Arsenic speciation in biological samples using high performance liquid chromatography (HPLC) coupled with inductively coupled plasma mass spectrometry (ICP-MS) and investigation of cellular uptake capabilities of arsenicals in different cell lines [Elektronische Ressource] / von Sasan Rabieh

Mozhgan

Sujets

• Biologie

Informations

• Publié par: universitat_duisburg-essen
• Publié le: 01 janvier 2007
• Nombre de lectures: 13
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Arsenic speciation in biological samples using high
performance liquid chromatography (HPLC) coupled with
inductively coupled plasma mass spectrometry (ICP-MS)
and
Investigation of cellular uptake capabilities of arsenicals in
different cell lines







Dissertation
zur Erlangung des Grades
“Doktor der Naturwissenschaften”
(Dr. rer. nat.)




am Fachbereich Chemie, Institut für Umweltanalytik
der Universität Duisburg-Essen
Essen, Deutschland




von

Sasan Rabieh, BSc, MSc (Hons)
geboren in Ahvaz, Iran





Essen, März 2007
































1.Gutachter: Prof. Dr. Alfred V. Hirner
2.Gutachter: Prof. Dr. Albert W. Rettenmeier
Vorsitzender des Prüfungsausschusses: Prof. Dr. R. Zellner

Tag der Disputation: 08. Mai 2007










DEDICATION





This thesis is dedicated to:



My supervisor, Prof. Dr. Alfred V. Hirner,

My parents,

&

My wife












Acknowledgements

I wish to express my gratitude to my supervisor, Prof. Dr. Alfred V. Hirner, for all his
support, friendship, advice, excellent guidance, and valuable discussion throughout the entire
projects of studies, particularly during the time of preparing the manuscripts and thesis. Dear
Alfred, without your extensive support and dedication I would not be where I am today. Once
again, thank you for everything.
I would also like to thank Prof. Dr. Albert W. Rettenmeier, my co-referee, for his
comments and advice in an attempt to improve the text of my thesis and also for an
opportunity to cooperate with his institute.
Thanks are also in order to Dr. Louise Hartmann, for all her support, assistance, advice,
and motivation during my studies.
I would also like to extend my gratitude to PD Dr. Elke Dopp for her excellent
cooperation, continuous support, and valuable discussion throughout our projects.
Many thanks to Prof. Dr. Jörg Matschullat (University of Mining and Technology,
Freiberg, Germany) for providing the Brazilian urine samples.
Special thanks to my colleagues, Roland Diaz-Bone and Jörg Hippler for all their help
and support since my arrival in Germany. I will never forget your much appreciated kindness
and assistance.
I would also like to thank other colleagues, Joachim, Jan, Lars, Jens, Frank, Sebastian,
Marc, Maria, Martin, Margareta and all other members of our institute for their support, and
help during my studies.
My special thanks to Dr. Ursula von Recklinghausen, Frau Zimmer, and Frau
Zimmermann at the Institute of Hygiene and Occupational Medicine, University Hospital of
Essen for all their cooperation and assistance throughout our projects, which contributed
greatly to their success.
I also have to thank Prof. Dr. Jörg Feldmann and his entire group at University of
Aberdeen, Scotland while visiting them.
I would also like to take this opportunity to thank Frau Birgit Wöstefeld for all her help
and assistances that she afforded me.
Financial support from the Deutsche Forschungsgemeinschaft (DFG), German research
foundation, is highly appreciated.
Lastly, I would like to express my sincere thanks to my parents, brothers, and sisters
for all their continuous support and encouragement as well as my wife, Mozhgan, for all her
support, encouragement, and love.
CONTENTS
List of Figures .........................................................................................................................IV
List of Tables...................................................................................................VI
Abbreviations.......................................................................................................................VIII
Publications.............................XI
1 Introduction ........................................................................................................................... 1
1.1 Inductively Coupled Plasma Mass Spectrometry in Environmental Analysis.......... 2
1.1.1 Instrumentation.................................................................................. 2
1.2 Liquid Chromatography ........................................................................................................ 4
1.2.1 Effecting Variables in Liquid Chromatography Separation...................................... 4
1.2.2 Characteristic Parameters of a Chromatogram.......................................... 5
1.2.3 Liquid Chromatographic Stationary Phases .............................................................. 6
1.2.3.1 Normal Phase Chromatography (NPC) 6
1.2.3.2 Reversed Phase Chromatography (RPC) .................................... 6
1.2.3.3 Reversed Phase Ion Pair Chromatography (IPC)............................................... 7
1.2.3.4 Ion Exchange Chromatography (IEC)........................ 7
1.3 High Performance Liquid Chromatography (HPLC)..................................................... 8
1.3.1 Instrumentation.......................................................................................................... 8
1.4 Hyphenated (Coupling) Techniques.................................................................................... 9
1.5 Definition of Metal Speciation .............................................................................................10
1.5.1 Sources of Arsenic in the Environment................................................................... 10
1.5.2 Biomethylation of Arsenic .............................................................. 12
1.5.3 Cytotoxicity and Toxicity................................................................ 14
1.5.4 Methylated Metabolites of Arsenic .......... 16
1.5.4.1 MMAs(III)................................................................................ 16
1.5.4.2 DMAs(III)........................ 16
1.5.4.3 TMAs(III)................................................................................. 17
1.5.5 Arsenic and Metabolites in Urine............. 17
1.6 Cell Biology ..............................................................................................................................18
1.6.1 Cell Membrane ........................................................................................................ 18
1.6.2 Organelles................................................................................................................ 18
1.6.2.1 Cell Nucleus .............................. 19
1.6.2.2 Ribosomes ......................................................................................... 19
1.6.2.3 Mitochondria ............................................................................ 19
1.6.2.4 Lysosomes ................................. 19
1.6.3 Movements of Substances Across Membranes....................................................... 19
1.7 Objective of the Present Work.............................................................................................20
2 Material and Methods......................................................................................................... 22
2.1 Chemicals and Reagents........................................................................................................22
2.2 Urine Samples..........................................................................................................................22
2.3 Cell Lines and Cell Culture Conditions.............................................................................23
2.3.1 Human UROtsa Cells .............................................................................................. 23
2.3.2 CHO-9 Cells ............................................. 24
2.3.3 HeLa S3 Cells.................................................................................. 24
I2.3.4. Hep G2 Cells .......................................................................................................... 25
2.4 Cellular Uptake of Arsenic by Different Cell Lines.......................................................25
2.4.1 Association of Arsenic with Intracellular Organelles in UROtsa Cells .................. 26
2.5 Instrumentation.......................................................................................................................29
3 Results................................................................................................................................... 31
3.1 Optimization of the HPLC Parameters .............................................................................31
3.1.1 Optimization of pH.......................................................................... 31
3.1.2 Optimization of the Concentration of TBAH........