Il capello come matrice per il monitoraggio della esposizione ad antiparassitari.

This Thesis is based mainly on the results of the timing analysis applied to the gamma-ray pulsars observed principally by the AGILE satellite and also extended, in some cases, to observations with the Fermi-LAT satellite and the MAGIC Cherenkov telescope. Aim of this extended study of pulsars at the high energies was to characterize their properties based, now, on a more statistically relevant sample, and be able to disentangle useful informations that can be key to explain the emission mechanisms in pulsars. THE SCIENTIFIC CONTEXT Pulsars are highly-magnetized, rapidly-rotating neutron stars. As explained in Chapter 1, they have been observed in the radio band over the past forty years, due to their highly anisotropic emission, which, when combined with the misalignment between the rotation and the magnetic axis, produces the pulsed emission we observed, also called the ”lighthouse effect”. Pulsars have been observed as gamma-ray emitters as well, but it is only in the past three years that their number hit the double digits and they started to yield their potential as keys to explain the neutron stars mechanisms. In Chapter 1, the basics of pulsar theory are given. While the phenomenological aspects have been widely studied thanks to extensive radio observations through the years, their electrodynamics represents an articulated field that is difficult to probe. The ”classical” models of magnetosphere predict the presence of regions of particles’ under-density (a ”gap”), inside an overall force-free magnetized area that surrounds the pulsar, where the particles can be accelerated and can produce the observed radiation, after a number of cascade processes. More ”modern” models of magnetosphere, with their roots in old predictions, discuss the hypothesis of a totally force-free magnetosphere. The discussed theories search for a confirmation in our gamma-ray observations, as the gamma-rays are the ones carrying away a good fraction of the rotational energy loss. THE NEW PERSPECTIVES OPENED BY THE MULTI-BAND OBSERVATIONS In April 2007 the Italian Space Agency launched the AGILE satellite for gamma-ray astronomy. About one year later, AGILE was joined in the observation of gammarays by the 16 times bigger Fermi-LAT satellite launched by NASA. AGILE and Fermi-LAT, with their wide field of view and large collective area, are particularly suited for the study of pulsars at high energies. Most recently, the window of veryhigh energy observations has opened up to pulsar studies and, in particular, by the MAGIC telescope, with the lowest up to now threshold for ground-based telescopes, at 25 GeV. Its observations are briefly described, together with AGILE and Fermi-LAT’s, in Chapter 2. The techniques for studying pulsars in the gamma-rays are also explained in Chapter 2, with the fundamental premise about the radio observations which were part of my analysis work, as they constitute the primary basis for the gammaray observations. The advances with respect to the observations of the previous generation gamma-ray instruments are highlighted. In particular, AGILE was able to take into account, for the first time in gamma-ray observations, the timing noise that affects young pulsars. In this way, the observations can be carried out for longer time spans without being affected by sensible light curve smearing. Thus, we could take advantage of the long time span, up to now the longest for gamma-ray observations, to increase the resolution of our light curves and see structures at the sub-millisecond level. GAMMA-RAY PULSARS AGILE and Fermi-LAT pulsar observations first concentrated on the known gamma-ray pulsars. As shown in Chapter 3, the apparently ”familiar” pulsars actually hid thriving new prospects for pulsar studies, as well as the new pulsars subsequently detected, described in Chapter 4. In these two Chapters, the properties of the gamma-ray emission ars analyzed for a number of pulsars, mainly using AGILE data, but also with Fermi-LAT and MAGIC observations. The light curves are investigated with increased resolution from previous observations and the spectral properties are addressed. The availability of a statistically significant sample of gamma-ray pulsars led us to draw some lines on the models. The classical polar cap model seems to be failing the test of gamma-ray observations for most of the present sample, and a simple explanation of which can be found in conservation laws arguments discussed in Chapter 4. At the same time it starts getting clear that a model that contemplates a single gap zone does not seem to be feasible to explain the observed pulse profiles. And, possibly, the entire gap theory should be combined with the more physical force-free models. Episodes of variability in pulsars have been observed and studied in this context. The Vela glitch of August 2007 was observed by AGILE in search for gamma-ray emission. The Crab pulsar could have a contribution to the emission from a newly observed third pulsar peak, that is less significant and much weaker than the canonical two, and could be due to giant pulses. AGILE observed the first gamma-ray millisecond pulsar but its emission only appeared in a restricted time interval, leading to the interesting possibility that pulsar emission might have some intrinsic variability. HIGH MAGNETIC FIELD PULSARS After the advent of Fermi-LAT, AGILE found its collocation in the gamma-ray astronomy in the characterization of the low-energy gamma-rays (from 30 to 100 MeV), where the collective areas of the two instruments is equivalent, but AGILE deals with much lower background. For this reason, we concentrated on those pulsars that show a low-energy cutoff, which were theorized to emit gamma-ray radiation through the exotic QED process of photon splitting. A detailed analysis of the two most significant cases is given in Chapter 5. We have found that the concurrence of a high magnetic field and an aligned geometry, could overcome the objections from Chapter 4 against inner magnetosphere emission and be, indeed, dominated by polar cap emission. Interestingly, this phenomenology, that is observed in pulsars that are similar to magnetars, may be observed in objects that are transitioning from pulsar to magnetar. THE ENVIRONMENT OF PULSARS Young pulsars are known to power a relativistic wind of particles that surrounds the pulsar and is best known as its Pulsar Wind Nebula (PWN). Important phenomena take place in the PWN and they are powered by the pulsar inside it. As discussed in Chapter 6, very high energy emission was already observed from PWN, but high energy emission was missing, in a spectral region where important constraints on the emission processes could be given. AGILE was the first satellite to detect GeV emission from a PWN apart from Crab, Vela X, and it was also the first to claim the unexpected flux variation in the Crab Nebula which underwent two intense flares in 2010 and 2011. In Chapter 6 we give a description of the events and a possible trail for an interpretation, although no clear picture can yet emerge from the observed events. CONCLUSIONS AND FUTURE PROSPECTS The multi-band approach that has been used for the observations described in this Thesis has proven valid for the exploitation of new science and the most useful approach for the comprehensive analysis of pulsar phenomena across the electromagnetic spectrum. As a completion to this work, the more comprehensive AGILE Pulsar Catalog is in preparation. It will comprise all the pulsars observed by AGILE and particularly focus on the low-energy tail of them, which present interesting properties that bridge pulsars and magnetars

Il capello come matrice per il monitoraggio della esposizione ad antiparassitari.

This Thesis is based mainly on the results of the timing analysis applied to the gamma-ray pulsars observed principally by the AGILE satellite and also extended, in some cases, to observations with the Fermi-LAT satellite and the MAGIC Cherenkov telescope. Aim of this extended study of pulsars at the high energies was to characterize their properties based, now, on a more statistically relevant sample, and be able to disentangle useful informations that can be key to explain the emission mechanisms in pulsars. THE SCIENTIFIC CONTEXT Pulsars are highly-magnetized, rapidly-rotating neutron stars. As explained in Chapter 1, they have been observed in the radio band over the past forty years, due to their highly anisotropic emission, which, when combined with the misalignment between the rotation and the magnetic axis, produces the pulsed emission we observed, also called the \u201dlighthouse effect\u201d. Pulsars have been observed as gamma-ray emitters as well, but it is only in the past three years that their number hit the double digits and they started to yield their potential as keys to explain the neutron stars mechanisms. In Chapter 1, the basics of pulsar theory are given. While the phenomenological aspects have been widely studied thanks to extensive radio observations through the years, their electrodynamics represents an articulated field that is difficult to probe. The \u201dclassical\u201d models of magnetosphere predict the presence of regions of particles\u2019 under-density (a \u201dgap\u201d), inside an overall force-free magnetized area that surrounds the pulsar, where the particles can be accelerated and can produce the observed radiation, after a number of cascade processes. More \u201dmodern\u201d models of magnetosphere, with their roots in old predictions, discuss the hypothesis of a totally force-free magnetosphere. The discussed theories search for a confirmation in our gamma-ray observations, as the gamma-rays are the ones carrying away a good fraction of the rotational energy loss. THE NEW PERSPECTIVES OPENED BY THE MULTI-BAND OBSERVATIONS In April 2007 the Italian Space Agency launched the AGILE satellite for gamma-ray astronomy. About one year later, AGILE was joined in the observation of gammarays by the 16 times bigger Fermi-LAT satellite launched by NASA. AGILE and Fermi-LAT, with their wide field of view and large collective area, are particularly suited for the study of pulsars at high energies. Most recently, the window of veryhigh energy observations has opened up to pulsar studies and, in particular, by the MAGIC telescope, with the lowest up to now threshold for ground-based telescopes, at 25 GeV. Its observations are briefly described, together with AGILE and Fermi-LAT\u2019s, in Chapter 2. The techniques for studying pulsars in the gamma-rays are also explained in Chapter 2, with the fundamental premise about the radio observations which were part of my analysis work, as they constitute the primary basis for the gammaray observations. The advances with respect to the observations of the previous generation gamma-ray instruments are highlighted. In particular, AGILE was able to take into account, for the first time in gamma-ray observations, the timing noise that affects young pulsars. In this way, the observations can be carried out for longer time spans without being affected by sensible light curve smearing. Thus, we could take advantage of the long time span, up to now the longest for gamma-ray observations, to increase the resolution of our light curves and see structures at the sub-millisecond level. GAMMA-RAY PULSARS AGILE and Fermi-LAT pulsar observations first concentrated on the known gamma-ray pulsars. As shown in Chapter 3, the apparently \u201dfamiliar\u201d pulsars actually hid thriving new prospects for pulsar studies, as well as the new pulsars subsequently detected, described in Chapter 4. In these two Chapters, the properties of the gamma-ray emission ars analyzed for a number of pulsars, mainly using AGILE data, but also with Fermi-LAT and MAGIC observations. The light curves are investigated with increased resolution from previous observations and the spectral properties are addressed. The availability of a statistically significant sample of gamma-ray pulsars led us to draw some lines on the models. The classical polar cap model seems to be failing the test of gamma-ray observations for most of the present sample, and a simple explanation of which can be found in conservation laws arguments discussed in Chapter 4. At the same time it starts getting clear that a model that contemplates a single gap zone does not seem to be feasible to explain the observed pulse profiles. And, possibly, the entire gap theory should be combined with the more physical force-free models. Episodes of variability in pulsars have been observed and studied in this context. The Vela glitch of August 2007 was observed by AGILE in search for gamma-ray emission. The Crab pulsar could have a contribution to the emission from a newly observed third pulsar peak, that is less significant and much weaker than the canonical two, and could be due to giant pulses. AGILE observed the first gamma-ray millisecond pulsar but its emission only appeared in a restricted time interval, leading to the interesting possibility that pulsar emission might have some intrinsic variability. HIGH MAGNETIC FIELD PULSARS After the advent of Fermi-LAT, AGILE found its collocation in the gamma-ray astronomy in the characterization of the low-energy gamma-rays (from 30 to 100 MeV), where the collective areas of the two instruments is equivalent, but AGILE deals with much lower background. For this reason, we concentrated on those pulsars that show a low-energy cutoff, which were theorized to emit gamma-ray radiation through the exotic QED process of photon splitting. A detailed analysis of the two most significant cases is given in Chapter 5. We have found that the concurrence of a high magnetic field and an aligned geometry, could overcome the objections from Chapter 4 against inner magnetosphere emission and be, indeed, dominated by polar cap emission. Interestingly, this phenomenology, that is observed in pulsars that are similar to magnetars, may be observed in objects that are transitioning from pulsar to magnetar. THE ENVIRONMENT OF PULSARS Young pulsars are known to power a relativistic wind of particles that surrounds the pulsar and is best known as its Pulsar Wind Nebula (PWN). Important phenomena take place in the PWN and they are powered by the pulsar inside it. As discussed in Chapter 6, very high energy emission was already observed from PWN, but high energy emission was missing, in a spectral region where important constraints on the emission processes could be given. AGILE was the first satellite to detect GeV emission from a PWN apart from Crab, Vela X, and it was also the first to claim the unexpected flux variation in the Crab Nebula which underwent two intense flares in 2010 and 2011. In Chapter 6 we give a description of the events and a possible trail for an interpretation, although no clear picture can yet emerge from the observed events. CONCLUSIONS AND FUTURE PROSPECTS The multi-band approach that has been used for the observations described in this Thesis has proven valid for the exploitation of new science and the most useful approach for the comprehensive analysis of pulsar phenomena across the electromagnetic spectrum. As a completion to this work, the more comprehensive AGILE Pulsar Catalog is in preparation. It will comprise all the pulsars observed by AGILE and particularly focus on the low-energy tail of them, which present interesting properties that bridge pulsars and magnetars

Development, validation and application of an HPLC-MS/MS method to quantify urinary mercapturic acids

Introduction Mercapturic acids are metabolic end products of some occupational and environmental toxicants such as volatile organic compounds. They are metabolites formed by the conjugation of an electrophilic compound with glutathione. These electrophilic metabolic intermediates are believed to be the active species able to react with DNA and responsible for the genotoxicity associated with parent compounds [1]. Mercapturates can be found in urine and, therefore, they can be considered useful non-invasive biomarkers of exposure. Although several analytical methods were reported for the analysis of single or small groups of mercapturates [2], only two papers describes the analysis of several mercapturates [3,4]. The aim of this work was to set up a LC-MS/MS method able to determine mercapturic acids derived from different toxicants. Experimental For the preparation of standard solution, the majority of standard compounds were purchased from Toronto Research Chemicals (Ontario, Canada), along with relative isotopically labelled standards. The complete list of analytes is reported in Table 1. The simple sample preparation developed includes dilution with formic acids (0.2 M), addition of an internal standard mixture of 16 deuterated analogs and filtration with 0.45 \u3bcm regenerated cellulose membrane filter (Agilent Technologies, Santa Clara, California). Analysis were carried out using a hybrid triple quadrupole/linear ion trap mass spectrometer (QTRAP 5500, AB Sciex, Monza, Italy) interfaced with an ultrahigh pressure liquid chromatograph (UHPLC, Agilent 1220, Cernusco sul Naviglio, Italy) equipped with a Betasil C18 column (150 x2.1 mm, 5 \u3bcm; Thermo Fisher Scientific, Rodano, Italy) and a pre-column BETASIL C18 (10 x 2,1 mm, 5\u3bc; Thermo Fisher Scientific, Rodano, Italy). Chromatographic separation was performed using a linear gradient with an aqueous mobile phase composed by an aqueous solution of ammonium formiate 5 mM and 0.1% formic acid and an organic mobile phase composed by acetonitrile. A complete validation was carried out: linearity, sensitivity, accuracy, precision, selectivity, matrix effect, recovery and process efficiency were evaluated according to both FDA guidelines and the considerations reported in the review written by Gonz\ue1lez and co-workers [5,6]. The method was then applied to the analysis of urine samples from adult subjects with different smoking habits: non-smokers, electronic cigarette smokers, and traditional tobacco smokers. Results Results from linearity assays showed that correlation coefficients (R2) were close to 1 for most of compounds, demonstrating optimal linear responses for the considered concentrations ranges, although a polynomial regression was necessary for AAMA since it showed a saturation at high concentrations. Limits of quantitation (LOQ) values were between 0.15 and 1 \u3bcg/L, except for HEMA and AAMA (1.93 and 1.30 \u3bcg/L respectively). Precision, evaluated as relative standard deviations (RDS), was below 15% for most analytes in both intra-day and inter-day tests. Accuracy was between 85 and 110 % of expected values, with few exceptions exceeding 120% at the lowest concentrations. Selectivity was verified by injection of a blank sample (synthetic urine) showing no chromatographic peak having an area at 20% of LOQ at the relative retention time and mass transition of compounds of interest. The same condition was verified analysing a blank sample immediately after the injection of the standard mixture at the highest concentration of the calibration curve, indicating the absence of carry-over. Results from the matrix effect, recovery and process efficiency tests were suitable in most of the cases, with some exceptions that were partially corrected using the internal standards. Results from urine samples of individuals with different smoking habit showed significant differences between smokers and non-smokers: 11 different mercapturic acids were significantly higher (P-value 640.005) in traditional tobacco smokers than in non-smokers (an example is illustrated in Figure 1). Conclusion In this work, we developed a method useful to quantify mercapturic acids in urine samples. The method was subjected to a complete validation and showed to be suitable for most of the considered analytes. Despite some critical issues with some analytes (in particular HEMA), it demonstrated to be an useful tool for fast determination of mercapturates. The first application carried out using human urine samples suggests that mercapturic acids are suitable biomarkers for toxicants in tobacco smoke. References 1. B.M. De Rooij, J.N.M. Commandeur, N.P.E; Biomarkers, 3 (1998), pp 239-303. 2. P.I. Mathias, C. B'hymer; Biomarkers, 21 (2016), pp 293-315. 3. K.U. Alwis, B.C. Blount, A.S. Britt, D. Patel, D.L. Ashley; Analytica Chimica Acta, 750 (2012), pp 152-160. 4. N. Pluym, G. Gilch, G. Scherer, M. Scherer; Analytical and Bioanalytical Chemistry, 407 (2015), pp 5463-5476. 5. FDA. Guidance for Industry - Bioanalytical Method Validation. (2013) Available at: https://www.fda.gov/downloads/Drugs/Guidances/ucm368107.pdf 6. O. Gonz\ue1lez, M.E. Blanco, G. Iriarte, L. Bartolom\ue9, M.I. Maguregui, R.M. Alonso; Journal of Chromatography A, 1353 (2014), pp10-27

A workflow for data integration, analysis, and metabolite annotation for untargeted metabolomics

Metabolomics is the youngest of the \u201comics\u201d disciplines and it is regarded as a promising approach to understand the metabolic changes that can occur in particular conditions and to identify new biomarkers. We present here a workflow for data integration, analysis, and metabolite annotation to be applied to untargeted metabolomic experiments. Data acquired with LC-MS/MS, operating in data dependent mode, are processed using the R-packages IPO and XCMS to perform feature detection, retention time correction and alignment. The data-table obtained is elaborated and submitted to statistical analysis using the on-line software MetaboAnalyst. Multivariate analysis, in particular principal component and partial least squares discriminant analysis are performed for data visualization. Univariate analysis, in particular T-test for pairwise and ANOVA for multi-groups comparison, are performed to detect significant features among groups. The software BEAMS, developed by the University of Birmingham, is then implemented for grouping adducts and isotopes, and to perform a first annotation. Metabolite annotation is finally completed by comparing the fragmentation pattern obtained from each parent ion corresponding to a significant feature with data stored in on-line databases as Metlin, and with the help of the software MS-FINDER, which performs in-silico fragmentation. We applied this workflow to an untargeted metabolomic experiment performed on 67 urine samples obtained from adult subjects with different smoking habits: non-smokers, electronic cigarette smokers, and traditional tobacco smokers. 117 features, out of 3613, were statistically different among groups. We estimated that they correspond to about 80 metabolites. We were able to putatively annotate compound classes of most of the significant metabolites (level 3 according to the \u201cProposed minimum reporting standards\u201d; Sumner et al., 2007) and to putatively annotate some of them (level 2). Among them, the glucuronide conjugated of 3-hydroxycotinine supports the validity of the proposed approach

Development and Application of an LC-MS/MS Untargeted Exposomics Method with a Separated Pooled Quality Control Strategy

Pooled quality controls (QCs) are usually implemented within untargeted methods to improve the quality of datasets by removing features either not detected or not reproducible. However, this approach can be limiting in exposomics studies conducted on groups of exposed and nonexposed subjects, as compounds present at low levels only in exposed subjects can be diluted and thus not detected in the pooled QC. The aim of this work is to develop and apply an untargeted workflow for human biomonitoring in urine samples, implementing a novel separated approach for preparing pooled quality controls. An LC-MS/MS workflow was developed and applied to a case study of smoking and non-smoking subjects. Three different pooled quality controls were prepared: mixing an aliquot from every sample (QC-T), only from non-smokers (QC-NS), and only from smokers (QC-S). The feature tables were filtered using QC-T (T-feature list), QC-S, and QC-NS, separately. The last two feature lists were merged (SNS-feature list). A higher number of features was obtained with the SNS-feature list than the T-feature list, resulting in identification of a higher number of biologically significant compounds. The separated pooled QC strategy implemented can improve the nontargeted human biomonitoring for groups of exposed and nonexposed subjects

Investigation of urine metabolites related to tobacco smoke chemicals using an untargeted metabolomic approach

Although thousands of different chemicals have been identified in cigarette smoke, the characterization of urinary metabolites derived from those compounds is still not completely achieved. The aim of this work was to perform an untargeted metabolomic experiment on a pilot cross-sectional study conducted on subjects with different smoking habits. Urine samples were collected from 67 adults; including 38 non-smokers, 7 electronic cigarette smokers, and 22 traditional tobacco smokers. Samples were analyzed by liquid chromatography/time-of flight mass spectrometer operating in data dependent mode. Data were processed using the R-packages IPO and XCMS to perform feature detection, retention time correction and alignment. The ANOVA test was used to detect significant features among groups. The software BEAMS (University of Birmingham) was implemented for grouping adducts and isotopes, and to perform a first annotation. Annotation was completed by comparing fragmentation patterns with on-line databases as Metlin, and using the software MS-FINDER. One hundred and seventeen features, out of 3613, were statistically different among groups. We estimated that they correspond to about 80 metabolites, for which we were able to putatively annotate about half. Among these, the identification of the glucuronide conjugated of 3-hydroxycotinine supports the validity of the proposed approach. Furthermore, several metabolites, mostly as sulfate conjugates, derived from chemicals known to be present in tobacco smoke, were annotated, among which the metabolite of methoxyphenol, acrolein, 1,3-butadiene, and crotonaldeide

Untargeted metabolomics in urine to investigate smoking exposure

Background: Although thousands of different chemicals have been identified in cigarette smoke, the characterization of urinary metabolites derived from those compounds is still not completely achieved. The aim of this work was to perform an untargeted metabolomic experiment on a pilot cross-sectional study conducted on subjects with different smoking habits. Methods: Urine samples were collected from 67 adults; including 38 non-smokers, 7 electronic cigarette smokers, and 22 traditional tobacco smokers. Samples were analyzed by liquid chromatography/time-of flight mass spectrometer operating in data dependent mode. Data were processed using the R-packages IPO and XCMS to perform feature detection, retention time correction and alignment. The ANOVA test was used to detect significant features among groups. The software BEAMS (University of Birmingham) was implemented for grouping adducts and isotopes, and to perform a first annotation. Annotation was completed by comparing fragmentation patterns with on-line databases as Metlin, and using the software MS-FINDER. Results: One hundred and seventeen features, out of 3613, were statistically different among groups. We estimated that they correspond to about 80 metabolites, of which we were able to putatively annotate about half. The identification of the mercapturic acids of acrolein, 1,3-butadiene, and crotonaldeide, chemicals known to be present in tobacco smoke, supports the validity of the proposed approach. With a lower level of confidence, we annotated the glucuronide conjugated of 3-hydroxycotinine and the sulfate conjugate of methoxyphenol; finally, with the lowest degree of confidence, several other sulfate conjugates of small molecules were annotated. Short discussion/conclusions: The proposed approach seems to be useful for the investigation of exposure to toxicants in humans

Abitudine al fumo di sigaretta e profilo di escrezione di acidi mercapturici urinari : confronto tra tabacco e sigaretta elettronica

Introduzione. Nel fumo di sigaretta sono presenti moltissime molecole, centinaia delle quali riconosciute cancerogene per l\u2019uomo. Il contenuto del fumo di sigaretta elettronica \ue8 meno noto e sulla sua pericolosit\ue0 per la salute umana \ue8 in corso un acceso dibattito. Molte sostanze cancerogene contenute nel fumo di tabacco, dopo assorbimento nel nostro organismo, subiscono biotrasfomazione a dare composti elettrofili che sono ritenuti responsabili della loro genotossicit\ue0/cancerogenicit\ue0. In molti casi questi intermedi reattivi vengono eliminati nelle urine come acidi mercapturici. Scopo. Scopo di questo lavoro \ue8 stato verificare se l\u2019abitudine al fumo di tabacco e la sigaretta elettronica rappresentano sorgenti di esposizione a composti elettrofili, eliminati nell\u2019urine come acidi mercapturici (MA). Metodo. Sono stati raccolti campioni di urina di soggetti adulti con diversa abitudine al fumo di sigaretta: 22 soggetti fumatori (FT) di sigaretta tradizionale, 7 soggetti fumatori di sigaretta elettronica (FE) e 38 soggetti non fumatori (NF). Nei campioni di urina \ue8 stata ricercata la presenza di 18 acidi mercapturici derivati dalla biotrasformazione di acroleina, acrilamide, acrilonitrile, benzene, 1,3-butadiene, crotonaldeide, N,N-dimetilformammide, etilene, ossido di etilene, cloruro di vinile, ossido di propilene, stirene, toluene nonch\ue9 agenti metilanti ed etilanti. Le determinazioni sono state eseguite con un metodo basato sulla cromatografia liquida accoppiata a spettrometria di massa. Risultati. Dal confronto dei livelli di MA nei soggetti con diverse abitudini al fumo sono state evidenziate differenze significative tra fumatori e non fumatori: 11 diversi acidi mercapturici erano significativamente pi\uf9 alti (valore p 640.005) nei fumatori tradizionali rispetto ai non fumatori. I livelli mediani di MA sono risultati variare tra

Un approccio metabolomico non mirato per indagare l'esposizione a sostanze tossiche nel fumo di sigaretta

Introduzione: Nel fumo di sigaretta siano state identificate migliaia di diverse sostanze chimiche pericolose; ci\uf2 nonostante la caratterizzazione dei metaboliti urinari di queste sostanze a seguito di esposizione nell'uomo \ue8 stata effettuata sono parzialmente. Obiettivo: Lo studio si propone di applicare un approccio metabolomico non mirato all'analisi di campioni di urina di soggetti con diversa abitudine al fumo, allo scopo di identificare i metaboliti derivanti da sostanze tossiche associati. Metodi: Sono stati raccolti campioni estemporanei di urina da 67 soggetti suddivisi in tre gruppi sulla base della loro abitudine al fumo: 38 soggetti erano non fumatori, 7 erano fumatori di sigaretta elettronica e 22 erano fumatori di tabacco. I campioni sono stati analizzati utilizzando la cromatografia liquida accoppiata ad uno spettrometro di massa con tempo di volo, raccogliendo i segnali degli ioni negativi. I dati sono stati processati utilizzando i pacchetti R IPA e MXCMS per correggere i tempi di ritenzione ed effettuare l'allineamento tra i cromatogrammi. Il test ANOVA \ue8 stato utilizzato per identificare gli elementi caratteristici che distinguono tra loro i gruppi. Il software BEAMS, sviluppato dall'universit\ue0 di Birmingham, \ue8 stato applicato per raggruppare gli addotti e gli isotopi riferiti ad una stessa sostanza ed effettuare una prima annotazione dei picchi. L'annotazione \ue8 stata completata confrontando gli spettri di frammentazione ottenuti da standard puri e con il database Metlin, usando il software MS-FINDER Risultati: Nei cromatogrammi ottenuti sono stati identificati complessivamente 3613 segnali, di cui 117 sono risultati diversi nei gruppi studiati. Questi segnali sono stati attribuiti a circa 80 diversi metaboliti, dei quali siamo riusciti ad annotarne putativamente circa la met\ue0. L\u2019identificazione, con un grado di confidenza pari a 1, degli acidi mercapturici dell\u2019acroleina, del 1,3-butadiene, e della crotonaldeide, sostanze risaputamene presenti nel fumo di tabacco, supportano la validit\ue0 dell\u2019approccio adottato (il grado di confidenza 1 si attribuisce alle molecole identificate con certezza per confronto con lo standard puro). Con un grado di confidenza minore (pari a 2) sono state identificati: il coniugato glucuronide della 3-idrossicotinina e il coniugato solfato del metossifenolo. Infine, con un grado di confidenza 3, sono state identificate numerose altre piccole molecole, escrete come coniugati solfati. Conclusione: L\u2019approccio proposto sembra utile per indagare l\u2019esposizione a miscele di sostanze tossiche nell\u2019uomo. Dato che l\u2019esposizione a miscele di sostanze chimiche, piuttosto che a singoli composti, \ue8 una caratteristica peculiare di molti ambienti di lavoro, si reputa che questo approccio apra interessanti prospettive per la medicina del lavoro