Development and applications of new analytical methodologies based on HPLC-ICP-MS for trace speciation analysis of selenium in biological samples

dc.contributor.advisor	Barbante, Carlo	it_IT
dc.contributor.author	Roman, Marco <1983>	it_IT
dc.date.accessioned	2011-07-02T09:29:58Z	it_IT
dc.date.accessioned	2012-07-30T16:04:27Z
dc.date.available	2011-07-02T09:29:58Z	it_IT
dc.date.available	2012-07-30T16:04:27Z
dc.date.issued	2011-05-05	it_IT
dc.identifier.uri	http://hdl.handle.net/10579/1099	it_IT
dc.description.abstract	New methods for the determination of seleno-proteins in human plasma/serum were developed by coupling miniaturized affinity HPLC systems to ORS-ICP-MS and ICP-SFMS detectors. A methods interlaboratory comparison allowed to provide for the first time the indicative reference concentration of individual seleno-proteins in the commercially available BCR-637 human serum. Two methods for seleno-proteins speciation in plasma/serum where applied to investigate their distribution in patients affected by type II diabetes and colorectal cancer, compared to healthy individuals. A new method was then developed for the speciation analysis of Se in rat colon tissues, based on two-dimensional HPLC coupled to ICP-QMS. Five species of Se were isolated, among which glutathione peroxidases 1 and 2, and thioredoxin reductase 1 were potentially identified by MALDI-TOF-MS. The method was transferred to human samples obtaining promising preliminary results.	it_IT
dc.description.abstract	Sono stati sviluppati nuovi metodi per la determinazione delle seleno-proteine in plasma/siero umani mediante accoppiamento di sistemi miniaturizzati di HPLC di affinità e detector ORS-ICP-MS e ICP-SFMS. Un confronto interlaboratorio tra metodi ha consentito di stimare per la prima volta la concentrazione di riferimento di seleno-proteine nel siero umano commerciale BCR-637. Due metodi per la speciazione di seleno-proteine in plasma/siero sono stati applicati per investigare la loro distribuzione in pazienti affetto da diabete di tipo II e cancro colonrettale, in confronto a soggetti sani. Un nuovo metodo è stato poi sviluppato per la speciazione delle seleno-proteine in tessuti di colon di ratto, basati su HPLC bidimensionale accoppiata con ICP-QMS. Cinque specie del selenio sono state isolate, tra cui glutatione perossidasi 1 e 2, e tioredoxina reduttasi 1 sono state potenzialmente identificate mediante MALDI-TOF-MS. I metodo è stato trasferito a campioni umani ottenendo promettenti risultati preliminari.	it_IT
dc.format.medium	Tesi cartacea	it_IT
dc.language.iso	en	it_IT
dc.publisher	Università Ca' Foscari Venezia	it_IT
dc.rights	© Marco Roman, 2011	it_IT
dc.subject	Inductively coupled plasma-mass spectrometry	it_IT
dc.subject	High performance liquid chromatography	it_IT
dc.subject	Speciation analysis	it_IT
dc.subject	Selenoproteins	it_IT
dc.subject	Isotope dilution analysis	it_IT
dc.subject	Analisi di speciazione elementare	it_IT
dc.title	Development and applications of new analytical methodologies based on HPLC-ICP-MS for trace speciation analysis of selenium in biological samples	it_IT
dc.type	Doctoral Thesis	it_IT
dc.degree.name	Scienze chimiche	it_IT
dc.degree.level	Dottorato di ricerca	it_IT
dc.degree.grantor	Scuola di dottorato in Scienze e tecnologie (SDST)	it_IT
dc.description.academicyear	2009/2010	it_IT
dc.description.cycle	23	it_IT
dc.degree.coordinator	Ugo, Paolo	it_IT
dc.location.shelfmark	D001029	it_IT
dc.location	Venezia, Archivio Università Ca' Foscari, Tesi Dottorato	it_IT
dc.rights.accessrights	openAccess	it_IT
dc.thesis.matricno	955499	it_IT
dc.format.pagenumber	XIII, 352 p.	it_IT
dc.subject.miur	CHIM/01 CHIMICA ANALITICA	it_IT
dc.description.tableofcontent	I. Biochemistry of selenium 1 I.1. Introduction 1 I.2. Biochemistry of selenium 2 I.3. Selenium metabolism in mammals 3 I.4. Selenium-containing proteins 7 I.4.1. Seleno-albumin 8 I.5. Se-proteins 9 I.5.1. Glutathione peroxidases family 10 I.5.2. Thioredoxin reductases 12 I.5.3. Se-protein P 15 References 18 II. Selenium and human health 29 II.1. Introduction 29 II.2. Selenium and nutrition 30 II.2.1. Essentiality and toxicity of selenium 30 II.2.2. Assessment of the selenium nutritional status 31 II.2.3. Recommended selenium intake levels 33 II.2.4. Current selenium intake levels 35 II.2.5. Selenium supplementation 37 II.3. The role of selenium in human diseases 38 II.3.1. Muscle disorders 39 II.3.2. Cardiovascular diseases 39 II.3.3. Hepatopathies 40 II.3.4. Renal failure 40 II.3.5. Neurological disorders 40 II.3.6. Immunity defence and Inflammatory disorders 42 II.3.7. AIDS and HIV 43 II.3.8. Endocrine disorders 44 II.3.9. Male fertility 44 II.3.10. Aging 44 II.3.11. Diabetes 45 II.3.12. Cancer 47 II.4. References 50 III. Overview of the analytical techniques for speciation of selenium in biological samples 73 III.1. Introduction 73 III.2. Sample preparation 74 III.2.1. Body fluids 75 III.2.2. Tissues 75 III.3. Separation and detection techniques 78 III.3.1. HPLC hyphenated to ICP-MS 79 III.3.2. GC hyphenated to ICP-MS 83 III.3.3. CZE hyphenated to ICP-MS 83 III.3.4. Other hyphenated techniques 84 III.3.5. Non-hyphenated techniques 85 III.4. Identification techniques and integrate approaches 86 III.5. References 90 IV. Instrumentation 105 IV.1. Introduction 105 IV.2. High Performance Liquid Chromatography 105 IV.3. Inductively coupled plasma-mass spectrometry 108 IV.3.1. Principles of the method 108 IV.3.2. Sample introduction system, ionization source and interface 110 IV.3.3. Octopole reaction system 112 IV.3.4. Mass analyzer 113 IV.4. Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry 115 IV.4.1. Matrix-assisted laser desorption/ionization 115 IV.4.2. Time-of-flight mass analyzer 116 IV.5. References 119 V. Statistics for data analysis in epidemiological studies 121 V.1. Introduction 121 V.2. Normality: S-W test 121 V.3. Paired groups comparison: t-test and K-S test 122 V.4. Multiple groups comparison 124 V.4.1. Analysis of variance 124 V.4.2. Kurskal-Wallis analysis of variance 124 V.5. Correlation 125 V.5.1. Linear correlation 125 V.5.2. Spearman’s rank correlation 125 V.6. Test for confounding factors: analysis of covariance 126 V.7. Multivariate analysis 126 V.7.1. Factor analysis 126 V.8. Multiple correspondence analysis 128 V.9. Logistic Regression Analysis 129 V.10. Survival Analysis 129 V.11. References 132 VI. Methods for the quantification of selenium by ICP-MS 135 VI.1. Introduction 135 VI.2. External calibration 136 VI.3. Internal standardization 137 VI.4. Standard addition 137 VI.5. Isotope dilution analysis 138 VI.5.1. Mathematical elaborations for IDA 139 VI.5.2. Isotopically enriched spike characterization 146 VI.5.3. Estimation of the optimum spike-to-sample ratio and choice of the enriched isotope 148 VI.5.4. Detection limits comparison for external calibration and isotope dilution 151 VI.5.5. Optimization of integration time for Se isotopic ratios measurement 152 VI.6. References 153 VII. Development of analytical methodologies for Se speciation in human plasma/serum 155 VII.1. Introduction 155 VII.2. Goals of the study 156 VII.3. Plasma/serum Se-proteins separation by 2AF-HPLC 157 VII.3.1. Columns preparation 158 VII.3.2. HPLC instrumental set-up 160 VII.4. Speciation of plasma/serum Se-proteins by 2AF-HPLC-SFMS 161 VII.4.1. Instrumentation and set-up 161 VII.4.2. Optimization of ICP-SFMS sample introduction system 162 VII.4.3. Final operating conditions 164 VII.4.4. Interferences elimination by SFMS in high resolution mode 165 VII.4.5. Accuracy assesment 167 VII.4.6. Determination of columns capacity 168 VII.4.7. Determination of columns recovery 169 VII.4.8. Analytical performance characteristics 170 VII.5. Speciation of plasma/serum Se-proteins by 2AF-HPLC-ORS-QMS 171 VII.5.1. Instrumentation and set-up 171 VII.5.2. Interferences elimination by ORS technology 172 VII.5.3. Study of plasma conditions 175 VII.5.4. Final operating conditions 182 VII.6. Methods interlaboratory comparison for assessment of Se-proteins indicative concentration in BCR-637 human serum CRM 183 VII.6.1. Comparison of the analytical methods 183 VII.7. Conclusions 188 VII.8. References 189 VIII. Study of human plasma Se-proteins concentration in type II diabetes mellitus 191 VIII.1. Introduction 191 VIII.2. Goals of the study 192 VIII.3. Patients and Methods 192 VIII.3.1. Patients and study protocol 192 VIII.3.2. Determination of clinical parameters 193 VIII.3.3. Determination of plasma Se-proteins 194 VIII.3.4. Complications 194 VIII.4. Results 194 VIII.4.1. Comparison between patients with type II diabetes and healthy subjects based on their Se-proteins status 196 VIII.4.2. Association between level of plasma Se-proteins and clinical parameters 199 VIII.4.3. Association between level of plasma Se-proteins and complications 203 VIII.5. Discussion 205 VIII.6. Conclusions 207 VIII.7. References 208 IX. Study of human serum Se-proteins concentration in colorectal cancer 211 IX.1. Introduction 211 IX.2. Goals of the study 212 IX.3. Patients and methods 213 IX.3.1. Patients and study protocol 213 IX.3.2. Serum samples collection 213 IX.3.3. Determination of serum Se-proteins and quality control 215 IX.4. Results 215 IX.4.1. Comparison between CRC patients and healthy subjects based on their Se-proteins status 215 IX.4.2. Association between level of serum Se-proteins and prognostic criteria in CRC patients 219 IX.4.3. Survival analysis 220 IX.5. Discussion 221 IX.6. Conclusions 223 IX.7. References 225 X. Development of methodologies for Se-proteins speciation in rat colon tissue 229 X.1. Introduction 229 X.2. Goals of the study 230 X.3. Experimental 231 X.3.1. Instrumentation 231 X.3.2. Reagents and materials 232 X.3.3. Samples collection 234 X.3.4. Procedures 234 X.4. Results and Discussion 238 X.4.1. Total Se determination validation 238 X.4.2. Evaluation of extraction efficiency 238 X.4.3. Optimization of the extraction procedure for species preservation 240 X.4.4. Separation of Se-species by SEC-HPLC 242 X.4.5. Separation of Se-species by AE-HPLC 245 X.4.6. Separation of Se containing species by AF-HPLC and AF-CE-HPLC 247 X.4.7. Identification by MALDI-TOF 250 Conclusions 255 X.5. References 256 XI. Development of methodologies for Se-proteins speciation in human colon tissue 261 XI.1. Introduction 261 XI.2. Goals of the study 262 XI.3. Experimental 262 XI.3.1. Instrumentation, reagents and materials 262 XI.3.2. Samples 263 XI.3.3. Procedures 263 XI.4. Results and Discussion 265 XI.4.1. Separation of Se-species by SEC-HPLC 265 XI.4.2. Separation of Se-species by AE-HPLC 267 XI.4.3. Separation of Se species by AF-HPLC 269 XI.4.4. Se-species quantification 270 XI.4.5. Chromatographic method characterization 270 XI.5. Conclusions 273 XI.6. References 274 General conclusions 277 Published Articles 281 Conference Acts 317 Attachments 319 References 334 Acknowledgements 351	it_IT
dc.identifier.bibliographiccitation	Roman, Marco. “Development and applications of new analytical methodologies based on HPLC-ICP-MS for trace speciation analysis of selenium in biological samples”, Ca' Foscari University of Venice, PhD Thesis, 23. cycle, 2011	it_IT
dc.degree.discipline	Spettrometria di massa	it_IT
dc.degree.discipline	Proteomica	it_IT