction sites on human hemoglobin with glyoxal and methylglyoxal. We found that there was a single glyoxal addition at Lys-11, Lys-139, Arg-92, His-20, His-45, His-50 on ?globin and at Lys-144, His-77, His-92, His-143, Cys-93 and Cys-112 on β-globin, when the concentration of glyoxal was 20 mM. When 0.5 mM of glyoxal and methylglyoxal react with hemoglobin under physiological conditions (pH7.4, 37℃) for 48 hr, we also found that there was a single glyoxal addition at Lys-11, His-20, His-45, His-50, Arg-92 and Lys-139 on ?globin and at His-77, His-92, Cys-93, Cys-112, His-143 and Lys-144 on β-globin. For methylglyoxal, a sigle addition was found at His-20 and Arg-92 on ?globin and at Cys-93, Lys-144 on β-globin. We have also confirmed that glyoxal and methylglyoxal form hydroimidazolone at Arg-92 of ?globin. Finally, we used 0.5 μM of glyoxal and methylglyoxal to react with hemoglobin under physiological conditions (pH7.4, 37℃) for 35 days, we also found a single glyoxal addition at Lys-11, His-20, His-45, His-50 on ?globin and at His-77, His-143, His-116, Cys-93 and Cys112on β-globin. For methylglyoxal addation, only addition product of Cys-93 on β-globin was found. In human blood hemoglobin, only glyoxal addation on β-globin was found.
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Wang, Yi-Ching, and 王薏菁. "Simultaneous Quantification of (1) Three Ethyl-Thymidine Adducts in Human White Blood Cells (2) 3-Hydroxypropylmercapturic Acid and 3-Hydroxy-1-methylpropylmercapturic Acid in Human Urine by Stable Isotope Dilution Capillary Liquid Chromatography /Nanospr." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/34ektb.
Full textAbstract:
碩士國立中正大學
化學暨生物化學研究所
99
O-Substitution by alkylating agents of cigarette smoke results in DNA adducts, which are considered to be major promutagenic lesion and may cause cancer formation. Animal studies indicated that, after exposure to ethylating agents, O4-ethylthymidine (O4-edT) was initially formed in very low levels, but it was poorly repaired and thus accumulated to biologically relevant levels. Studies also show that ethylated adducts in certain tissues and urine are higher in smokers than in nonsmokers. In this study, we have developed a highly sensitive and quantitative assay for simultaneous detection and quantification of O2-edT, N3-edT, and O4-edT adducts by isotope dilution capillary liquid chromatography nanospray ionization tandem mass spectrometry (capillary LC-NSI/MS/MS) under the highly selective reaction monitoring (H-SRM) mode. Typically, [13C10,15N2]O2-edT, [13C10,15N2]N3-edT, and [13C10,15N2]O4-edT were added to iodoethane-treated calf thymus, human placenta, or human leukocyte DNA as internal standards, and the mixture was subjected to enzyme hydrolysis to form the nucleosides. The edT adducts in DNA hydrolysate were enriched by a reversed phase solid-phase extraction column before analysis by capillary LC-NSI/MS/MS.The detection limits of O2-edT, N3-edT, and O4-edT injected on-column are 18.5 amol, 37.0 amol, and 37.0 amol, respectively. Levels of O2-edT, N3-edT, and O4-edT in iodoethane-treated calf thymus DNA are 5.7, 77.2 and 8.0 in 108 normal nucleotides, respectively, but none of these adducts are detected in human placental DNA. Levels of O2-edT, N3-edT, and O4-edT are 11.2 ± 23.4, 12.8 ± 16.8, 10.5 ± 21.7 in 108 normal nucleotides in smokers white blood cells (WBC) DNA (n=9), comparing with 0.4 ± 1.3, 9.0 ± 19.3, 1.7 ± 4.1 in nonsmokers WBC DNA (n=9). In order to increase the detection sensitivity, we try to derivatizate edT adducts. Four derivatization agents have been tried, but none of them are successful. The main reasons might be the poor and reactivity of edT and dT.
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chang, Ya-lan, and 張雅嵐. "Analysis of (1) Glyoxal-Induced DNA Cross-Links in Human White Blood Cell DNA and (2) Etheno Adducts and 8-Hydroxy-2’-deoxyguanosine in Human Urine by Stable Isotope Dilution Liquid Chromatography-Nanospray Ionization Tandem Mass Spectrometry." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/57758824654267224051.
Full textAbstract:
碩士國立中正大學
化學暨生物化學研究所
100
There are many reactive chemicals in our living environment which could reactive with DNA forming DNA adducts. There are two parts in my thesis. In the first part, we developed a highly sensitive and specific quantitative assay for simultaneous detection and quantification of dG-gx-dA and dG-gx-dC cross-links by stable isotope dilution nanoflow liquid chromatography nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) under the highly selective reaction monitoring (H-SRM) mode. Levels of dG-gx-dC and dG-gx-dA in human placenta DNA are 5.92 and 6.39 in 108 normal nucleotides, respectively. Levels of dG-gx-dC and dG-gx-dA are 1.44 ± 1.17 and 1.70 ± 1.08 in 108 normal nucleotides ,respectively, in 11 smokers’ white blood cell (WBC) DNA, and those in 9 nonsmokers’ WBC DNA are 0.81 ± 0.62 and 0.79 ± 0.85 in 108 normal nucleotides, respectively. Thus, it is clinically feasible using this highly sensitive assay to investigate the potential of these cross-links as low-invasive biomarkers for glyoxal-induced DNA damage and in disease development and prevention. In the second part, we developed a highly specific and sensitive method for simultaneous quantification of etheno DNA adducts: εAde, εCyt, 1,N2-εGua, and 8OHdG in human urine by stable isotope dilution capillary LC-NSI/MS/MS under the H-SRM mode. The urine concentration of εAde, εCyt, 1,N2-εGua, and 8OHdG are 52.2 ± 82.4, 21.2 ± 24.7, 76.6 ± 53.9 pg/mL and 2.82 ± 1.52 ng/mL in 4 smokers’ urine samples, respectively, and those in 8 nonsmokers’ urine samples are 40.8 ± 44.8, 55.6 ± 89.5, 69.6 ± 116.7 pg/mL and 2.64 ± 2.67 ng/mL, respectively. This capillary LC-NSI/MS/MS method provides a useful assay in measuring etheno and oxidative adducts as noninvasive biomarkers in cancer risk assessment.
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劉俊廷. "(1)Simultaneous analysis of multiple DNA adducts in human blood DNA by stable isotope dilution nanoflow liquid chromatography-nanospray ionization tandem mass spectrometry (2)Analysis of glyoxal-, methylglyoxal-induced post-translational modifications of human hemoglobin by nanoflow liquid chromatography-nanospray ionization tandem mass spectrometry." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/tx7995.
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Porter, Brendan. "Adaptation and acclimation of red alder (Alnus rubra) in two common gardens of contrasting climate." Thesis, 2011. http://hdl.handle.net/1828/3760.
Full textAbstract:
Red alder (Alnus rubra Bong.) is the only tree in British Columbia and the Northwest US to engage in actinorhizal symbiosis to fix atmospheric nitrogen. This study was conducted to explore the plasticity in growth and physiology among 58 17-year-old red alder families in response to variation in climate in two common garden plots, one at Bowser, BC and one at Terrace, BC. Physiological assessments included height and diameter growth, bud flush, water use efficiency as measured by δ13C, cold hardiness as measured by controlled freezing and electrolyte leakage, autumn leaf senescence, and instantaneous and seasonally integrated rates of nitrogen fixation as measured by acetylene reduction and natural abundance δ15N isotope analysis, respectively. Significant differences were identified among families for growth (height and diameter), bud burst stage, leaf senescence, cold hardiness, and bud nitrogen content. No significant differences among families were identified for water use efficiency as measured by δ13C, or for rates of nitrogen fixation as measured by either acetylene reduction or natural abundance δ15N. This study identified possible adaptive differences among red alder genotypes, especially in traits such as bud flush timing, cold hardiness, or nitrogen fixation and their respective contributions to growth. These differences often reflected a tradeoff between growth and the ability to tolerate an extreme environment. Cold hardiness results indicate that red alder families are well adapted to their climate of origin, and may not be able to acclimate sufficiently to a northward assisted migration of genotypes. Nitrogen fixation results demonstrated gaps in our current knowledge of Frankia distribution and impact on the actinorhizal symbiosis in British Columbia.Graduate
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Seibert, C., B. R. Davidson, B. J. Fuller, Laurence H. Patterson, W. J. Griffiths, and Y. Wang. "Multiple-approaches to the identification and quantification of cytochromes P450 in human liver tissue by mass spectrometry." 2009. http://hdl.handle.net/10454/6179.
Full textAbstract:
Here we report the identification and approximate quantification of cytochrome P450 (CYP) proteins in human liver microsomes as determined by nano-LC-MS/MS with application of the exponentially modified protein abundance index (emPAI) algorithm during database searching. Protocols based on 1D-gel protein separation and 2D-LC peptide separation gave comparable results. In total, 18 CYP isoforms were unambiguously identified based on unique peptide matches. Further, we have determined the absolute quantity of two CYP enzymes (2E1 and 1A2) in human liver microsomes using stable-isotope dilution mass spectrometry, where microsomal proteins were separated by 1D-gel electrophoresis, digested with trypsin in the presence of either a CYP2E1- or 1A2-specific stable-isotope labeled tryptic peptide and analyzed by LC-MS/MS. Using multiple reaction monitoring (MRM) for the isotope-labeled tryptic peptides and their natural unlabeled analogues quantification could be performed over the range of 0.1-1.5 pmol on column. Liver microsomes from four individuals were analyzed for CYP2E1 giving values of 88-200 pmol/mg microsomal protein. The CYP1A2 content of microsomes from a further three individuals ranged from 165 to 263 pmol/mg microsomal protein. Although, in this proof-of-concept study for CYP quantification, the two CYP isoforms were quantified from different samples, there are no practical reasons to prevent multiplexing the method to allow the quantification of multiple CYP isoforms in a single sample.