Applicazione della spettrometria di massa nello studio delle pneumopatie ostruttive del bambino

Abstract Background and purposes The obstructive pulmonary diseases in the world – among which asthma and the chronic obstructive pulmonary disease (COPD) – have been growing constantly. In 2006 about 46 million persons suffered from asthma, and 9.5 millions were children; the COPD has also been growing constantly and, despite the fact that the incidence increases with age, it looks like several factors and previous childhood diseases can influence the subjects’ respiratory function and the evolution of the disease. Despite the many studies that have been carried out, it is not clear yet what are the mechanisms on which these respiratory diseases are based, and therefore further investigations must be made. Mass spectrometry, a sensitive and robust analytic technique, has found more and more applications in the clinical-diagnostic field, thanks to its capacity to determine the metabolites in the biological samples with a mass value or to identify their structure through fragmentation with extremely sensitive, specific and selective characteristics. The possibility to analyze complex blends, thanks to combination with gas or liquid chromatographic techniques, reduced analysis time, the qualitative identification but above all the quantitative dosages of several biomarkers in a single analysis, has made this technique preferable for clinical studies. Especially, besides the classical target approach used to quantify already known or presumably proper to certain pathologies specific compounds, over the past years a new type of approach has developed, the so called untargeted approach, known as “metabolomic”, which sees the study of the entire metabolic profile of a biological sample, by means of highly sensitive and precise analytic techniques, such as high resolution mass spectrometry (HMS). This approach allows the characterization of the general metabolic profile of a group of subjects with a specific pathology as compared to a group of control and extracts the discriminating variables, thanks to multivariate statistic analysis techniques. The untargeted analysis, which is not based upon prior hypotheses, can open the doors to new etiopathological hypotheses, besides helping identify the diagnostic and prognostic biomarkers. The purpose of this PhD thesis was to study the infant obstructive respiratory diseases by applying both of these approaches to urine or exhaled breath condensate (EBC) samples. The EBC is a biofluid which, given the extremely non invasive method of collecting it, represents an ideal biological matrix for studies of the kind, but, since the metabolite concentration is extremely low, is at the same time an analytically complex matrix. Methods and results In the first study, the HRMS metabolomic analysis of the EBC was employed to investigate upon the biochemical-metabolic profile of the EBCs of teenagers diagnosed with bronchopulmonary dysplasia (BPD)from birth as compared to a group of healthy teenagers. The samples were analyzed with the LTQ-OrbiTrap mass spectrometer and processed by means of the multivariate statistic analysis. The results have demonstrated that it is possible to discriminate the BPD group from the control group by marking out the EBC samples, obtaining a robust OPLS-DA model (R2: 0.95, Q2:0. 82), indicating how the teenagers who were affected by BPD from birth have a different biochemical-metabolic profile. The individuation of the glycerophospholipids, among the several discriminating variables, has suggested a possible involvement of the alveolar surfactant at the basis of the minor respiratory function in the BPD affected patients, even years later after the severe phase of the disease. The second study employed the HRMS metabolomic in order to obtain a first biochemical-metabolic characterization of children with frequent bronchospasm or with wheezing in preschool age, in view of a perspective study which would help appreciate which of these children will develop asthma when growing up, and, a posteriori, consider whether prognostic biomarkers exist. The preliminary results of this part of the study have been encouraging: thanks to the analysis of the urine samples with the hybrid Q-TOF mass spectrometer associated to the UPLC, we have had a clear distinction of the subjects affected by wheezing as compared to healthy children, through an OPLSA- DA model. The preliminary analysis has allowed to obtain a clear distinction between children who suffered from wheezing and healthy children, highlighting some discriminating molecules. The study is still ongoing since it presupposes the evaluation of the same subjects 18 and 36 months later. The third part of the study was related to the use of the target MS and was meant to evaluate and quantify some oxidative stress indicators in EBC samples belonging to subjects affected by asthma. A UPLC-MS/MS method was developed and validated for the quantitative analysis of the dimethyl arginine, ADMA and SDMA, in samples of EBC. The analysis was conducted by means of MRM technique in quadrupole mass spectrometry associated to UPLC, with a on line sample enrichment system. The analytic system has come out robust (r2>0.992, %Bias <3% intra- CV% inter-intra assay =20%, recovery between 97 and 102%), rapid (5 minutes chromatographic run) and sensitive, also suitable for the analysis of diluted biological samples, such as the EBCs. The quantification of these metabolites, conducted on samples of asthmatic and healthy children, has pointed out a rise of the ADMA in the EBCs of the asthmatic subjects, indicating a role of the ADMA in the tissue damage of the respiratory tract which typically characterizes the bronchial asthma. Conclusions This research has demonstrated that instrumentally highly sensitive mass spectrometry, which has allowed us to obtain significant information even from small quantities of samples and from extremely diluted matrixes, can be successfully applied to the study of obstructive respiratory diseases, making the follow up studies easier, and also helping medical doctors identify possible markers useful to the diagnosis and/or new therapeutic targets

Neonatal Urine Metabolic Profiling and Development of Childhood Asthma

none9Urine metabolomics case-control studies of childhood asthma have demonstrated a discriminative ability. Here, we investigated whether urine metabolic profiles from healthy neonates were associated with the development of asthma in childhood. Untargeted metabolomics by liquid chromatography-mass spectrometry was applied to urine samples collected at age 4 weeks in 171 and 161 healthy neonates born from mothers with asthma from the COPSAC2000 and COPSAC2010 cohorts, respectively, where persistent wheeze/asthma was prospectively diagnosed using a symptom-based algorithm. Univariate and multivariate analyses were applied to investigate differences in metabolic profiles between children who developed asthma and healthy children. Univariate analysis showed 63 and 87 metabolites (q-value 0.60. Database search enabled annotation of three discriminative features: a glucoronidated compound (steroid), 3-hydroxytetradecanedioic acid (fatty acid), and taurochenodeoxycholate-3-sulfate (bile acid). The urine metabolomics profiles from healthy neonates were associated with the development of childhood asthma, but further research is needed to understand underlying metabolic pathways.noneChawes, Bo L; Giordano, Giuseppe; Pirillo, Paola; Rago, Daniela; Rasmussen, Morten A; Stokholm, Jakob; Bønnelykke, Klaus; Bisgaard, Hans; Baraldi, EugenioChawes, Bo L; Giordano, Giuseppe; Pirillo, Paola; Rago, Daniela; Rasmussen, Morten A; Stokholm, Jakob; Bønnelykke, Klaus; Bisgaard, Hans; Baraldi, Eugeni

Spectrum of mutations in Italian patients with familial hypercholesterolemia: New results from the LIPIGEN study

Background Familial hypercholesterolemia (FH) is an autosomal dominant disease characterized by elevated plasma levels of LDL-cholesterol that confers an increased risk of premature atherosclerotic cardiovascular disease. Early identification and treatment of FH patients can improve prognosis and reduce the burden of cardiovascular mortality. Aim of this study was to perform the mutational analysis of FH patients identified through a collaboration of 20 Lipid Clinics in Italy (LIPIGEN Study). Methods We recruited 1592 individuals with a clinical diagnosis of definite or probable FH according to the Dutch Lipid Clinic Network criteria. We performed a parallel sequencing of the major candidate genes for monogenic hypercholesterolemia (LDLR, APOB, PCSK9, APOE, LDLRAP1, STAP1). Results A total of 213 variants were detected in 1076 subjects. About 90% of them had a pathogenic or likely pathogenic variants. More than 94% of patients carried pathogenic variants in LDLR gene, 27 of which were novel. Pathogenic variants in APOB and PCSK9 were exceedingly rare. We found 4 true homozygotes and 5 putative compound heterozygotes for pathogenic variants in LDLR gene, as well as 5 double heterozygotes for LDLR/APOB pathogenic variants. Two patients were homozygous for pathogenic variants in LDLRAP1 gene resulting in autosomal recessive hypercholesterolemia. One patient was found to be heterozygous for the ApoE variant p.(Leu167del), known to confer an FH phenotype. Conclusions This study shows the molecular characteristics of the FH patients identified in Italy over the last two years. Full phenotypic characterization of these patients and cascade screening of family members is now in progress

Twelve Variants Polygenic Score for Low-Density Lipoprotein Cholesterol Distribution in a Large Cohort of Patients With Clinically Diagnosed Familial Hypercholesterolemia With or Without Causative Mutations

: Background A significant proportion of individuals clinically diagnosed with familial hypercholesterolemia (FH), but without any disease-causing mutation, are likely to have polygenic hypercholesterolemia. We evaluated the distribution of a polygenic risk score, consisting of 12 low-density lipoprotein cholesterol (LDL-C)-raising variants (polygenic LDL-C risk score), in subjects with a clinical diagnosis of FH. Methods and Results Within the Lipid Transport Disorders Italian Genetic Network (LIPIGEN) study, 875 patients who were FH-mutation positive (women, 54.75%; mean age, 42.47±15.00 years) and 644 patients who were FH-mutation negative (women, 54.21%; mean age, 49.73±13.54 years) were evaluated. Patients who were FH-mutation negative had lower mean levels of pretreatment LDL-C than patients who were FH-mutation positive (217.14±55.49 versus 270.52±68.59 mg/dL, P<0.0001). The mean value (±SD) of the polygenic LDL-C risk score was 1.00 (±0.18) in patients who were FH-mutation negative and 0.94 (±0.20) in patients who were FH-mutation positive (P<0.0001). In the receiver operating characteristic analysis, the area under the curve for recognizing subjects characterized by polygenic hypercholesterolemia was 0.59 (95% CI, 0.56-0.62), with sensitivity and specificity being 78% and 36%, respectively, at 0.905 as a cutoff value. Higher mean polygenic LDL-C risk score levels were observed among patients who were FH-mutation negative having pretreatment LDL-C levels in the range of 150 to 350 mg/dL (150-249 mg/dL: 1.01 versus 0.91, P<0.0001; 250-349 mg/dL: 1.02 versus 0.95, P=0.0001). A positive correlation between polygenic LDL-C risk score and pretreatment LDL-C levels was observed among patients with FH independently of the presence of causative mutations. Conclusions This analysis confirms the role of polymorphisms in modulating LDL-C levels, even in patients with genetically confirmed FH. More data are needed to support the use of the polygenic score in routine clinical practice

Sistemi a biomasse per l'abbattimento dell'inquinamento puntiforme da fitofarmaci e determinazione del Koc: terbutilazina

studio dell'efficacia di tre biobed sperimentali per l'abbattimento dell'inquinamento puntiforme studiando l'andamento della terbutilazina in due anni di sperimentazion

Applicazione della spettrometria di massa nello studio delle pneumopatie ostruttive del bambino

Abstract Background and purposes The obstructive pulmonary diseases in the world – among which asthma and the chronic obstructive pulmonary disease (COPD) – have been growing constantly. In 2006 about 46 million persons suffered from asthma, and 9.5 millions were children; the COPD has also been growing constantly and, despite the fact that the incidence increases with age, it looks like several factors and previous childhood diseases can influence the subjects’ respiratory function and the evolution of the disease. Despite the many studies that have been carried out, it is not clear yet what are the mechanisms on which these respiratory diseases are based, and therefore further investigations must be made. Mass spectrometry, a sensitive and robust analytic technique, has found more and more applications in the clinical-diagnostic field, thanks to its capacity to determine the metabolites in the biological samples with a mass value or to identify their structure through fragmentation with extremely sensitive, specific and selective characteristics. The possibility to analyze complex blends, thanks to combination with gas or liquid chromatographic techniques, reduced analysis time, the qualitative identification but above all the quantitative dosages of several biomarkers in a single analysis, has made this technique preferable for clinical studies. Especially, besides the classical target approach used to quantify already known or presumably proper to certain pathologies specific compounds, over the past years a new type of approach has developed, the so called untargeted approach, known as “metabolomic”, which sees the study of the entire metabolic profile of a biological sample, by means of highly sensitive and precise analytic techniques, such as high resolution mass spectrometry (HMS). This approach allows the characterization of the general metabolic profile of a group of subjects with a specific pathology as compared to a group of control and extracts the discriminating variables, thanks to multivariate statistic analysis techniques. The untargeted analysis, which is not based upon prior hypotheses, can open the doors to new etiopathological hypotheses, besides helping identify the diagnostic and prognostic biomarkers. The purpose of this PhD thesis was to study the infant obstructive respiratory diseases by applying both of these approaches to urine or exhaled breath condensate (EBC) samples. The EBC is a biofluid which, given the extremely non invasive method of collecting it, represents an ideal biological matrix for studies of the kind, but, since the metabolite concentration is extremely low, is at the same time an analytically complex matrix. Methods and results In the first study, the HRMS metabolomic analysis of the EBC was employed to investigate upon the biochemical-metabolic profile of the EBCs of teenagers diagnosed with bronchopulmonary dysplasia (BPD)from birth as compared to a group of healthy teenagers. The samples were analyzed with the LTQ-OrbiTrap mass spectrometer and processed by means of the multivariate statistic analysis. The results have demonstrated that it is possible to discriminate the BPD group from the control group by marking out the EBC samples, obtaining a robust OPLS-DA model (R2: 0.95, Q2:0. 82), indicating how the teenagers who were affected by BPD from birth have a different biochemical-metabolic profile. The individuation of the glycerophospholipids, among the several discriminating variables, has suggested a possible involvement of the alveolar surfactant at the basis of the minor respiratory function in the BPD affected patients, even years later after the severe phase of the disease. The second study employed the HRMS metabolomic in order to obtain a first biochemical-metabolic characterization of children with frequent bronchospasm or with wheezing in preschool age, in view of a perspective study which would help appreciate which of these children will develop asthma when growing up, and, a posteriori, consider whether prognostic biomarkers exist. The preliminary results of this part of the study have been encouraging: thanks to the analysis of the urine samples with the hybrid Q-TOF mass spectrometer associated to the UPLC, we have had a clear distinction of the subjects affected by wheezing as compared to healthy children, through an OPLSA- DA model. The preliminary analysis has allowed to obtain a clear distinction between children who suffered from wheezing and healthy children, highlighting some discriminating molecules. The study is still ongoing since it presupposes the evaluation of the same subjects 18 and 36 months later. The third part of the study was related to the use of the target MS and was meant to evaluate and quantify some oxidative stress indicators in EBC samples belonging to subjects affected by asthma. A UPLC-MS/MS method was developed and validated for the quantitative analysis of the dimethyl arginine, ADMA and SDMA, in samples of EBC. The analysis was conducted by means of MRM technique in quadrupole mass spectrometry associated to UPLC, with a on line sample enrichment system. The analytic system has come out robust (r2>0.992, %Bias <3% intra- CV% inter-intra assay =20%, recovery between 97 and 102%), rapid (5 minutes chromatographic run) and sensitive, also suitable for the analysis of diluted biological samples, such as the EBCs. The quantification of these metabolites, conducted on samples of asthmatic and healthy children, has pointed out a rise of the ADMA in the EBCs of the asthmatic subjects, indicating a role of the ADMA in the tissue damage of the respiratory tract which typically characterizes the bronchial asthma. Conclusions This research has demonstrated that instrumentally highly sensitive mass spectrometry, which has allowed us to obtain significant information even from small quantities of samples and from extremely diluted matrixes, can be successfully applied to the study of obstructive respiratory diseases, making the follow up studies easier, and also helping medical doctors identify possible markers useful to the diagnosis and/or new therapeutic targets.Background e obiettivi Le pneumopatie ostruttive nel mondo, tra cui asma e bronco pneumopatia cronica ostruttiva (COPD), sono in continua crescita. Nel 2006 circa 46 milioni di persone erano affette da asma, di cui 9.5 milioni erano bambini; anche la COPD è in continuo aumento e, anche se l’incidenza cresce con l’età anagrafica, sembra che diversi fattori e malattie pregresse dell’infanzia, possano influenzare la funzionalità respiratoria dei soggetti e l’evolvere della patologia. Nonostante i molti studi condotti, i meccanismi alla base di queste malattie respiratorie non sono ancora del tutto chiariti, e necessitano pertanto di ulteriori indagini. La spettrometria di massa, tecnica analitica sensibile e robusta, trova sempre più applicazioni in ambito clinico-diagnostico, grazie alla capacità di determinare i metaboliti presenti nei campioni biologici dai valori di massa o identificarne la struttura tramite frammentazione con caratteristiche di estrema sensibilità, specificità e selettività. La possibilità di analisi di miscele complesse, grazie all’accoppiamento con tecniche cromatografiche gassose o liquide, i tempi di analisi ridotti, l’identificazione qualitativa ma soprattutto i dosaggi quantitativi anche di numerosi biomarcatori in una singola analisi, ha reso questa una tecnica di elezione per studi clinici. In particolare oltre al classico approccio target per quantificare specifici composti già noti o che si suppone siano caratteristici di una patologia, negli ultimi anni si è sviluppato un nuovo tipo di approccio untargeted, noto come “metabolomica”, che vede lo studio dell’intero profilo metabolico di un campione biologico, attraverso tecniche analitiche altamente sensibili e ad elevata capacità, quali la spettrometria di massa ad alta risoluzione (HMS). Tramite questo approccio è possibile caratterizzare il profilo metabolico generale di un gruppo di soggetti con una determinata patologia rispetto ad un gruppo di controllo ed estrapolare le variabili discriminanti, grazie a tecniche statistiche di tipo multivariato. L’analisi untarget, non basata su ipotesi a priori, può aprire la strada a nuove ipotesi eziopatologiche oltre ad aiutare nell’identificazione di biomarkers diagnostici e prognostici. Obiettivo di questo dottorato era studiare le pneumopatie ostruttive in età pediatrica mediante l’applicazione di entrambi questi approcci, su campioni di urine o condensato di aria espirata (EBC). L’EBC è un biofluido che, data la sua raccolta assolutamente non invasiva, costituisce una matrice biologica ideale per studi di questo tipo, ma essendo la concentrazione dei metaboliti estremamente bassa, risulta al tempo stesso una matrice analiticamente complessa. Metodi e risultati Nel primo studio, l’analisi metabolomica HMS dell’EBC è stata impiegata per indagare il profilo biochimico-metabolico degli EBC di adolescenti con diagnosi di broncodisplasia alla nascita (BPD) rispetto a quello di un gruppo di adolescenti sani. I campioni sono stati analizzati con lo spettrometro di massa LTQ-OrbiTrap e processati tramite analisi statistica multivariata. I risultati ottenuti hanno mostrato che è possibile discriminare il gruppo BPD dal gruppo di controllo attraverso la caratterizzazione dei campioni di EBC, ottenuta con un modello OPLS-DA robusto (R2:0.95, Q2:0.82 ), mostrando un differente profilo biochimico-metabolico negli adolescenti con BPD alla nascita. L’individuazione di glicerofosfolipidi, tra le variabili discriminanti, ha suggerito un possibile coinvolgimento del surfattante alveolare alla base della minore funzionalità respiratoria dei soggetti con BPD, anche dopo anni dalla fase acuta della malattia. Il secondo studio ha previsto l’utilizzo della metabolomica HRMS per ottenere una prima caratterizzazione biochimica-metabolica dei bambini con broncospasmo ricorrente o wheezing in età prescolare, in vista di uno studio prospettico per valutare quali di questi bambini svilupperà asma con la crescita, e considerare a posteriori se esistano dei biomarkers prognostici. I risultati preliminari di questa parte dello studio sono stati promettenti: grazie all’analisi di campioni di urina con lo spettrometro di massa ibrido Q-TOF associato ad UPLC, si è ottenuta une netta differenziazione dei soggetti con wheezing rispetto ai bambini sani, mediante un modello OPLSA- DA. L’analisi preliminare ha permesso di ottenere una netta separazione tra bambini con wheezing e bambini sani, mettendo in evidenza alcune molecole discriminanti. Lo studio è ancora in corso in quanto prevede la valutazione degli stessi soggetti a 18 e 36 mesi. La terza parte dello studio relativa all’utilizzo della MS target prevedeva la valutazione e quantificazione di alcuni indicatori di stress ossidativo in campioni di EBC di soggetti asmatici. È stato sviluppata e validata una metodica UPLC-MS/MS per l’analisi quantitativa di dimetilarginine, ADMA e SDMA, in campioni di EBC. L’analisi è stata condotta tramite tecnica MRM in spettrometria di massa quadrupolare associata ad UPLC, con sistema di arricchimento on-line del campione. Il sistema analitico è risultato robusto (r2>0.992, %Bias <3% intra- CV% inter-intra assay =20%, recovery% tra 97 e 102%), rapido (corsa cromatografica di 5 minuti), e sensibile, adatto anche ad analisi di campioni biologici diluiti quali gli EBC. La quantificazione di tali metaboliti condotta su campioni di bambini asmatici e sani, ha mostrato un aumento di ADMA negli EBC dei soggetti asmatici, indicando un ruolo dell’ADMA nel danno tissutale delle vie aeree tipico dell’asma bronchiale. Conclusioni Questa ricerca ha dimostrato che la spettrometria di massa ad elevata sensibilità strumentale, grazie alla quale è stato possibile ottenere informazioni significative anche da piccole quantità di campione e da matrici estremamente diluite, può essere applicata con buoni risultati alla studio di pneumopatie ostruttive, rendendo più facili studi di follow up, oltre che aiutare il medico nell’individuare possibili marcatori utili alla la diagnosi e/o nuovi target terapeutici

Airway metabolic anomalies in Adolescents with Bronchopulmonary Dysplasia: new insights from the metabolomic approach

Objectives To assess a group of adolescents with bronchopulmonary dysplasia (BPD) from a biochemicalmetabolic standpoint, applying the metabolomic approach to studying their exhaled breath condensate (EBC). Study design Twenty adolescents with BPD (mean age 14.8 years) and 15 healthy controls (mean age 15.2 years) were recruited for EBC collection, exhaled nitric oxide measurement, and spirometry. The EBC samples were analyzed using a metabolomic approach based on mass spectrometry. The obtained spectra were analyzed using multivariate statistical analysis tools. Results A reliable Orthogonal Projections to Latent Structures-Discriminant Analysis model showed a clear discrimination between cases of BPD and healthy controls (R2 = 0.95 and Q2 = 0.92). The search for putative biomarkers identified an altered complex lipid profile in the adolescents with BPD. Conclusions The metabolomic analysis of EBC distinguishes cases of BPD from healthy individuals, suggesting that the lung of survivors of BPD is characterized by long-term metabolic abnormalities. The search for putative biomarkers indicated a possible role of an altered surfactant composition, which may persist far beyond infancy.JRC.F.4-Fraud Detection and Preventio

Metabolomic Profile of Amniotic Fluid and Wheezing in the First Year of Life—A Healthy Birth Cohort Study

Objectives: To apply metabolomic analysis of amniotic fluid in a discovery cohort to see whether a specific biochemical-metabolic profile at birth is associated with the subsequent onset of wheezing over the first year of life. Study design: This prospective exploratory study was conducted in a healthy term-born Dutch cohort recruited at 2 hospitals in Utrecht (UMCU, Utrecht, and Diakonessenhuis, Utrecht), The Netherlands. A metabolomic approach based on mass spectrometry was applied to analyze 142 amniotic fluid samples collected at birth. The infants were followed up during their first year of life with recording any respiratory symptoms daily, and they were classified according to the onset of wheezing. Results: Orthogonally constrained projection to latent structures discriminant analysis was used to investigate differences in the metabolic profiles of the infants with (n = 86) and without (n = 56) wheezing. A search of the available databases for amniotic fluid metabolites identified by stability selection, combined with pathway analysis, highlighted the possible metabolic perturbations involved in this condition. The model built using 16 relevant variables with plausible biological significance, showed an area under the curve of 0.82 (P <.001) and an area under the curve calculated by 7-fold full cross-validation of 0.72 (P =.003), with the steroid hormone biosynthesis and the 2-phenylalanine metabolism emerging as probably perturbed pathways. Conclusions: Infants who will or will not experience wheezing in their first year of life have distinct amniotic fluid metabolomic profiles at birth. Changes occurring in biochemical-metabolic pathways in late intrauterine life may have a pathogenic role in early-onset wheezing