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Invertebrates Analysis by Capillary Electrophoresis

Permanent Link: http://ufdc.ufl.edu/UFE0024340/00001

Material Information

Title: Invertebrates Analysis by Capillary Electrophoresis
Physical Description: 1 online resource (133 p.)
Language: english
Creator: Sohn, Do
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: capillary, invertebrates, neurotransmitters
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Nervous systems are of biological machines, where have evolved over millions of years. Thus, tracking the lineages and constraints that have molded nervous systems provides an opportunity to understand signal molecules and their metabolites in variety of marine organisms having key positions in the evolutionary tree of life. In particular basal animals, including placozoa, ctenophores, cnidarians, sponges relatively simple organization compared to other known animals. While there have been extensive studies on genome, physiology, histochemistry, and regeneration, direct microchemical data are limited on basal animals. In order to provide direct evidence for the presence of neurotransmitters and their metabolites, we have identified and characterized major low molecular weight potential signaling molecules and their metabolites in marine organisms. Capillary electrophoresis (CE) techniques with laser-induced fluorescence (LIF) and contactless conductivity detection (CCD) were used for in-depth studies of the metabolites of the neurotransmitters and nitric oxide (NO). Since NO is a highly reactive signaling molecule, indirect detection methods were used. For example, L-Arginine and L-Citrulline, a precursor and co-product of NO, respectively, were analyzed with CE-LIF. Also, nitrite and nitrate, both major oxidation products of NO, were analyzed with CE-CCD. The results of this study will be used in conjunction with key research questions to fill numerous gaps in our understanding of diversity and evolution of signal molecules and the development of integrative systems in animals.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Do Sohn.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Tan, Weihong.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024340:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024340/00001

Material Information

Title: Invertebrates Analysis by Capillary Electrophoresis
Physical Description: 1 online resource (133 p.)
Language: english
Creator: Sohn, Do
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: capillary, invertebrates, neurotransmitters
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Nervous systems are of biological machines, where have evolved over millions of years. Thus, tracking the lineages and constraints that have molded nervous systems provides an opportunity to understand signal molecules and their metabolites in variety of marine organisms having key positions in the evolutionary tree of life. In particular basal animals, including placozoa, ctenophores, cnidarians, sponges relatively simple organization compared to other known animals. While there have been extensive studies on genome, physiology, histochemistry, and regeneration, direct microchemical data are limited on basal animals. In order to provide direct evidence for the presence of neurotransmitters and their metabolites, we have identified and characterized major low molecular weight potential signaling molecules and their metabolites in marine organisms. Capillary electrophoresis (CE) techniques with laser-induced fluorescence (LIF) and contactless conductivity detection (CCD) were used for in-depth studies of the metabolites of the neurotransmitters and nitric oxide (NO). Since NO is a highly reactive signaling molecule, indirect detection methods were used. For example, L-Arginine and L-Citrulline, a precursor and co-product of NO, respectively, were analyzed with CE-LIF. Also, nitrite and nitrate, both major oxidation products of NO, were analyzed with CE-CCD. The results of this study will be used in conjunction with key research questions to fill numerous gaps in our understanding of diversity and evolution of signal molecules and the development of integrative systems in animals.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Do Sohn.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Tan, Weihong.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024340:00001


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INVERTEBRATES ANALYSIS BY CAPILLARY ELECTROPHORESIS By DOSUNG SOHN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009 1

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2009 Dosung Sohn 2

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To my family, Youngmi, Mia, and Aiden 3

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ACKNOWLEDGMENTS I am really grateful that I ha d the opportunity to have Dr. We ihong Tan as my PhD advisor. Working under his supervision has been an invaluab le experience. His hard work, determination, commitment to the advancement of science, and kindness has been very inspiring to me. I thank him for his patience, encourag ement, and clever suggestions during my five-year doctoral research. I am also very grat eful to my graduate committee me mbers, Dr. Leonid L. Moroz, Dr. Charles Martin, Dr. Ben Smith, and Dr. Nico le A. Horenstein for their valuable recommendations. I highly appreciate Dr. Moroz s friendship and generosity. He always put me in challenging tasks and gives me a chance to devel op myself toward to a creative researcher. His smile and kind comments filled my heart with great joy many times. I am very pleased with my lab mates, Thomas Ha, Sami Jezzni, and Colin Medlin Their enthusiasm, happiness, good hearts, and commitment to research provide the lab with a nice environment, where anybody would feel welcome and prompted to work. I also thank my close friends whose names are too many to list. Their friendship made my stayi ng in this country, aw ay from my family and loved ones, much more pleasant than it would have been wit hout them. I was also supported by my mother, Youngja Lee, my Father, Dongw oon Sohn, and my sisters. Finally, I am indebted to the Department of Chemistry at University of Florida in Gainesville for giving me the opportunity to purs ue a PhD degree. The financial support from the National Institutes of Health is also gratefully acknowledged. 4

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4LIST OF TABLES ...........................................................................................................................8LIST OF FIGURES .........................................................................................................................9ABSTRACT ...................................................................................................................... .............11 CHAPTER 1 INTRODUCTION ................................................................................................................ ..13Historical Background ............................................................................................................13Separation Technology for Metabolomics ..............................................................................14Separation Methods .........................................................................................................14Detection Methods ...........................................................................................................15Capillary Electrophoresis and its Application to Metabolomics ............................................16Capillary Electrophoresis Fundamentals .........................................................................16Capillary Zone Electrophoresis .......................................................................................16Micellar Electrokinetic Chromatography (MEKC) .........................................................20Chiral Separation by CE ..................................................................................................24Capillary Isotachophoresis (ITP) .....................................................................................27Capillary Electrochromatography ...................................................................................28Capillary Gel Electrophoresis ..........................................................................................29Capillary Isoelectric Focusing .........................................................................................29Fluorescence Detection in CE ................................................................................................29Conductivity Detection in CE .................................................................................................3 2Basic Principles ...............................................................................................................32Contactless Conductivity Detectior (CCD) .....................................................................33Nitric Oxide in Living Organisms ..........................................................................................35Neurotransmission ...........................................................................................................36Vasodilation .................................................................................................................. ...37Phosphorylation ............................................................................................................... 37Immune System ...............................................................................................................38Measurement of the Activity and Concentra tion of Nitric Oxide and Its Metabolites ...38Invertebrates for Neurochemistry Analysis ............................................................................43Cnidaria ...................................................................................................................... .....44Porifera ...................................................................................................................... ......46Ctenophora .................................................................................................................... ..47Placozoa ...........................................................................................................................47Mollusca ...................................................................................................................... ....48Dissertation Overview ......................................................................................................... ...49 5

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2 DEVELOPMENT AND EVALUATION OF CE COUPLED WITH LASERINDUCED FLUORESCENCE (LIF) DETECTION FOR THE ASSAY OF AMINO ACIDS AND NEUROTRANSIMITTERS ............................................................................50Introduction .................................................................................................................. ...........50Methods and Materials ...........................................................................................................53Reagents ...................................................................................................................... ....53CE Instrumentation ..........................................................................................................53Data Analysis ...................................................................................................................55Results and Discussion ........................................................................................................ ...56Optimization of CE Separation Conditions .....................................................................56Analytical Calibration .....................................................................................................59Glu and Asp Enantiomer Separation ...............................................................................60Conclusion .................................................................................................................... ..........623 DEVELOPMENT AND EVALUATION OF CE COUPLED WITH CONTACTLESS CONDUCTIVITY DETECTION TO IMPROVE THE ANION ASSAY .............................63Introduction .................................................................................................................. ...........63Methods and Materials ...........................................................................................................65Instrumentation ............................................................................................................... .65Reagents ...................................................................................................................... ....65Animals ....................................................................................................................... .....65Hemolymph and Ganglia .................................................................................................66Chloride Cleanup by Solid-Phase Extraction ..................................................................66Separation and Analysis ..................................................................................................67Results and Discussion ........................................................................................................ ...68Solid-Phase Microextrac tion (SPME) Cleanup ...............................................................68Optimization of Separation ..............................................................................................69Analytical Performance ...................................................................................................70Conclusion .................................................................................................................... ..........744 NITRIC OXIDE (NO) SINGNALIN G IN TRICHOPLAX ADHAERENS ..........................75Introduction .................................................................................................................. ...........75Methods and Materials ...........................................................................................................77Chemicals and Reagents ..................................................................................................77Animal Culture ................................................................................................................ 78NOS Inhibitor Incubation ................................................................................................78Amino Acids Microanalysis using CE with LIF .............................................................78Nitrite/Nitrate Microanalysis using CE with Contactle ss Conductivity ..........................79Behavior Tests .................................................................................................................80Data Analysis ...................................................................................................................81Results and Discussion ........................................................................................................ ...81Amino acid analysis by CE-LIF ......................................................................................81Nitrite and Nitrate Analysis by CE-Conductivity ...........................................................83Locomotory phases in Trichoplax ...................................................................................85 6

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NO as a modulator of locomotion ...................................................................................86Glycine as a chemoattractant in Trichoplax ....................................................................87Conclusion .................................................................................................................... ..........885 USING CE FOR METABOLOMIC PROF ILING OF THE BASAL ANIMALS: CTENOPHORES, CNIDARIANS PLACOZOA, AND SPONGES ....................................90Introduction .................................................................................................................. ...........90Methods and Materials ...........................................................................................................91Chemicals and Reagents ..................................................................................................91Sample Preparation ..........................................................................................................91Amino Acids Microanalysis using CE with LIF .............................................................92Nitrite/Nitrate Microanalysis using CE with Contactle ss Conductivity ..........................93Data Analysis ...................................................................................................................93Results and Discussion ........................................................................................................ ...94Neurotransmitters and Their Metabolites in Basal Animals ...........................................94Glu and Asp Enantiomer Analysis in Basal Animals ......................................................96Nitrite and Nitrate Assay in the Basal Animals ...............................................................97Conclusion .................................................................................................................... ..........996 COMPARATIVE ANALYSIS OF MULLUSCA: SQUID, NAUTILUS, AND APLYSIA CALIFORNICA ..................................................................................................102Introduction .................................................................................................................. .........102Methods and Materials .........................................................................................................104Chemicals and Reagents ................................................................................................104Sample Preparation ........................................................................................................104Amino Acids Microanalysis using CE with LIF ...........................................................105Nitrite/Nitrate Microanalysis using CE with Contactle ss Conductivity ........................106Data Analysis .................................................................................................................106Results and Discussion ........................................................................................................ .107Squid Axoplasm Analysis ..............................................................................................107Nautilus Analysis ...........................................................................................................109Aplysia californica Analysis ..........................................................................................112Conclusion .................................................................................................................... ........114LIST OF REFERENCES .............................................................................................................116BIOGRAPHICAL SKETCH .......................................................................................................133 7

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LIST OF TABLES Table page 2-1 Correlation coefficients, RSDs, and LOD .........................................................................602-2 Correlation coefficients, RSDs, and LOD .........................................................................623-1 Description of hemolymph and cen tral nervous system ganglia of Aplysia californica ...715-1 Metabolite concentratio ns in basal animals. ....................................................................1005-2 Land D Glu and Asp con centrations in basal animals ................................................101 8

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LIST OF FIGURES Figure page 1-1 A) Electric double layer created by negatively charged su rface and nearby cations. B) Predominance of cations in diffuse part of the double layer produces net electroosmotic flow toward the cathode when an external field is applied .......................171-2 Migration of uncharged compounds in MEKC .................................................................221-3 Migration schemes for cationic enantiome rs in CE using cyclodextrins (CDs) as chiral selectors ...................................................................................................................261-4 Energy level diagram for a typical molecule .....................................................................301-5 Schematic diagrams of conductivity detectors ..................................................................341-6 Schematic of some of the physiologically relevant reactions of NO and NO-derived species in aqueous, aerobic solution ..................................................................................361-7 Phylogenetic tree of Metazoan relationships .....................................................................442-1 Schematic optical layout of the fluorescence detection system .........................................542-2 Electropherograms of standard amino acids (1M) ..........................................................562-3 Electropherograms of standard amino acids depending on various SDS concentrations ................................................................................................................ ....572-4 Electropherograms of standard amino aci ds depending on different pH conditions .........582-5 Calibration curves of standard amino acids .......................................................................592-6 Electropherograms of Glu and Asp enantiomers ...............................................................613-1 Custom-made and factory-made chloride clean-up kit ......................................................673-2 Sample recovery graph ......................................................................................................693-3 Calibration curves of nitrite and nitrate .............................................................................703-4 Electropherograms of standard solutions (650nM of all anions), hemolymph, and central ganglia in Aplysia californica ................................................................................723-5 Nitrite and nitrate concentrations of hemolymph and central ganglia in Aplysia californica ..........................................................................................................................734-1 Electropherograms and concentration profiling of Trichoplax adhaerens ........................82 9

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4-2 Electropherograms and Arg-to-Cit ratios of Trichoplax adhaerens upon treatment with NOS inhibitors ...........................................................................................................834-3 Electropherograms of controls and Trichoplax upon NOS inhibitors ...............................844-4 Trichoplax behavioral analysis (Control) ..........................................................................854-5 Trichoplax behavior analysis (NO modulators) .................................................................864-6 Trichoplax behavior analysis (Glycine) .............................................................................875-1 Electropherograms of basal animals ..................................................................................945-2 Electropherograms of Glu and Asp enantiomers in the basal animals ..............................975-3 Nitrite and nitrate electrophe rograms of the basal animals ...............................................986-1 Electropherograms and concentration profiling of Squid ................................................1076-2 Nitrite and nitrate electropherograms and concentration profile .....................................1086-3 Electropherograms and concentration prof ile of Glu and Asp enantiomers in the Squid axoplasm ................................................................................................................1096-4 Electropherograms and concentration profiling of Nautilus ............................................1106-5 Nitrite and nitrate electropherograms and concentration profile .....................................1106-6 Electropherograms and concentration prof ile of glu and asp enantiomers in the Nautilus ............................................................................................................................1116-7 Electropherogram and concentration profiles of Aplysia californica chemosensory cells ......................................................................................................................... .........1136-8 Electropherograms and concentrat ion profiles of embryonic cells of Aplysia californica ........................................................................................................................114 10

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Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy INVERTEBRATES ANALYSIS BY CAPILLARY ELECTROPHORESIS By Dosung Sohn December 2009 Chair: Weihong Tan Major: Chemistry Over millons of years nervous systems of biological machines have evolved. Thus, tracking the lineages and constraints that have molded nervous systems provides an opportunity to understand signal molecules and their metabolite s in variety of marine organisms having key positions in the evolutionary tree of life. In particular basal animals, including placozoa, ctenophores, cnidarians, sponges relatively simp le organization compared to other known animals. While there have been extensive st udies on genome, physiology, histochemistry, and regeneration, direct microchemical da ta are limited on basal animals. In order to provide direct evidence for the presence of neurotransmitters and their metabolites, we have identified and characterized major low molecular weight potential signaling molecules and their metabolites in marine organi sms. Capillary electrophoresis (CE) techniques with laser-induced fluorescence (LIF) and contac tless conductivity detection (CCD) were used for in-depth studies of the metabolites of the ne urotransmitters and nitr ic oxide (NO). Since NO is a highly reactive signaling molecule, indirect detection methods were used. For example, LArginine and L-Citrulline, a precursor and co-product of NO, respectively, were analyzed with CE-LIF. Also, nitrite and nitrate, both major oxi dation products of NO, we re analyzed with CECCD. 11

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12 The results of this study will be used in conjunction with key research questions to fill numerous gaps in our understanding of diversity and evolution of signal molecules and the development of integrative systems in animals.

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CHAPTER 1 INTRODUCTION Metabolomics is the systematic study of small molecules in living organisms (Xiayan & Legido-Quigley 2008). The metabolome represents the collection of all metabolites (i.e. intermediates and products of metabolism) in a biological system. Metabolomic profiling can often show the physiological differences in a cel l, in cases where mRNA gene expression data and proteomic analyses provide insufficient information. One of the challenges of systems biology and functional genomics is to combine proteomic, transcriptomic, and metabolomic information to give a more complete understand ing of living organisms. This research was undertaken to gain metabolomic information on marine organisms by studying the signaling molecules. Historical Background The development of metabolomics began in 1970, when Arthur Robinson investigated Pauling's ideas that biological variability could be explained on the basis of far wider ranges of nutritional requirements than what was generally recognized (Pauling et al. 1971). In analyzing the chromatographic patterns of urine from vita min B6-loaded subjects, it was realized that the patterns of hundreds or thousands of chemical c onstituents in urine contained a considerable amount of information, including identification of several dis eases, determination of living conditions, and physiological age. It was expected that body fluid an alysis could be optimized to create a low-cost, information-rich, medically-r elevant means of measuring metabolically-driven changes in functional state, even when the chemi cal constituents are all in the normal range. This information on the functional status of a complex biological system resi des in the quantitative and qualitative pattern of metabolites in body fluids. 13

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The name metabolomics was proposed in the 1990s (Oliver et al 1998), and in 2004 a society was formed to promote its study. Many of the bioanalytical methods used for metabolomics have been adapted from exis ting biochemical techniques. There are two characteristics common to metabolomic research. Firs t, effort is made to profile metabolites with as little bias as possible towards a specific me tabolite or group of metabolites. Second, large numbers of metabolites are profiled at the same time, instead of being analyzed individually. The field of metabolomics exploded in the early 2000s, particularly as a result of efforts by researchers from the Max Planck Institute for Plant Physiology, and this research set the framework for metabolomics-scale investigations (Fiehn et al. 2000, Fernie et al. 2004). The Human Metabolome Project, led by Dr. David Wishart, completed the first draft of the human metabolome, consisting of a database of approximately 2500 metabolites, 1200 drugs and 3500 food components (Wishart et al. 2007). Similar projects have been underway for several plant species, most notably Medicago truncatula and Arabidopsis thaliana for several years. Separation Technology for Metabolomics There are four important issues to be addressed for metabolite analysis: efficient and unbiased extraction of metabolites from biologi cal tissues, separation and detection of the analytes by chromatographic or other methods, and identification and quantification. Separation Methods Gas chromatography (GC), especially when coupled with mass spectrometry (MS), is one of the most widely used and powerful met hods for separation and analysis (Pasikanti et al. 2008). It offers very high chromatographic resolution, but chemical derivatization is needed to increase the volatility of many biomolecule s. Modern instruments are capab le of 2D chromatography via a short polar column after the main analytical column. Although this me thod provides increased resolution, some large and polar meta bolites cannot be analyzed by GC. 14

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Compared to GC, high performance li quid chromatography (HPLC) has lower chromatographic resolution, but it does have the advant age that components do not have to be volatile, so that a much wide r range of analytes can potentially be measured (Wilson et al. 2005). Capillary electrophoresis (CE) has higher theoreti cal separation efficiency than HPLC, and is suitable for use with a wider range of metabol ite classes than is GC, but as with all electrophoretic techniques, CE is most appropriate for charged analytes (Xiayan & LegidoQuigley 2008). Detection Methods Mass spectrometry (MS) is an important mean to identify and quantify metabolites after separation by GC, HPLC, or CE. GC-MS is the most common combination of the three and was the first to be developed (Xiayan & Legido-Quig ley 2008). In addition, mass spectral fingerprint libraries exist or can be developed to allow identification of a metabolite according to its fragmentation pattern (Zou et al. 2008). There is also a number of studies which use MS as a stand-alone technology; the sample is infused directly into th e mass spectrometer with no prior separation, and the MS serves to both separate and to detect metabolites (Prakash et al. 2007). Nuclear magnetic resonance (NMR) spectroscop y is the only detection technique which does not rely on separation of the analytes, and the sample can thus be recovered for further analyses. All kinds of small molecule metabolit es can be measured simultaneously (Tukiainen et al. 2008). Practically, however, it is much less sensitive than mass spectrometry-based techniques, and NMR spectra can be very difficult to inte rpret for complex mixtures. MS and NMR are by far the two leading tec hnologies for analyzing metabolites. Other methods of detection that have been used include electrochemi cal detection coupled to HPLC (Parrot et al. 2007) and radiolabeling combined with thin-layer chromatography (Rogers et al. 1996). 15

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Capillary Electrophoresis and its Application to Metabolomics Capillary Electrophoresis Fundamentals Capillary electrophoresis is a relatively new an alytical separation technique that has found extensive use in clinical chemistry (Patterson et al. 2008, Gates et al. 2007, Bakry et al. 2007). Typical applications include analysis of peptides, prot eins, drugs, drug metabolites, carbohydrates, biological extracts an d small molecules. Capillary el ectrophoresis can be divided into six main groups according to the separati on mode: capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC) capillary isotachophoresis (CITP), capillary electrochromatography (CEC), capillary gel elec trophoresis (CGE), and capillary isoelectric focusing (CIEF). All these are elec trically driven techniques, mean ing that applied voltage rather than pressure is the driving force for separation (Landers 1996). Capillary Zone Electrophoresis This is probably the most commonly used se paration mode in capillary electrophoresis. In this high resolution analytical se paration technique, analytes ar e separated according to their electrophoretic mobilities in an el ectric field applied to a separati on capillary filled with a buffer solution. CZE employs narrow bore (20-100 m I.D.) capillaries, which can be made of Teflon, glass or fused silica. A typical CZE experiment is carried out as follows. The separation capillary is immersed in inlet and outlet vials, both contai ning a buffer solution. After the capillary is filled with this solution by a pressure injection, the inlet vial is repl aced by a sample vial. Following electrokinetic injection of the sample, the inlet end is placed back in to the buffer vial, and voltage is then applied betw een the two capillary ends. Analytes migrate along the capillary at different velocities, which ar e mainly determined by their charges and charge-to size ratios. Th e net or apparent ve locity is given by 16

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app = ep + eo (1.1) where, ep is the analyte electr ophoretic velocity and eo is the velocity of the electroosmotic flow (EOF). These two parameters can also be expres sed in terms of mobility values as follows app = app E (1.2) app = ep + eo (1.3) where ep and eo represent the analyte electrophoretic mobility and the EOF mobility, respectively. The eo is a result of the bulk flow due to the movement of hydrated buffer ions along the capillary in the presence of an electric field, and is primarily determined by the charge density on the capillary surface. Figure 1-1. A) Electric double layer created by negatively charged su rface and nearby cations. B) Predominance of cations in diffuse pa rt of the double layer produces net electroosmotic flow toward the cathode when an external field is applied 17

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In the case of fused silica capillaries, this charge is a function of the pH of the buffer solution. For example, at low pH values (2-3) the silanol (SiOH) groups are protonated, and therefore the surface charge and eo are negligible. As the pH of th e buffer solution is raised, the silanol groups are ionized to SiOand H3O+, leading to an increase in negative charges on the capillary wall. This negatively charged surface at tracts buffer cations, forming two main layers (Figure 1-1). The first layer is fixed adjacent to the wall, but the second layer (farther from the wall) is mobile. It is the moveme nt of this latter layer that gi ves rise to the EOF (Harris 2007). The electroosmotic mobility is defined as eo (1.4) where is the zeta potential (potentia l across the two layers), and and are the dielectric constant and viscosity of the buffer solution, respectively. The zeta po tential is given by 4 (1.5) where is the thickness of the diffuse double layer and is the charge per unit surface area. Unlike mechanically driven flows, the electroosmo tic flow has a flat flow profile. This means that the velocity of the fluid is constant along the radial axis of the capillary, which is the main reason for the high separation efficiencies observe d in CE. Because the electroosmotic flow has a great impact on separation, a number of strategies have been developed to control its magnitude and direction. The electrophoretic mobility is an intrinsi c property of the analyte and is given by r qep 6 (1.6) where q is the charge of the analyte and 6r is the friction coefficient ( f ). 18

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The apparent mobility, app, of a particular species can be calculated from experimental data using the following relationship t d net appL V t L E u (1.7) where unet is the velocity of the species, E is the electric field, Ld is the length of column from injection to the detector, Lt is the total length of the column from end to end, V is the voltage applied between the two ends, and t is the time required for solute to migrate from the injection end to the detector. The quality of the separation is determined by a number of factors relating to the buffer, the characteristics of the capilla ry, the mode of inj ection (hydrodynamic or electrokinetic), and the applied voltage. The buffer identity, pH, and concentration, as well as the presence of additives or modifiers (i .e. organic solvents, surfactants, and urea) all play key roles (Landers 1996). The capillary dimensions (i nternal diameter and length) a nd the method used to modify the surface are also important. High efficiencies are achieved when analyte adsorption is prevented. When using bare fused silica capillaries, high ionic strength buffers (e ither acidic or basic) ar e typical. For coated capillaries, the use of neutral pH buffers is pos sible. The inclusion of additives in the buffer solution may alter the analyte mobilities and modify the capillary su rface (Landers 1996). As a general trend, high separation voltages and long capillaries with small internal diameters provide high separation efficiencies. However, care must be exercised when applying high voltages to avoid Joule heating produced by high currents. Joule heating results in temperature gradients and bubble formation, both of which are detrimental to the separation efficiency (Landers 1996). 19

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The volume of sample injected is determ ined by the injection mode employed. For hydrodynamic injection, th e volume is given by tL tpd V 1284 (1.8) where p is the pressure difference al ong the length of the capillary, d is the capillary inner diameter, t is the injection time, is the buffer viscosity, and Lt is the total length of the capillary. For electrokinetic injection, the moles of sample injected is given by s beoepsEt Cr Q 2 (1.9) where, Q is the moles of sample injected, r is the radius of the capillary, Cs is the sample concentration, b is the conductivity of the buffer, and s is the conductivity of the sample (Landers 1996). With this inject ion mode, the analyte electrophoret ic mobility impacts the moles of sample injected. Therefore, unlike hydr odynamic injection, elec trokinetic injection discriminates according to electrophoretic mobilitie s. When the sample is dissolved in a low ionic strength solution such as water, electrokinetic injecti on is preferred over hydrodynamic injection, since it typically results in higher sepa ration efficiencies as a consequence of stacking effects by which analyte is focused into narrow bands at the start of the capillary (Harris 2007). Injection volumes should be maintained within 0.2% of the capillary volume to prevent bandbroadening due to column overloading (Landers 1996). Micellar Electrokinetic Chromatography (MEKC) In MEKC, the buffer contains a surfactant in su fficient concentration to form micelles. The separation relies on partitioning of the analytes between the buffer solution and the micelles, also called a pseudostationary phase. Interaction of an alytes with the micelle s occurs via hydrophobic, ionic, or hydrogen bonding forces. This techniqu e has been applied to the separation of both 20

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neutral and charged compounds. In the case of neutral compounds, separation is based on partitioning solely, while for charged compounds, separation is determined by partitioning as well as electrophoretic mobility (Landers 1996). Micelles: Surfactants are amphiphilic molecules th at contain a hydrophobic moiety and a polar or ionic head group. They can be recognized by the charge of the head group (i.e. nonionic, anionic, cationic, and zwitterioni c surfactants) or by the variations in the nature of hydrophobic moiety (i.e. hydrocarbon, bile salts, and fluor ocarbon surfactants). Above a critical micelle concentration (CMC), surfactants begin to form aggregates that are in dynamic equilibrium with the monomers in the bulk aqueous solution. The nu mber of monomer surfactants in the aggregate and the shapes of size of micelles vary greatly be tween surfactants. For example, surfactants with alkyl chains form roughly spheri cal micelles with diameters betw een 3 and 6nm and aggregation numbers of 30 and 100. Migration in MEKC: Figure 1-2 illustrates the typical migration scheme for uncharged compounds in MEKC using an anionic surfactant and a cationic surfactant in an uncoated fused silica capillary. Figure 1-2A shows an aninoic mi celle in a bare silica capillary with ionized surface (SiO-). Although anions naturally are attracted to the anode, the EOF velocity is stronger than the electrophoretic velocity and the anionic micelles are carried toward the cathode. When cationic micelles are used, the capillary wall is co ated with the positively charged surfactants with oftentimes leads to a revers al in the direction of the EOF (Figure 1-2B). It is therefore necessary to reverse the polarity of the electrodes in the CE set up to ensure the elution of the cationic micelles and consequently the uncha rged solutes through the detection window. 21

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Figure 1-2. Migration of uncha rged compounds in MEKC using A) anionic and B) cationic pseudostationary phases. C) Schematic diag ram of separation of molecules depending on S1 and S2 differential partitioning into the pseudostationary phase Uncharged solutes: As in chromatography, the retention factor, k', in MEKC is defined as the ratio of the number of moles of solute in the micellar pseudostationary phase, nmc, and that in the bulk aqueous phase, naq. The retention factor is directly proportional to the micelle-water partition coefficient, Pmw, and the phase ratio, by 22

PAGE 23

k'= aq mcn n= Pmw (1-10) The retention factor in MEKC can be determined from migration time data using Eq 1-11. k' = mc r eo eort t t tt 1 (1-11) where teo is the elution time of an unretained solute and tmc is the migration time of micelles. This is very similar to the equation for the retention factor in conventional chromatography, with the exception of the additional term (1-tr/tmc) in the denominator. This term indicates the existence of an elution window, because the stationary phase in MEKC is ac tually mobile. If tmc approaches infinity (i.e., stationary micelles), the extra te rm in the denominator is omitted and the retention factor equation becomes the same as that in conventional chromatography. Charged solutes: In addition to partitioning into mi celles and migrating at the micellar mobility, charged compounds possess electrophoretic mobilities of their own in the bulk aqueous solvent. As a result, the observed retention time also includes the time that solute migrates electrophoretically in the bulk aqueous phase, to. In calculating the retention factor, this electromigration time should be taken into account: k' = mc r o ort t t tt 1 (1-12) Resolution: The fundamental resolution equation for uncharged solutes in MEKC has the same format as that for conventional chromat ography, which includes three terms related to efficiency, selectivity, and reten tion. In addition there is a fourth representing the existence of an elution window: 23

PAGE 24

R = 1 2 21 1 1 1 4 k t t t t k k Nmc eo mc eo (1-13) Again, in the case of sta tionary micelles (i.e., if tmc ~ ), the fourth term drops out and the equation is identical to that in conventional chromatography. Surfactant concentration: The primary role of surfactant c oncentration is to adjust the retention factor to within th e optimum range for better reso lution. The relationship between retention factor, k', and surfactant con centration can be described as follows: k' = mw sf sfP CMCC CMCC 1 (1-14) where is surfactant molar volume; Csf is the total surfactant concentration; CMC is the critical micelle concentration; and Pmw is the partition coefficient of a solute between an aqueous phase and micelles. At low micelle concentrations, the second term in th e denominator becomes negligible and a linear relationship between the re tention factor and surf actant concentration can be described as follows: k' = Pmw (Csf CMC) (1-15) Chiral Separation by CE In a chiral separation, a chiral selector (e.g., a cyclodextrin) is used as the pseudostationary phase instead of micelles. The principle of chiral separation can be explained by the following two chemical equilibria E1 + CS E1CS (1-16) K1 E2 + CS E2CS (1-17) K2 24

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where E1 and E2 are two enantiomers in a racemic mixt ure; CS is a chiral selector; and K1 and K2 are the binding constants between the chiral se lector and the enantiomers, respectively. The electrophoretic mobility () of an enantiomer at a given concen tration of the chir al selector is expressed as ][1 ][CSK CSKcf (1-18) where f and c are the electrophoretic mobilities at the concentrations of the ch iral selector at 0 and respectively; and [CS] is the equilibrium concentration of the chiral selector. The relationship between the mobility difference ( ), or separation selectivity, and the concentration of the chiral selector can be expressed by ][1][1 ][2 1CSKCSK CSKcf (1-19) As can be seen from equation 1-19, is proportional to the mobility difference of the racemate in the free (f) and totally complexed (c) forms, and their binding constant difference ( K). No chiral separation can be achieved if there is no complexation between enantiomers and the chiral selector. In addition, the two enan tiomers should bind to the chiral selector to different extents in order to be separated. Therefore, the choice of a chiral selector is crucial for chiral separation, because it controls three terms: K, K, and (f c). The other experimental factor that influences is the selector concentration, [CS], which should be optimized in order to achieve the deserved separation. The resolution equation in CZE is also valid in chiral separation as Rs = eo avgN 4 (1-20) 25

PAGE 26

where N is the number of theoretical plates; avg is the average electrophoretic mobility of the two enantiomers; and eo is electrophoretic mobility. Chiral resolution can be improved by enhancing the capillary efficiency ( N ), maximizing separa tion selectivity ( ), optimizing retention ( ), and controlling the EO F. In order to maximize several parameters such as the type and concentration of chiral selector, as we ll as the pH (for ionizable solutes) must be optimized. Other experimental conditions, such as the buffer ionic strength and temperature can also play roles through their e ffects on retention separation, as suggested in Eq.1-20. Two general migration schemes are recognized in CE : co-electroosmotic flow (co-EOF), where the ions and EOF migrate in the same direction as the EOF, and counter electroosmotic flow (counter-EOF), where the ions migrate in the opposite direction to the EOF. Figure 1-3. Migration sc hemes for cationic enantiomers in CE using cyclodextrins (CDs) as chiral selectors A) co-EOF, B) counter-EOF. s = electrophoretic mobility of the free form of enantiomer; CD = electrophoretic mobility of an anionic CD Figure 1-3 shows diagrams of these two schemes for positively charged enantiomers. In the case of co-EOF, for example, basic racemates are positively charged at a lower pH range and 26

PAGE 27

there is a weak EOF from the anode to the cathode. The co-EOF setup has been the most commonly used scheme for separations of basic racem ates with different types of chiral selectors. In the counter-EOF case, however, higher resolution can be achieved as the ( avg + eo) in the denominator of the resolution equation become s smaller. According to Eq. 1-20, higher resolution is achieved if the analytes migrate in the direc tion opposite to the EOF. This is achieved at the expense of longer analysis times. In certain situations, ch iral separation can be achieved by controlling the EOF even when other pa rameters, such as sel ector concentration of pH are not at optimum values. Capillary Isotachophoresis (ITP) This technique uses a discontinuous buffer sy stem. The sample is sandwiched between a leading and a terminating electrolyte having hi gher and lower mobilities that the analytes, respectively. After voltage is app lied, a non-uniform electric field is established in the capillary. Analytes and the leading and terminating electrol ytes start to migrate at different velocities, eventually forming focused zones. When equilibr ium is reached, all zones move at the same velocity. The initial concentration of the anal yte determines the length of the focused zone (Khaledi 1998). ITP is a nonlinear electr ophoretic technique used in the separation of a variety of ionic compounds, from small molecules and metal ions (Beckers 1995) to large molecules, like proteins (Stowers et al. 1995). Unlike linear zone electrophoresis in which the separating solute bands continually spread by diffusi on or dispersion, ITP forms self-s harpening, adjacent zones of substantially pure solute. In ITP a multianalyte sample is usually introduced between the leading electrolyte (LE, containing lead ing ion) and the terminating electrolyte (TE, containing terminating ion). The leading ion, the terminati ng ion, and the sample components must have the same charge polarity, and the sample ions must have electrophoretic mobilities smaller than the 27

PAGE 28

leading ion but larger than the terminating ion. After applicati on of a fixed electric current, sample components move forward behind the leadi ng ion and in front of the terminating ion and form discrete, contiguous zones in order of th eir electrophoretic mobilit ies. Then, following a brief transient period where the discrete solute zones are formed, the ITP stack assumes a fixed concentration profile with a constant velocity moving in the direction of the leader. The method is self-resharpening, i.e., the stacked zones can quickly recover their shape after a dispersive event. Kohlrausch developed the basic theory of IT P 110 years ago, but it did not receive much attention until the deve lopment of capillary elec trophoresis in the 1970's. Since then, ITP, along with zone electrophoresis (ZE) and isoelectric focusing (IEF), have become indispensable analytical tools, especially for high resolution and rapid analysis of biological samples. ITP is also an extremely powerful method to concentrate samples. No matter how low the sample concentration is, it can be concentrated to a plat eau concentration which, in the ideal case, is described by the following equation: RA RL B A LAcc (1-21) where A is an analyte, R is c ounterion, and L is leader ion. Capillary Electrochromatography This technique is considered a hybrid of LC and CZE, combining the separation efficiency of CZE and the selectivity of LC. Voltage, rather than pressure, is used as the driving force for the mobile phase. Because of the flat solvent fron t, the separation efficiency is improved. Like micellar electrokinetic chromatography, the sepa ration mechanism for neutral compounds is due to analyte partitioning between the mobile phase and the stationary phase, whereas for charged 28

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compounds, an additional parameter (electrophoretic mobility) must be taken into account. Both packed columns and coated columns can be used.(Landers 1996) Capillary Gel Electrophoresis This technique is carried out in a capillary filled with a gel, wh ich may or may not be covalently bound to the cap illary. Analytes separate due to a sieving mechanism. This method is widely applied to the separation of compounds having very similar charge/size ratio.(Landers 1996) Capillary Isoelectric Focusing In this technique, analytes separate according to their isoel ectric points. A typical CIEF experiment is performed as follows. The capillary is filled with the sample solution containing ampholytes (compounds that can act as either acid or base) having a range of pI values. One end of the capillary is immersed in an acidic solu tion, and the other in a basic solution (anode and cathode, respectively). After voltage is applied, ampholytes star t to migrate and form a pH gradient within the capillary. Analytes migrate in this pH gradient and focus at the positions where their pI equals the pH. Once all the anal ytes reach their equilibrium positions, focused analytes are moved along the capillary and detected by applying an external hydrodynamic force.(Landers 1996) Fluorescence Detection in CE Background: When a molecule absorbs ultraviolet or visible radiation, a higher electronic state is populated. There are a number of decay processes to repopulate th e ground state; if the molecule emits light, the radiation is termed F luorescence (Figure 1-4). The wavelength of the emitted radiation is longer (or lower energy) th an that of the excitation radiation and is characteristic of the compound of interest. 29

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Figure 1-4. Energy level diag ram for a typical molecule A critical parameter in fluorescence is the fluorescence quantum yield ( f), which is defined by f = a fI I (1-22) where If is the number of quanta emitted and Ia is the number of quanta absorbed. At low concentrations, the fluorescence in tensity (F) from an excited compound is linearly dependent on the concentration of the compound as described F = kIa( f) cLV (1-23) where k is Boltzman constant, is the absorbance coeffici ent for the compound, c is the concentration of the compound, L is the path length of the cell, and V is the illuminated volume. Although the fluorescence signal is pathlength dependent, the S/N ratio is not strictly pathlength dependent. Background fluorescence a nd solvent Raman scattering are the major contributors to the background signal. As a smalle r-diameter capillary is used, the fluorescence signal from a particular concen tration of analyte decreases bu t so does the spectral background. Thus, the reported fluorescence detection limits are the 10-100fM range for a wide range of pathlengths. 30

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Benefits of fluorescence detection: There are two major advantages of fluorescence detection for CE, namely sensitivity and selectivity. Typically a fl uorescence detector can provide a minimum limit of detection that is 2 to 3 orders of magnitude be tter than that of an absorbance detector. When a fluorescence measur ement is performed, the observed signal is compared to a sample that does not fluorescence (the mobile phase), so that the background is very close to zero. In contrast, when an absorban ce detector is used, tran smission of radiation of the sample is compared to the transmission of the blank. At low concentrations, the difference between the transmission of the sample and the transmission of the blank is small (i.e. the background is not small, relative to the signal) and the error in the measurement can become very significant. When an absorbance detector is used to dete ct the compounds of inte rest in the detection window, the measurement is based on a single wa velength, while two wavelengths are used for fluorescence detection. If two compounds happen to co-migrate and have the same absorbance wavelength but different emission wavelength (o r does not fluoresce), a fluorescence detector can readily provide useful analytical data. Labeling of analytes: A large number of compounds (e.g. many aromatic compounds) show native fluorescence, and dire ct quantification is very si mple. If, however, a compound does not exhibit native fluorescence, it may be very straightforward to generate a fluorophore by reacting it with a fluorogenic reagent (Bardelmeijer et al. 199 7). This technique is commonly used in the determination of the concentra tion of amino acids, carboxyl ic acids and similar compounds. The detection of amino acids is an especially good example of this technique. Precolumn derivatization can provide a fluorophore fo r all of the common am ino acids, while only 31

PAGE 32

three (phenylalanine, tryrosin e and tryptophan) exhibit native fluorescence, this fluorescence detection of amino acids via their fluores cent derivatives is very commonly used. Conductivity Detection in CE Basic Principles In conductivity detection, the solution resistance R( ) is calculated from its conductance G (S), defined as G = 1/R. The value for G may also be determined fr om the ratio of the specific conductance (S cm-1) and the cell constant kcell (cm-1) and can be given by (Guijt et al. 2004) cellk G (1-24) In a conductometric cell, it is impossible to m easure only R, since the electronic measurement setup will effectively be networ k of capacitors and resistors. To correct for differences between different measurement setups, the cell c onstant is used for determination of G. There are several important aspects which characterize conductivity detection in CE. Signal response in conductivity de tection is principally related to the equivalent conductivity of a solute. Analyte ions displace background co -ions during electrophoret ic separations by equivalent to their charge. Thus, the response arises from the difference in conductivity between the analytes and the background electrolyte (B GE) co-ions. For optimum S/N ratio, the conductivity difference between the analyte and the electrolyte must be as large as possible. There are two ways to achieve the above condi tion. In the first, the sample ion zones exhibit conductivities larger than the BGE. Thus, positive analytical response signals are obtained even at equal concentratio ns of analyte and electrolyte co -ions, but this gain in response gives peak asymmetry. In the second case, electrolyte systems with matching equivalent conductivities of the sample ions and BGE co-i ons are used. A higher ionic strength of the sample zone compared to the electrolyte is, however, required to obtain a positive analytical 32

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response. On the contrary, this counteracts the ge neral principles of CE, which require the use of electrolytes with a higher ionic st rength compared to the sample zone in order to take advantage of electrostacking effects. An efficient way to so lve this contradiction is the use of amphoteric buffers (Beckers 2003). These buffers produce electrolyte systems with low background conductivities and can thus be us ed at relatively high ionic strengths. As a consequence, electrodispersive effects are reduced, despite mismatching electrophoretic mobilities of buffer co-ions and analytes. Contactless Conductivi ty Detector (CCD) Although several commercial in strument manufacturers inte nded to market CEs with conductivity detectors, only one supplier introduced an end-co lumn conductivity detector (Crystal CE from Thermo CE) (Haber et al. 1998). The instrument us es a specifically designed detection cell (wall jet arrangement) with a fi xed capillary to ensure constant detection conditions. However, problems can arise from the direct contact of the separation electrolyte with the measuring electrode, as contamination of the electrodes may occur from electrolyte additives or sample components. Furthermore, the capillary set is expensive because of the special alignment with the detection cell. Other ambitious instrumental attempts, such as suppressed conductivity detection fo r CE unfortunately did not b ecome commercially available products (Dasgupta & Bao 1993). In order to avoid the aforementi oned obstacles with contact conductivity detectors, contactless conductivity detector s offer some remedies. The detection signal is not obtained transversa lly across the capillary, but rather in the longitudinal dimension (Figure 1-5) The major perspective of this technique is that there is much less limitation with respect to the inner diameter of the capillary compared to the techniques presented in the 1970s for isotachophoretic purposes. Thus, a contact less conductivity 33

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detector can be used with capillaries havi ng small inner diameters and in miniaturized instrumentation, such as in chip-based separation systems. Figure 1-5. Schematic diagrams of conductivity de tectors. A) Contact de tector based on Thermo Crystal 1000. B) CCD using two tubula r electrodes. The electrode length, l, and the gap between the electrodes, d, are indicated (top) and th e simplified equivalent circuitry (down) In a capacitively coupled contactless conductivi ty detector, two stainl ess steel tubes which act as the electrodes are placed around a fused-silica capillary in a ce rtain distance from each other (Figure 1-5B). By applying an oscillating frequency in the range between 20 and 900 kHz, a capacitive transition occurs between the actuator electrode and the liquid inside the capillary. After having passed the detection gap between the electrodes, a second capacitive transition between the electrolyte and the pi ck-up electrode occurs. Thus, this scheme represents a series combination of a capacitor, an ohmic resistor and a second capacitor. By using suitable amplifier electronics, conductivity changes of the electrolyte inside the capillary can be monitored. Usually, the electr odes are placed on an insulated socket to ensure a rigid construction with a constant electrode distance. Th e socket is then shielded by being placed in a grounded metal housing. To reduce noise and capaciti ve leakage, especially when the electrodes are positioned very close to each other, a grounded shielding, usually made of a thin metal sheet or foil, can be placed between the electrodes. 34

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Nitric Oxide in Living Organisms Nitric oxide (NO) was identifie d in 1987 as an endothelial-der ived relaxing factor (EDRF) causing vasodilatation in smooth muscles (Ignarro et al. 1987), and it has been shown to participate in modulation of neural functions in the brain, i mmune defense, and learning and memory (Garthwaite & Boulton 199 5, Nathan & Shiloh 2000, Katzoff et al. 2002). Enzymatically produced NO is synthesized from L-arginine and oxygen, with L-citrulline as a co-product. Nitric oxide synthase (NOS) catalyzes the reaction, and various co-factors such as NADPH, flavin mononucleotide (FMN ), flavin adenine dinucleoti de (FAD), tetrahydrobiopterin (BH4), and calcium/calmodulin (Ca2+/CaM) are involved (Murad 1999). As depicted in Figure 1-6, the physiologica l chemistry of NO and NO-derived species includes many interrelated and interdependent processes (Fukuto 2000) In oxygen-containing aqueous solutions, nitric oxide scarcely produce n itrate; however, nitric oxide gas reacts with oxygen to form NO2 gas, which dimerizes to N2O4, subsequently yielding both nitrite and nitrate (Ignarro et al. 1993). The oxidation of NO by O2 to NO2 in an aqueous system is written as follows: 4NO + O2 + 2H2O 4NO2 + 4H+ (1-25) The rate equation for the loss of NO from reaction (1-25) is d[NO]/dt = 4k[NO]2[O2] with k = 26 M-2s-1, which means NO degradation in an aerob ic, aqueous solution is not linear with respect to the NO concentr ation (Lewis & Deen 1994). For example, assuming O2 concentrations around 200 M, a 10 M solution of NO degrades to half it s original concentr ation in about 1 minute whereas a 10nM solution takes over 70 hour s. This is true for NO in pure aqueous solutions, but in the presence of biological tissues the half-lif e of NO is 3~5 seconds. This difference can be attributed to many chemical interactions in cells or tissues with oxygen, superoxide anion, other oxygen-derived radicals, and oxyhem oproteins (Ignarro 1990). In 35

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addition, oxyhemoglobin, one of th e oxyhemoproteins, reacts with NO2 -, yielding methemolgobin and NO3 -. Mn + H+ -1RSH RSH RSH RSH CuI CuII RSH Metal(Mn) R H2O O2 NO HbFeII-O2 NO O2 NO OONO ONOONO 2NO2 N 2O4 N O2 + NO3 + 2H+ N 2O3 2NO2 + 2H+ R-NO -OONO 1 or 2e-oxidation chemistr y ONOOCOOO2NOCO2 HCO3 + NO3 + H+ HbFeIII + NO3 RS-NO + NO2 + H+ RSSR + HNO CO2 RS-NO + RSH RS+ NO RSSR + N2O + H2O +H2O -H2O [Mn-1-NO+ Mn-NO] H2O NO2 Figure 1-6. Schematic of some of the physiologically relevant reactions of NO and NO-derived species in aqueous, aerobic solution Neurotransmission Nitric oxide also serves as a neurotransmitter between nerve cells, as part of its general role in redox signaling. Unlike most other neurotransmitters, which only transmit information from a presynaptic to a postsynaptic neuron, the small, uncharged, and fat-soluble nitric oxide molecule can diffuse widely and it readily enters cells (Ignarro 1990). Thus, it can act on several nearby neurons, even on those not connected by a synapse. At the same time, the short half-life of NO means that such action will be restricted to a limited area, without the necessity for enzymatic breakdown or cellular reuptake (Igna rro 2000). Nitric oxide is also highly reactive with other free radicals, lipid s, and proteins. 36

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It is conjectured that this process may be involved in memory through the maintenance of long-term potentiation (LTP) (Katzoff et al. 2002). Nitric oxide is an im portant non-adrenergic, non-cholinergic (NANC) neurotransmitter in various parts of the gastrointestinal tract, and it causes relaxation of the gastrointestinal sm ooth muscle (Boeckxstaens & Pelckmans 1997). Dietary nitrate is also an important sour ce of nitric oxide in mammals. Green, leafy vegetables and some root vegetables have high co ncentrations of nitrate. When eaten, nitrate is concentrated in saliva (about 10 fold) and is redu ced to nitrite on the surf ace of the tongue by a biofilm of commensal facultativ e anaerobic bacteria (Lundberg et al. 2004). This nitrite is swallowed and reacts with acid and reducing substa nces in the stomach (such as ascorbate) to produce high concentrations of nitric oxide. Th e purpose of this NO production is thought to be prevention of food poisoning by both sterilization of swallowed food and maintenance of gastric mucosal blood flow. A similar mechanism is thought to protect the skin from fungal infections, where nitrate in sweat is reduced to nitrite by skin commensal organisms and then to NO on the slightly acidic skin surface (Lundberg et al. 2004). Vasodilation Nitric oxide is of critical importance as a mediator of vasodilation in blood vessels. Release of NO is induced by several factors, an d once synthesized, it resu lts in phosphorylation of several proteins that cause sm ooth muscle relaxation. The vasodilatory actions of nitric oxide play a key role in renal contro l of extracellular fluid homeostasis and are essential for the regulation of blood flow and blood pressure (Yoon et al. 2000). Phosphorylation Nitric oxide, a highly reactive fr ee radical, then diffuses into the smooth muscle cells of the blood vessel and interacts with soluble guanylate cyclase to stimulate generation of the second messenger, cyclic GMP (3,5 guanosine monop hosphate), from guanosine triphosphate (GTP). 37

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The soluble cGMP activates cyclic nucleoti de dependent protein kinase G (PKG), which phosphorylates a number of proteins that regulate calcium concen trations, calcium sensitization, cell hyperpolarization through potassium channe ls, and actin filament and myosin, dynamic alterations to result in smooth muscle relaxation (Lincoln et al. 2006). Immune System Macrophages, certain cells of the immune sy stem, produce nitric oxide in order to kill invading bacteria. In this case, the nitric oxide synthase is in ducible NOS (iNOS). Under certain conditions, this can backfire. For example, fulm inant infection (sepsis) causes the inducible isoform of nitric oxide synthase to be expressed, resulting in ex cess production of nitric oxide by macrophages, leading to vasodilata tion, probably one of the main causes of hypotension in sepsis (Victor et al. 2004). Measurement of the Activity and Concentration of Nitric Oxide and Its Metabolites Among the various analytical methods for dete ction of NO, major four approaches are discussed: (1) separation techni ques including CE coupled with various detectors; (2) optical methods including fluorescence microscope; (3) electrochemical methods including cyclic voltammetry, amperometric sensors, and analyteselective exchange potentiometric sensors; and (4) immunohistochemistry and in situ hybridization. Separation techniques: Capillary electrophoresis coupled with laser induced fluorescence (LIF) detection provides low detection limits, high efficiency, and ultra small sample consumption, and thus allows single cell analysis (Lapainis et al. 2007, Miao et al. 2005). CELIF has been successfully employed to m easure NO directly in single neurons in Lymnaea stagnalis by derivatizing NO with 4,5-di aminofluorescein (DAF-2) (Kim et al. 2006). In addition to measuring NO itself, CE-LIF has been appl ied for measuring NOS-related metabolites in single cells. Arginine-to-citrulline (Arg/Cit) concentration ratios have identified several neurons 38

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and neuronal structures in the CNS of Lymnaea stagnalis and Aplysia californica as nitrergic, or presumed nitrergic (Moroz et al. 2005, Floyd et al. 1998). Cit can be converted back to Arg via the intermediate argininosuccinate (ArgSu c). ArgSuc levels in single neurons of A. californica have been measured and compared to Arg and Cit levels (Ye et al. 2007). Furthermore, amino acids, including L-arginine and its me tabolites in human serum plasma (Causse et al. 2000) and microdialysis samples, have been an alyzed by CE-LIF (Powell & Ewing 2005). Capillary electrophoresis coupled with conductivity has been employed to analyze the oxidative products of NO: nitrite (NO2 ) and nitrate (NO3 ). In the CNS of Pleurobranchaea californica NO2 and NO3 levels vary from millimolar levels in nitrergic neurons to undetectable levels in many NOS-negative neurons (Moroz et al. 1999). In rat dorsal root ganglia, endogenous levels of nitrate (231 M) and nitrite (24-96 M) were found (Boudko et al. 2002). These concentrations exceeded those previous ly found in neuronal tissue homogenates using different techniques. Microchip CE with electrochemical (EC) detect ion has been developed to determine nitrite by direct amperometric detection, following a re duction of nitrate to nitrite by Cu-coated Cd granules (Kikura-Hanajiri et al. 2002). The utility of this method was demonstrated by monitoring the amount of nitrat e and nitrite produced from 3morpholinosydnonimine (SIN-1), a metabolite of the vasodilator molsidomine and a nitric oxide-releasing compound. Ultraviolet absorption is the traditional way to quantify analytes, and it is the common CE detection mode for the measurement of NO metabolites in biological samples. UV absorbance (214nm) detection was first used with CZE to detect nitrate and nitr ite simultaneously in biological samples (Meulemans & Delsenne 1994). Also, the use of CE in the measurement of nitrite and nitrate in human urine was demons trated by Morcos and Wiklund, who found that 39

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hydrodynamic sample injection was free from the interference of urin e concentration, pH, sodium, and chloride observed when electr okinetic injection was used (Morcos & Wiklund 2001). Recently, the high-throughput determination of nitrite and nitrate in biol ogical fluids using an electrophoretic lab-on-a-chip (microchip capillary electrophoresis MCE) technique was developed (Miyado et al. 2008). The addition of a zwitterionic additive, 2% (w/w) 2-(Ncyclohexylamino)ethane sulfonic ac id (CHES), into the running bu ffer reduced the adsorption of protein onto the surface of channel and allowed co mplete separation of nitrite and nitrate in human plasma within 1 min. Furthe rmore, nitrate levels were mon itored in the rat vitreous cavity using in vivo low-flow push-pull perfusion sampling and the results showed a significant difference in different locations (Pritchett et al. 2008). Infusion of N(G)-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor, with physiological saline led to a significant decrease (35%) in the observed nitrate level. Optical methods: Intracellular imaging of NO in bi ological systems has been performed using different types of fluores cent indicators probed via fluorescence microscopy. With the high sensitivity of fluorescent dyes, fluorescence mi croscopy provides the advantages of high temporal, spatial and three-dimensional resolution (Ye et al. 2008). Since NO doe s not fluoresce itself, the key to NO fluorescence imaging is the use of chemical probes with a direct, fast, sensitive and selective response. For this purpo se, many fluorescent dyes have been designed and applied to NO measurements in biological systems. A group of probes based on ophenylenediamine indicators are th e most widely used, for example, the diaminofluoresceins (DAFs). The two aromatic vicinal amine groups of DAF react with NO in the presence of O2 to form a highly fluorescent triazole product (Nagano & Yoshimura 2002), which has been used in many NO studies including cells (Kojima et al. 1999, Saini et al. 2006, Yukawa et al. 2005), sea 40

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urchin gametes (Kuo et al. 2000), and cultured cell lines (Arundine et al. 2003, PereiraRodrigues et al. 2005). There are also metal-based NO sensors that wo rk by the mechanism of spin exchange or selective ligand dissociation. Spin exchange utilizes the activation mechanism of guanylate cyclase (GC) with NO. In GC, the imidazole gr oup of the histidine resi due coordinates to the heme-iron, but NO binds to the heme-iron more tightly, thus displacing the imidazole group and activating the enzyme (Soh et al. 2001). The probe consists of 2,2,6,6-tetramethylpiperidine-Noxyl (TEMPO) labeled with acridine to imitate the imidazole moiety and Ndithiocarboxysarcosine (DTCS)-Fe(II) mimic the he me-iron complex. Acridine fluoresces itself, but its fluorescence is quenched in the acridine-TEMPO complex. When the NO interacts with the Fe(II) in the DTCS-Fe(II), the fluorescence from the acridine moiety is recovered. Also, the copper fluorescein complex (CuFL) shows NO-triggered fluorescence enhancement by a different mechanism (Lim et al. 2006). NO reduces the Cu(II) of CuFL to Cu(I), forming NO+, which immediately turns on the fluorescence. NO produced in live neurons and macrophages was monitored in a concentratio nand time-dependent manner. Furthermore, genetically encoded fluorescent proteins capable of reacting with NO have been introduced (Namiki et al. 2005, Pearce et al. 2000, Sato et al. 2006). Interestingly, a fluorescent cyclic guanosine monophosphate (cGMP) indicato r protein, named CGY, was developed. By connecting CGY to soluble guanylyl cyclase (sGC) to form chimera proteins (NOA-1) in the cells being investigated, in nano molar levels of NO were measured in vascular endothelial cells (Sato et al. 2005). There was 1nM NO basal produc tion in each endothelial cell and a 0.5nM increase with physiological stimuli, such as vasoactive hormone, or with a shear stress to mimic the blood stream. 41

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Electrochemical methods: Several electrochemical techniques can be employed to measure NO, but the amperometric and voltamme tric methods have been the most popular. Amperometry monitors the redox current in the picoampere range produced by NO oxidation at a fixed potential. Fast-scan cyclic voltammetry (FSCV) is performed by holding the microelectrode at a constant potential versus a reference electrode, followed by a rapid increase in potential, and an immediat e return back to the holding potential (K ita & Wightman 2008). With a response time of less than a few seconds and high sensitivity, these methods provide fast, quantitative measurement of small fl uctuations in NO concentration. Microelectrodes have been used to elucid ate a number of neurobiological functions, including neurotransmission processes and mechanis ms at single cell levels. Hulvey and Martin designed a microfluidic device that utilizes a reservoir-based approach for endothelial cell immobilization and integrated embedded carbon ink microelectrodes fo r the detection of extracellular NO release (Hulve y & Martin 2009). Also, the a dvantages of platinized carbonfiber microelectrodes for the direct and in s itu electrochemical det ection of NO released by neurons in rat cerebella slices were examined (Amatore et al. 2006). Furthermore, different types of carbon fibers (Textron, Amoco, Courtaul ds) and carbon nanotubes covered with Nafion/ophenylenediamine (o-PD) were evaluated for NO measurement in th e presence of major interfering molecules in the brain (asc orbate, nitrite and dopamine) (Santos et al. 2008). With continued efforts to improve the sensitivity, selectivity and reliability of the NO-sensitive electrodes, these NO sensors will be able to monitor NO produc tion continuously and in realtime at the cellular level. Immunohistochemistry: The nicotinamide adenine dinuc leotide phosphate diaphorase (NADPH-d) histochemical technique has been combined with the immunohistochemical 42

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visualization of various specific components of the NO signaling pathway, such as NOS, citrulline (Anctil et al. 2005), and cGMP. These studies have produced a wealth of data on various signaling functions for NO in the central nervous systems and peripheral tissues of the main bilaterian vertebrate and invertebrate an imal groups (Moroz 2001). Due to the ability of NOS to transfer electrons from the NADPH electron donor to a compe titor tetrazolium salt, NADPH-d activity can be used as a histochemical marker for NOS activity in vertebrate and invertebrate tissue preparations. This technique, which consists of the conversion of a soluble tetrazolium salt to an insoluble visible formazan, remains one of the most convenient procedures for the screening of NOS-c ontaining cells (Cristino et al. 2008). NADPH-d labeling has been used for the study of central motoneurons in the predatory Pleurobranchaea californica and in peripheral putative se nsory cells of the herbivorous Aplysia (Moroz & Gillette 1996). In both species NO also plays a critical role in memory formation (Katzoff et al. 2002). Also, NADPH-d activity has also been observed in putative ectodermal sensory cells of Aglantha tentacles. The neurites of these cells run very close to the swimming pacemaker and probably activate slow swimming in Aglantha (Moroz et al. 2004). Although, NADPH-d label is very useful for localizing NOS activities, it is recommended that more than one procedure be used, because antibodies which recognize invertebrate NOS isoforms are not yet available. Invertebrates for Neurochemistry Analysis Phylogeny is the fundamental product of e volution and a phyloge netic hypothesis is essential to understand biological phenomena. Much of this research has relied upon morphological characters, genes, and protein products contai ned within animal cells. The phylogeny presented here is a relatively conservative guess based upon various published studies (Schierwater et al. 2009b, Philippe et al. 2009, Dunn et al. 2008, Claus 2008). As shown in 43

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figure 1-7, there are quite a few unresolved bran ches, and therefore metabolomic information on marine organisms by studying the signaling mol ecules will help to know the relationship between animals. Figure 1-7. Phylogenetic tree of Metazoan relationships. Dashed lin es indicates the controversial data analysis. Cnidaria Cnidaria is a phylum containing most of animals found exclusively in marine environments and their distinguishi ng feature is the presence of cn idocytes, specialized cells that they use mainly for capturing prey (Claus 2008). Th eir bodies of cnidaria consist of mesoglea, a non-living jelly-like substance, sand wiched between two layers of epithelium that are mostly one cell thick (Kozloff 1990a). They have two ba sic body forms: swimming medusae and sessile polyps, both of which are radially symmetrical with mouths surr ounded by tentacles that bear cnidocytes. Both forms have a single orifice and body cavity that are used for digestion and respiration. Many cnidarian spec ies produce colonies that are single organisms composed of 44

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medusa-like and/or polyp-like zooi ds. Cnidarians' activities are coordinated by a decentralized nerve network and simple receptors. Severa l free-swimming Cubozoa and Scyphozoa possess balance-sensing statocysts, and some have simple eyes. All cn idarians reproduce sexually. Many have complex lifecycles with as exual polyp stages and sexual medusae, but some omit either the polyp or the medusa stage. Over the past twenty years, a number of bi ochemical studies have begun to document the presence of non-peptidergic neurotransmitters in the Cnidaria (Kass-Simon & Pierobon 2007). Among the hydrozoans, H. vulgaris was found to contain dopa mine and noradrenaline; Chlorohydra viridissima contained dopamine, norepine phrine, and 5-HT; and in Polyorchis, dopamine was identified in nerve-rich ti ssue by HPLC and gas chromatography/mass spectrometry (Chung et al. 1989). A number of studies have now produced str ong biochemical evidence for the presence of receptors to glutamate, gamma-aminobutyric acid (GABA), and glycine in cnidarians. Receptor binding studies on H. vulgaris have shown that glutamate binds to crude membrane fractions (Bellis et al. 1991). These studies gave the first indicat ion of a putative glutamate receptor in Hydra which may mediate an action independent of the glutathione (GSH) feeding response. GABA receptors, whose biochemical and pharmaco logical properties compare with those of mammalian ionotropic GABA receptors, have also been demonstrated in H. vulgaris (Pierobon et al. 1995). The first invertebrate receptors to glycine have been observed and characterized in membrane preparations from H. vulgaris (Pierobon et al. 2001). Biochemical, histochemical, and physiological data for NO as a cnidarian intercellular messenger have begun to accumulate. Biochemical studies give evidence for nitric oxide involvement in the GSH feeding response of H. vulgaris (Colasanti 1997). In Aglantha, a high 45

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level of nitrite was detected in the tentacles but not in the gonads or other predicted NOSnegative areas. The level of nitrite in Aglantha tentacles exceeded th e levels of nitrate, suggesting a high background le vel of NO formation (Moroz et al. 2004). In all, the accumulating biochemical findings and the still sparse molecular data, are consistent with the idea that peptides, the classi cal slow and fast transmitters, and nitric oxide may be primary neuronal messengers in the Cnidaria. Porifera The sponges or poriferans are an imals of the phylum Porifera. Their bodies consist of an outer thin layer of cells, called the pinacoderm, a nd an inner mass of cells and skeletal elements, called the choanoderm (kozloff 1990d). Sponges do not have nervous, diges tive or circulatory systems. Instead, most of these organisms rely on maintaining a constant water flow through their bodies to obtain food and oxygen and to remove wastes, and the shapes of their bodies are adapted to maximize the efficiency of the wate r flow. All are sessile aquatic animals and, although there are freshwater spec ies, most are marine species. In a recent study, the Amphimedon genome was shown to contain 36 families of genes known to encode proteins of th e post-synaptic density (Sakarya et al. 2007). So, even though it has no neurons, this sea sponge synthesizes an almost complete set of post-synaptic density proteins. A comparison of the DNA sequences from the 36 sea sponge genes with the homologous sequences from humans, Drosophila melanogaster (fruit flies) and Nematostella vectensis (a cnidarian with a simple nervous system ) revealed striking similarities between the genes in all four species. This suggests that in the sea sponge these proteins interact in exactly the same way as they do in the human post-synaptic density. Amphimedon has nearly all the components required to make a post-synaptic density. Only a few of the human postsynaptic 46

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density genes are missing from the sea sponges genome, in particular those encoding ion channel receptors for the neurotransmitter glutamate. Ctenophora Ctenophores have no brain or cen tral nervous system, but instead have a nerve network that forms a ring around the mouth and is densest ne ar structures such as the comb rows, pharynx, tentacles, and the sensory complex furthest fr om the mouth (Kozloff 1990b). The largest single sensory feature is the aboral organ at the opposit e end from the mouth. Its main component is a statocyst, a balance sensor consis ting of a statolith, a solid part icle supported on four bundles of cilia, called "balancers", that sense its orientat ion. The statocyst is protected by a transparent dome made of long, immobile cilia. In Mnemiopsis, acetlycholine and beta-adr energic mechanisms were observed in a study of the pharmacological activities associated with lumine scence control (Anctil 1985). Placozoa The Placozoa are the simplest in structure of all multicellular an imals (Metazoa). They consist of the single species, Trichoplax adhaerens. A common name does not yet exist for the taxon; the scientific name li terally means flat animals. Trichoplax are very flat creatures around a millimeter in width, which lack any organs or internal structure (Schierwater 2005). They comprise three cellular layers. Their top and bottom epitheloid la yers are identical and possess cilia used in locomotion. Although Trichoplax has no nervous system, it has behavi oural responses to environmental stimuli, and sensitivity to the neuropeptide (Arg-Phe-NH2) RFamide has been reported (Schuchert 1993). In the Trichoplax genome, DOPA decarboxylase and DBH-like monooxygenase (which are involv ed in dopamine, noradrenaline and adrenaline synthesis in adrenergic cells) and putative vesicular amine transporters (which are used for neurotransmitter 47

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uptake) are present as well as putative neurotra nsmitter and neuropeptide receptors (Srivastava et al. 2008). There are also four putative opsin genes, which possess a crucial lysine residue in the seventh transmembrane domain and are thought to function in light reception. Eighty-five members of the class 3 G-protei n-coupled receptor (GPCR) fam ily (unrelated to other GPCR families by sequence), including putative metabot ropic glutamate receptors, are also found. Transmembrane proteins important in nerve cond uction (multiple candidate ionotropic glutamate receptors) and in neurotransmitter release and uptake (for example, sodium neurotransmitter symporter) are encoded by the genome. Mollusca Molluscs are highly diverse in size, anatomi cal structure, behavior and habitat, and representatives of the phylum live in a wide rang e of environments, including marine, freshwater, and terrestrial biotopes (Kozloff 1990c). The phylu m Mollusca is typically divided into nine or ten taxonomic classes. Cephalopod, such as squid, cuttlefish and octopus, are among the most neurologically advanced of all i nvertebrates. Either the giant squid or the colossal squid is the largest known invertebrate species. The gastropods (snails and slugs) are by far the most numerous molluscs in terms of classified specie s, and they account for 80% of the total number of classified species. Regarding the cephalopods, NO is an integral component in the complex mechanisms implicated in the initiation and maintenance of th e symbiont infection of the light organ of the Hawaiian bobtail squid, E. scolopes (Davidson et al. 2004). The production of NO accompanies light organ embryogenesis, reaching highest levels in the light or gan of newly hatched animals, and NO also regulates the number of bacteria. In addition, NO acts as a major signal molecule in the molluscan nervous system (Moroz et al. 2000). It is involved in the activation of the motor 48

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49 network of feeding in Aplysia and Pleurobranchaea and plays a critical role in the formation of multiple memory processes in Aplysia (Katzoff et al. 2002). Dissertation Overview Chapter 2 describes the development of a CE -LIF for the detection of nitric oxide metabolites, such as L-arginine and L-citrullin e and other neurotransmitters. The method uses a borate buffer with a surfactant to identify and quantify neurotransmitters in marine animals. In addition, enantiomers of amino acids are analyzed by cyclodextrin (CD). Chapter 3 presents the design and evaluation of conductivity systems for capillary zone electrophoresis using a dynamic cap illary coating technique. Appli cation of these systems to the analysis of nitrite and nitrat e in complex samples such as Aplysia californica is demonstrated. Chapter 4 describes nitr ic oxide signaling in Trichoplax adhaerens. The previously developed CE systems are utilized for analyzing the NO-metabolites of Trichoplax. In addition, results of behavior tests are de scribed, in which NO donors are used to monitor the response to the pharmacological applications. Chapter 5 presents the total analysis of the NO metabolites of basal animals such as sponges, ctenophores, cnidarians, and placozoa using CE-LIF/CCD. Chapter 6 presents the comparative analys is of the NO metabo lites of cephalopods, including squid, nautilus, and Aplysia, using CE-LIF/CCD.

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CHAPTER 2 DEVELOPMENT AND EVALUATION OF CE COUPLED WITH LASER-INDUCED FLUORESCENCE (LIF) DETECTION FOR THE ASSAY OF AMINO ACIDS AND NEUROTRANSIMITTERS Introduction During the last decade, amino acid neurotransm itters have been the focus of considerable attention in biomedical research, medical diagno stics, clinical chemistry, and the pharmaceutical industry, because they play essential roles in co ntrol and regulation of va rious functions in the central and peripheral nervous system (Boulat et al. 2001, Li et al. 2008, Poinsot et al. 2008, Sheeley et al. 2005, Trapp et al. 2004, Ummadi & Weimer 2002, Xu et al. 2009). The most studied amino acid neurotransmitters are glutamic acid (Glu), aspartic acid (Asp), gammaaminobutyric acid (GABA), gl ycine (Gly), taurine (Tau), arginine (Arg), and citrulline (Cit). As the major excitatory neurotransmitters in the mammalian central nervous system (CNS), Glu and Asp are present in more than half of all CN S synapses. This underscores their important involvement in learning, memory, sleep, movement, and feeding (Rawls et al. 2006, Antzoulatos & Byrne 2004). GABA, Gly, and Tau are the inhibitory neurotransmitters in the CNS (Piepponen & Skujins 2001). In f act, as many as 10-40% of nerve terminals in the hippocampus and cerebral cortex may use GABA as a neurotransmitter to transmit closure signals (Takayama & Inoue 2004). Another inhibitory transmitter, Gl y, plays key roles in postsynaptic inhibition, sensorimotor function, and abnormal startle responses (Kopp-Scheinpflug et al. 2008). The inhibitory amino acid Tau is an osmoregulator and neuromodulator (Saransaari & Oja 2006). In addition, Arg and Cit are the key amino acids in th e indicators of nitric oxide (NO) activity and are important in a urea cycle (Moroz et al. 1999). Moreover, Arg is a metabolic precursor in the formation of creatine, polyamines, the excitatory neurotransmitt er L-glutamate, the 50

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neuromodulator L-proline, the putative ne urotransmitter agmatine, and the putative immunomodulator, arginine-contain ing tetrapeptide tuftsin (Boudko 2007). The development of microanalytical methods for assaying complex heterogeneous systems is an essential and key step in our understa nding of neuronal chemis try and functions. While such signal molecules and related molecular ma rkers have been well characterized in model organisms (Drosophila and C. elegans) (Davies 2006, Strange 2003), l ittle information exists about the specific signaling molecules patter ning the nervous system in many marine invertebrate species. For some lineages, such as ctenophores, sponges, placozoa and even many bilaterian phyla, such informati on does not exist at all (Srivastava et al. 2008, Sakarya et al. 2007, Claus 2008, Dunn et al. 2008). Among the many different types of interc ellular signaling molecules, the gaseous radical nitric oxide is of appreciable interest because of its critical role in modulation of neuronal activity (Namiki et al. 2005). Endogenous nitric oxide is a coproduct with citrulline of the oxidation of arginine by NO synthase (NOS). Thus, an alysis of the potential signaling molecules in biological samples may pr ovide insight into the nervous systems of marine animals. While the primary objective of this study invol ved the identification and characterization of key potential signaling molecules of low molecular weight and their metabolites, our effort was also focused on the analysis of D-Aspartic acid (D-Asp). This ami no acid derivative acts as a specific agonist at the N-met hyl-D-aspartic acid (NMDA) recepto r, and therefore mimics the action of the neurotransmitter glutamate on that receptor (D'Aniello 2007). In contrast to glutamate, NMDA binds to and regulates this part icular receptor only, but not other glutamate receptors. As such, we are interested in the activity of both D-Asp and Glu in these basal animals. Especially, D-Asp is found in the central nervou s system in a variety of animals, including 51

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mammals (Sakai et al. 1998, D'Aniello 2007) and mollusks (Miao et al. 2006, Song et al. 2006). In addition, D-Glutamic acid (D-Glu) naturall y presents in many microbes (Glavas & Tanner 2001) as well as in plants and animals (Corrigan 1969, Kera et al. 1996). Kera et al. reported that D-Glu may play a role in Aplysia central nervous systems (Quan & Liu 2003). CE is one of the most powerful separation techniques known to date, because it provides high separation efficiencies, short analysis times, low operation costs, small-volume compatibility, and applicability to a wide va riety of species, incl uding inorganic compounds, organic acids, proteins, peptides, amino acids, and neurotransmitters (Kostal et al. 2008, Xiayan & Legido-Quigley 2008, Guzman et al. 2008). In order to detect amino acid neurotransmitters and their enantiomers at the levels that are pres ent in marine animals using CE, highly sensitive detection methods such as LIF are very impor tant. In CE, the LIF de tector with a HeliumCadmium (HeCd) laser emitting at 325 nm is the one most frequently used. However, most of the excitatory or inhibitory neurotransmitters in the CNS do not fluorescence when excited at 325 nm. Thus, they must be derivatized with a chromophore or a fluorophore to improve both the selectivity and the sensitivity for their determin ation. Analyte derivatization in CE has been performed pre-, on-, and postcolumn using a variet y of reagents such as fluorescamine, 3-(4carboxybenzoyl)-2-quinolinec arboxyaldehyde (CBQCA), fluoresce in 5-isothiocyanate (FITC), and naphthalene-2,3-dicarboxaldeh yde (NDA) (Quan & Liu 2003, Chen et al. 2005, Bergquist et al. 1996). o-Phthalaldehyde (OPA) was chosen as the de rivatization reagent in this work because it is a fluorogenic reagent and reacts with analytes within a few seconds. The aim of this study is to develop a robust CE-LIF method for the sensitive and selective determination of amino acid neurotransmitters and their enantiomers in the marine animals after derivatization with OPA. In our experiments, optimization of the derivatization and separation 52

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conditions was carefully invest igated. The optimized separation was based on MEKC using SDS as a surfactant and also employing -CD as a chiral selector. Methods and Materials Reagents All solutions were prepared with Milli-Q water (Milli-Q filtration system, Millipore, Bedford, MA) to minimize the presence of impuriti es. Borate buffer (30mM, pH 9.5) was used for sample preparation. All solutions were filtered using 0.2 m membrane filters to remove particulates. The buffers were degassed by ultrasonication for 10min to minimize the chance of bubble formation. A 75mM OPA / -mercaptoethanol ( -ME) stock solution was prepared by dissolving 10mg of OPA in 100 L of methanol and mixing w ith 1mL of 30mM borate and 10 L of -ME. OPA and -ME were stored in a refrigerator, and fresh solutions were made weekly. Stock solutions (10mM) of amino acids and neurotransmitters were prepared by dissolving each compound in the borate buffer. L-2-aminoadipic aci d (Sigma), dissolved in 30mM borate buffer (pH 9.5), was used as internal standard and made fresh for all experiments. Unless specified, all chemicals were obtained from Sigma (St. Louis, MO) and were reagent quality or better. CE Instrumentation CE coupled with a ZETALIF detector (Picomet rics, France) was used for the assay of amino acids. The Picometrics ZETALIF detector is a single excitation LIF detector, which is modular and external to the CE instrument. This detector is based on a confocal microscope setup, where a ball lens concentr ates the laser light into the ca pillary, and the fluorescence is collected by the same ball lens which has a ve ry high numerical aperture (Figure 2-1). The emission is then passed through a series of filte rs and the fluorescence is then measured by a photomultiplier tube (PMT). The use of a ball lens is very useful because it allows the use of a very stable excitation beam and the collection is bette r than without a ball lens using a simple 53

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microscope (Rodat et al. 2008). In addition, this setup can be easily used with UV lasers. In this work a multiline helium-cadmium laser (325nm ) from Melles Griot, Inc. (Omnichrome Series56, Carlsbad, CA) was used as the excitation source. Before the PMT, the fluorescence was filtered. All instrumentation and high-voltage CE power supply were controlled using a DAx 7.3 software. Figure 2-1. Schematic optical layout of the fluorescence detection system All experiments were conducte d using a 75cm length of 50m I.D. 360m O.D. fused silica capillary (Polymicro T echnologies, Phoenix, AZ). Th e 30mM borate and 30mM sodium dodecyl sulfate (SDS) electrolyte (adjusted to pH 10.0 with NaOH) was used for a separation 54

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buffer for amino acids analysis, and 15mM bor ate and 10mM beta-cyclodextrin electrolyte (adjusted to pH 10.0 with NaOH) was used for a separation buffer for chiral analysis of both Glu and Asp. For pre-column derivatization method, 1 L of OPA was incubated at 20oC for 6 minutes in a 0.5mL PCR tube with 18 L sample and 1 L internal standard. For separation steps, the capillary inner wall was successively washed with 1M NaOH for 2mins, Milli Q water for 3mins, and the separation buffer for 2mins by applying pr essure (1900mbar) to the inlet vial. Then a sample was loaded using electrokinetic injecti on (8kV for 12s). The separation was performed under a stable 20kV voltage at 20 C. Data Analysis Once an electropherogram was acquired, peak s were assigned by relative electrophoretic mobility and confirmed by spiking corresponding standards into the sample. Five-point calibration curves (peak area vs. concentration) of analytes were constructed for quantification using standard solutions. The 3 method was used to determine the limit of detection (LOD): LOD = mblank 3 2-1 where m is the slope of the calibration line and blank is the standard deviation of the blank (n=5). The reproducibility and accuracy of the method were evaluated by calculating relative standard deviation (RSD) for each analyte. In order to obta in the peak area, a baseline is constructed and subtracted using the derlim al gorithm of DAx software versi on 7.3 (Van Mierlo Software Consultancy, the Netherlands). A statistical data analysis is performed by Sigma Plot software (SPSS, Inc., Richmond, CA). 55

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Results and Discussion Optimization of CE Separation Conditions At first, CZE was used to separate OPA neurotransmitter derivatives because of its simplicity and speed in practical applications. However, as shown in figure 2-2, complete resolution of a mixture of amino acids was difficu lt in CZE, because the se paration is critically dependent on the charge-to-mass ratio of analyt es. While CZE allowed resolution of Arg, Glu, and Asp, the peaks for the others overlapped. Figure 2-2. Electropherograms of standard am ino acids (1M). Separation was conducted in 50m I.D. and 360m O.D. capillary w ith 30mM borate, pH 9.4 at 20kV A good alternative to CZE is offered by MEKC, in which the addition of a surfactant to the running electrolyte provides a two-phase chroma tographic separation medium, an aqueous phase and a micellar pseudophase. This makes MEKC very attractive for the differentiation of analytes with similar physico-chemical properties, particularly the amino acids (Iadarola et al. 2008). Therefore, MEKC was employed in this rese arch to separate the labeled amino acid neurotransmitters. Effect of surfactant concentration: SDS is an anionic surfactant that has been widely used in MEKC for amino acid analysis (Paez et al. 2000, Siri et al. 2006, Tivesten & Folestad 56

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1997, Zhu et al. 2005). Moreover, the concentration of su rfactant has a significant effect on the separation selectivity by adjusting the partition of analytes betw een the micellar pseudophase and the aqueous phase. Thus the effect of SDS concen tration on resolution was first examined in this work. SDS was added to the borate buffer at several different concentrations above its critical micelle concentration, including 10, 25, 30, and 50mM (Figure 23). Because the Arg peak was not observed within the 20-minute migration time window, so it wa s necessary to record data with extended time. The analyte separation clear ly improved with increas ing concentrations of SDS, probably due to increased micelle analyte in teraction, but when the concentration of SDS was more than 50mM, amino acids could not be distinguished from the baseline. Also, excessively high SDS concentrations (more than 50mM) resulted in longer migration times and larger currents without significant improvement of separation. As a result, 30mM was finally chosen as the optimized concentration of SDS used (Figure 2-3B). Figure 2-3. Electropherograms of standard amino acids depending on A) various SDS concentrations and B) difference between 10 and 30mM SDS. Peaks: Citrulline (Cit), Serine (Ser), 5-Hydroxytryptophan (5-HTP), -Aminobutyric acid (GABA), Tyrosine (Tyr), Glycine (Gly), Taurine (Tau), Phenlyalanine (Phe), Dopamine (DA), Tryptophan (Trp), 3,4-dihydroxy-L-phenylalanin e (L-DOPA), Internal standard (I.S.), Glutamate (Glu), Aspartate (Asp), Arginine (Arg), Octopamine (Oct), Serotonin (5HT) 57

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Effect of buffer pH: Different pH values of the runni ng buffer can influence the mobility of analytes by changing the charges on the anal ytes and the capillary wall. The influence of pH values of boric acid buffer in the range of 9. 4-10.0 was studied. It was found that increasing the pH can significantly improve the re solution of these derivatives, especially for labeled Tyr and GABA (Figure 2-4A). When the pH of borat e buffer was 10.0, OPA labeled Tyr and GABA were reasonably separated, but they migrated toge ther at pH 9.4. Nevertheless, a further increase of buffer pH prolonged the migration time a nd resulted in co-migration of Phe and DA. Considering both the resolution and the analysis time for 16 deseired analytes, pH 10.0 was adopted for the CE separation. Figure 2-4. Electropherograms of standard amino acids. A) Depe nding on different pH conditions, 9.4 and 10.0, respectively. B) Optimized CE conditions: 30mM Borate, 30mM SDS, pH 10.0, 20kV Influence of separation voltage: Under the above optimum c onditions, the influence of separation voltage (15-20 kV) was tested. It was found that sixteen OPA-labeled amino acid neurotransmitters were baseline separated wh en the voltage reached 20 kV. But a further increase in voltage resulted in a baseline fluctu ation, whereas a decrease of voltage prolonged the 58

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time of analysis. Based on the above optimized procedure, the following running buffer was used for the separation of the OPA derivatives: 30mM borate (pH 10.0) containing 30 mMSDS. The voltage applied was 20 kV. Figure 2-4B show s a typical electropher ogram of amino acid neurotransmitters of interest under the optimized conditions. Analytical Calibration Standard solutions of amino acid neurotransmitters were analyzed by CE-MEKC-LIF under the optimal derivatization and separation conditions mentioned above. The corresponding calibration curves were constructed by plotting pe ak area versus the analyte concentration. The correlation coefficients for these neurotransmitters were from 0.9989 to 0.9998, and the LODs (S/N = 3) ranged from 5.57nM for Phe to 149nM for Cit. Figure 2-5. Calibration curves of standard amino acids. (n=5) The reproducibility (expressed in RSD) test wa s carried out by repeating four sequential runs within-day and between-day using 100nM standard amino acid neurotransmitter solutions (Table 2-1). It was found that to ensure the reproducibility of the method, a rinsing sequence consisting of NaOH, water, and running buffe r should be performed between each run to eliminate the adsorption of analytes onto the capillary wall. The within-day RSDs for the OPA 59

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derivatives ranged from 1.2% for Arg to 2.89% fo r 5-HT in peak areas. The between-day RSDs were found to be less than 4.9% for peak areas. The observation shows excellent reproducibility of the migration times, and good reproducibility of the peak areas. Table 2-1. Correlation coefficients, RSDs, and LOD Analyte r RSD (%, n=4) LOD (nM) Within-day Between-day Peak area Peak area Cit 0.9985 1.37 3.07 149 Ser 0.9963 1.64 3.55 62 5-HTP 0.9961 1.62 3.92 73 Tyr 0.9992 1.62 3.42 10.7 GABA 0.9991 2.06 3.37 76.1 Gly 0.9923 2.01 3.94 128 Tau 0.9953 1.98 3.63 95.8 Phe 0.9982 2.05 2.85 5.57 DA 0.9853 1.91 4.26 69.2 Trp 0.9959 2.17 2.91 7.14 Glu 0.9962 2.84 3.75 22.3 Asp 0.9972 2.73 3.38 16.5 Arg 0.9674 1.20 3.81 113 Oct 0.9562 1.42 4.89 48 5-HT 0.9213 2.89 3.67 85 Glu and Asp Enantiomer Separation pH studies: Since the interactions between -CD and OPA-derivatives are pH dependent, various pHs were tested to optimize the sepa ration of the Glu enantiomer. However, Asp enantiomer showed less dependence on pH (data not shown). The L and D forms of Glu closely migrated at pH 9.0, but they were resolved with increasing pH, and were completely separated at pH 10.0 (Figure 2-6A). The hydroxyl groups of -CD become negative in a basic condition, and with a 20kV separation voltage the EOF is towards the negative electrode, while -CDs move in the opposite direction (Juvancz et al. 2008). Thus, the more an an alyte interacts with the -CD, the slower the complex moves to the negative elec trode. Figure 2-6B shows that the interaction strength between -CD and analytes are L-Asp > D-Asp > D-Glu > L-Glu. 60

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Figure 2-6. Electropherograms of Glu and Asp enantiomers. A) Glu enantiomer separation depending on pH ranging from 9.0 to 10.0. B) 1 M of L-Glu and D-Asp, and 100nM of D-Glu and L-Asp at pH 10.0. C) Standa rd calibration curves. Samples were loaded using electrokinetic injecti on (8kV for 12s), and then analyzed under a stable 20kV voltage at 20oC in 50m I.D. and 360m O.D. cap illary with 15mM borate and 10mM -CD Performance tests: Calibration curves with five point s were constructed by injecting a series of standard mixtures c overing the tested concentration range (Figure 2-6C). Equations were obtained by least-squares li near regression analysis of the peak area versus analyte concentration. Table 2-2 summarizes the results of the determina tion of reproducibility regarding accuracy, within-day and day-to-day precision assa ys and LOD. The intra-assay precision of the method based on within-day repeatability was pe rformed by replicate injections (n=4), where 61

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62 ) Analyte r RSD (%, n=4) LOD (nM) peak areas were measured. Statistical evaluation provided the relative stan dard deviations (RSD at different concentrations. The inter-assay precision (between-day variat ion) of the method was established by triplicate measurements of each co ncentration over a period of 3 different days. The measured concentrations had RSD values <4%. Table 2-2. Correlation coefficients, RSDs, and LOD Within-day Between-day Peak a k area L-Glu 0.9967 48 rea Pea 2.21 3.07 D-Glu 0.9961 105 2.01 3.21 D-Asp 0.9775 2.89 3.92 45 L-Asp 0.9781 2.75 3.63 51 Conclusion In this article, a robust CELIF analy for the analysis of amino acid neuro OPA. solution, tical method transmitters and enantiomers was proposed based on chemical derivatization with The OPA derivatives were base line separated in 30 mM borate buffer (pH 10.0), containing 30 mM SDS. The LOD for neurotransmitters was as low as 5.57nM. The practical utility of the proposed method will be demonstrated in chap ters 4 to 6 by the detection of amino acid neurotransmitters and enantiomers. With its high sensitivity, excellent selectivity, high re and good repeatability, this approach can detect amino acid neurotransmitters released from a complex biological sample.

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CHAPTER 3 DEVELOPMENT AND EVALUATION OF CE COUPLED WITH CONTACTLESS CONDUCTIVITY DETECTION TO IMPROVE THE ANION ASSAY Introduction Nitric oxide (NO) produced by nitric oxide synthase (NOS) has been known to play an important role in vascular homeostasis, neur otransmission, and immunological host defense mechanisms (Ignarro 2000). Due to the extremely short physiological half life of this gaseous free radical, alternative strategies for the detection of the reac tion products of NO biochemistry have been developed (Boudko 2007). Both detecti on and quantification of NO-metabolites are crucial to understanding health and disease. The major pathway for NO metabolism is the stepwise oxidation to nitrite (NO2 -) and nitrate (NO3 -) (Ignarro et al. 1993). In plasma or other physiological fluids or buffers NO is oxidized almost completely to nitrite, where it remains stable for several hours (Moroz 2001), but NO and nitrite are rapidly oxidized to nitrate in whole blood by certain oxyhemoproteins (oxyhemoglobin or oxymyoglobin) (Ignarro et al. 1993). On the other hand, it is now found that nitrite or nitrate can be redu ced to NO in various ways and the mechanisms have been extensively review ed (Lundberg & Weitzberg 2005). The nitrite and nitrate determination can not only reflect NO pr oduction but may also serv e as an alternative source of NO. Therefore detection and quantification of nitrite a nd nitrate provides an index of NO bioavailability or production. In addition to the role of NO in the biological pathways, nitrite is now considered a central homeostatic molecule in NO bi ology and may serve as an important signaling molecule (Bryan et al. 2005, Bryan 2006). It was demonstr ated that plasma nitrite le vels progressively decrease with increasing cardiovascular risk load (Kleinbongard et al. 2006). Furthermore, the determination of the nitrate levels from the vitreous humor of patients having diabetic retinopathy suggested that NO was involved with the pathology of this disease (Gao et al. 2007). 63

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Therefore it is important to car efully and accurately analyze all nitrite and nitrate in biological samples. However, the lack of data on the spatial distri bution of nitrites and nitrates in the nervous system greatly limits our understanding of NO-me diated pathways. The major problem is the absence of adequate analytical approaches for the microchemical analysis of nitrite and nitrate in small samples, which would represent specific metabolic domains of neuronal tissues. Capillary electrophoresis (CE) has been successfully used to analyze subnanoliter sample volumes in a large number of bioanalytical applications (Boudko 2007, Tseng et al. 2007). In particular this method is advantageous for neuronal microchemi cal analysis, because of the extreme cellular and chemical heterogeneity of neuronal samp les (Ye et al. 2008). Reduction of the sample volume enhances both the spatial and temporal re solution of analytical assays (Boudko et al. 2002). At a certain level of vol ume reduction, sampling also b ecomes nondestructive to cells, which benefits cellular physiology, biomedical res earch and therapeutic dia gnostics (Bakry et al. 2007). Most of these applications utili ze a UV-based detec tion technique (Gao et al. 2004), although it is obviously not a method of preferen ce due to its relatively low sensitivity. The technique of choice to detect inorganic and small organic ions is conductivity detection (CD), which provides approximately 10 times better lim its of detection (LOD). Several ion assays using ion chromatography and CE with CD have been described and a commercial CD system suitable for CE integration has re cently become available (Boudko et al. 2002). Here we describe a high-reso lution CZE technique for the sa mpling and evaluation of ionic profiles in small and specific samples from Aplysia californica. Our goals were 1) to evaluate an approach for anion analysis of biological tissue samples based on the combination of capillary zone electrophoresis and contactle ss conductivity detecti on (CCD), 2) to optimize this technique 64

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for the determination of the nitric oxide-related metabolites, nitrite and nitrate, and 3) to optimize this technique for the automatic analysis of ultrasmall samples so that it would be suitable for the analysis of small neuronal clusters and indi vidual neurons. The described CZE technique is effective for the indirect monito ring of alterations in NO producti on from small ganglia or even individual neurons in sp ecific neuronal regions. Methods and Materials Instrumentation A computerized GPA100 (Groton biosystems) CE with a robotic sample injector coupled to a TraceDec contactless conductivity detector (I nnovative Sensor Technologies GmbH, Strasshof, Austria) was used. Separation was performed using a 70 cm (50 m I.D.360 m O.D.) fused-silica capillary (Polymicro Technol ogies, AZ, USA). DAx 7.3 data acquisition and analysis software (Van Mierlo Software Consul tancy, Netherlands) was used to control the CE and acquisition board, as well as fo r data recording and analysis. Reagents Analytical-grade chemicals were obtained from Sigma (St. Louis, MO, USA). The background electrolyte was an ar ginine-borate buffer with adde d tetradecyltrimethylammonium hydroxide (TTAOH) (25mM arginine, 81.5mM borat e and 0.5mM TTAOH, pH 9.0) to modify the electroosmotic flow (EOF). The TTAOH wa s prepared by converting the bromide salt (TTAB) into the hydroxyl form us ing a styrene-based, anion-exch ange resin cartridge (On-Guard A, Dionex, CA, USA). The bu ffer was filtered through a 0.2 m membrane. Fresh electrolyte was prepared daily and degassed with combined vacuum-ultrasonic agitation prior to use. Animals Aplysia californica were obtained from either the Aplysia Research Facility (Miami, FL) or Marinus Scientific (Long Beach, CA), depending on the animal size (100-400g). After arrived, 65

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they were stored in 40-400 liter aquaria with circulating fresh sea water at 15-17 C until use. Feeding for animals was regulated to pr event interferences from ingested food. Hemolymph and Ganglia Ganglia (multiple clusters of neurons) and hemolymph (a combination of the fluids blood and lymph in invertebrates) were collected from Aplysia. In detail, all animals were pre-stored in a cold room at 4 C for 90 minutes to minimize the animal s inking while obt aining hemolymph. Afterwards, 1-2mL hemolymph was collected us ing a disposable syringe and passed through a nylon-membrane (0.2-m pore size, Millipore) syringe fi lter to remove debris. Next, Aplysia californica were anesthetized via injection of isotonic MgCl2 (337mM) equal to 50~60% of their weight, prior to removal of the central nervous system (CNS). After th e CNS was removed from the animal, it was incubated in 1% Protease IX (S igma, P-6141) in filtered artificial sea water (ASW: 460mM NaCl, 10mM KCl, 55mM MgCl2, 11mM CaCl2, 10mM HEPES, pH 7.6) at 37 C for 30 min to loosen the connective tissue of the neuronal sheath. Then, the ganglia were washed in fresh ASW three times to remove excess pr otease and were pinned down on a Sylgard (Dow Corning)-coated Petri dish containing ASW. Inner connective tissue sheaths surrounding the ganglia were removed manually using tungsten fo rceps and scissors under a stereomicroscope (WPI, Sarasota, FL, USA). The sample volume (V) was calculated from dimensions of the neuron under an Olympus SZX12 stereomicroscope assuming ganglia are spherical (V = 4r3/3). Chloride Cleanup by Solid-Phase Extraction The native chloride peak in most biological samples is so larg e that it masks the nitrite and nitrate peaks. To improve nitrite determination, chloride anions were removed by passing the sample through a silver-form sulfonated styren e-based resin using la boratory made microcartridges suitable for cleanup of 20 L samples by the spin-enfor ced solid-phase extraction 66

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(SESPE) technique. Resin was obtained from an OnGuard-Ag cartridge (Dionex, Sunnyvale, CA, USA). The cartridge was pre-cleaned by adding 1mL of 1M NaOH and 1mL of 18M Milli-Q water. Approximately 4.0 mg of th e resin was back-loaded into 0.1-10 L filter tips (USA Scientific, FL, USA), which were then used as SESPE cartridges. The resin-containing filter tips were inserted into larger 200 L tips to prevent surface contamination and to separate waste acquired during spinning (Figure. 3-1). Then the unit was centrifuge d using an Eppendorf centrifuge, and evaporated in air for overnight at room temperatur e. Pretreated cartridges were inserted into PCR tubes loaded with 20 L of diluted neuronal samples and spun for approximately 10s at 1000rpm, allowing sample passage through the cleanup column. Sodium fluoride (100nM) was then added to the final assay sample to generate an internal reference peak, which was used to identify ions. Figure 3-1. Custom-made and fact ory-made chloride clean-up kit Separation and Analysis A sample set, along with a set of gradually diluted standards, was injected with the CE unit and analyzed in the automatic mode using identi cal capillary tr eatment and separation conditions. The sample was introduced into the capillary by isotachophoreti c-stacking injec tion (-5kV for 12s) into a preloaded plug of high-mobility el ectrolyte (pressure-loaded 12mM LiOH, 50mbar 67

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for 12s). The separation conditi ons were: -15kV, 20C, running time 15min. The capillary was precleaned with 1M NaOH (1700mbar for 2 min) washed with DW (1700mbar for 2min) and loaded with buffer (1700mbar fo r 2min) prior to each run. Anions were identified by absolute retention times relative to fluoride. Some samples were analyzed a second time afte r spiking with nitrite or nitrate st andards to validate the quantification of these ionic species. The data analysis combined high-frequency cut-off filtering and baseline reconstruction/subtraction with a five-data point moving average algorithm. This was followed by an automatic peak identification and qua ntification protocol, which depended upon an initially created standard database Ion concentrations were determ ined from relative peak areas and calibration slopes using DAx 7.3 software. Finally, electropherograms were exported into text file format (*.txt) and assembled into representative graphs with SigmaPlot 10.0 (Systat Software Inc. CA, USA). Data were further anal yzed according to the procedures specified in Chapter 2. Results and Discussion Solid-Phase Microextraction (SPME) Cleanup A typical problem in nitrite/nitrate determinatio ns in biological matrices with CZE and CD is interference from the high natural concentratio ns of chloride anions, which mask and reduce the injected quantity of other ions (Boudko et al. 2002). Bromide, chloride, nitrite and nitrate slightly differ in their electrophoretic mobilitie s; thus, high chloride activities in marine biological samples interfere with th e electrokinetic injection and detection of nitrites and nitrates known to be present at much lower concentrat ions (Stratford 1999). The commercial chloride clean-up kit requires relatively large sample volumes to be treated for sufficient sample recovery (Boudko 2007). 68

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A simple cleanup procedure for the treatment of 20 L liquid samples which utilizes cartridges prepared by back -loading disposable 0.1-10 L filter tips (USA Scientific) with Dionex OnGuard-Ag resin (Figure 3-1). These cartridges were effective for the treatment of 100to 10,000-fold diluted neuronal samples. The cartri dge performance is summarized in Figure 3-2. Nitrite and nitrate showed good r ecovery after the samp les were filtered with the chloride removal kit. Average % recoveries (RSD) are: ch loride (6.4.0), nitrit e (91.5.2), and nitrate (123.8.4). Figure 3-2. Sample recovery graph. The concentration of each sample was 1 M. n=5 Optimization of Separation Although 1000-fold dilution of neuronal samples allowed effective chloride removal with the described micro-cartridge, ad ditional dilution of samples was necessary to obtain a sufficient volume for consistent sample loading in the automatic injection mode s. We found that a 20 L sample in the standard sampling vial is suffici ent for consistent ion quantification with the GPA100 and TraceDec system, whereas a 10 L sample is insufficient. Sample dilution also produces CE peak sharpening due to ion stacking at the samplecarrier electrolyte interface and therefore provides better LOD values. High field stacking pre-concentr ation is the commonly 69

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used optimization strategy for C ZE analysis of diluted ion samp les, leading to a 50-1000-fold increase in sensitivity (Shihabi 2000). To increase the analytical performance for the analysis of diluted neuronal tissue samples, the EOF-modi fying additive TTAOH was used. The leading electrolyte (12mM LiOH), a plug of high ion mobili ty, was also preloaded to further increase the efficiency of ITP stacking and therefore improve the LOD value. The calibration curves were constructed fo r sodium nitrite and sodium nitrate in concentrations of 0.1-10 M in ultrapure water (Figure. 3-3) The regression lines of the peak area versus standard concentrations were linear w ith correlation coefficients r = 0.9950 for nitrite and r = 0.9928 for nitrate and limits of detection of 13.3nM for NO2 and 32.4nM for NO3 -. Figure 3-3. Calibration curves of A) nitrite and B) nitrate. All st andard solutions were prepared in ultrapure water. n=5 Analytical Performance Hemolymph and ganglia (buccal, cerebral, Left (L)-pleural, Right (R)-pleural, L-pedal, Rpedal, and abdominal) were sized under stereomi croscope, as shown in Table 3-1. Pedal ganglia showed the largest size, and bu ccal ganglia were the smallest. 70

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The electropherograms of 8 standard anions and each ganglion sample are shown in Figure 3-4. The average migration times (tMSD) for chloride, nitrite, a nd nitrate were 3.5.03min, 3.7.05min, and 3.8. 05min, respectively. Table 3-1. Description of hemolymph a nd central nervous system ganglia of Aplysia californica Full Name Image Description Hemolymph Obtained 1-3mL right before dissection Filtered by nylon-membrane (0.2-m pore size) Color: violet Animal size: ~130g Buccal Ganglia Diameter: 750 m Total volume: 0.442L Cerebral Ganglia Diameter: 1050 m Total volume: 1.21L Pleural Ganglia Diameter: 1200 m Left or right ganglion volume: 0.905L Pedal Ganglia Diameter: 1800 m Left or right ganglion volume: 3.05L Abdominal Ganglia Diameter: 1200 m Volume: 1.81L 71

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The selectivity of the method ( ) and the resolution values (Rs) were calculated as = 1.06 and Rs = 2.2 for nitrite/chloride, and also = 1.03 and Rs = 1.0 for nitrate/nitrite. In hemolymph, nitrate was detected in the absence of nitrite, because nitrite may have been rapidly oxidized by oxyhemoproteins or present at very low concentration. In addition, HPO4 2 is present at all electropherograms from ga nglia, whereas in hemolymph it is not, indicating that this anion may come from cell homogenates. Figure 3-4. Electropherograms of standard solu tions (650nM of all anions), hemolymph, and central ganglia in Aplysia californica. All animal samples were 10,000-fold diluted. The baseline was subtracted and reconstructed Nitrite and nitrate concentrations were deri ved from standard calibra tion curves and they were in mM ranges, as shown in figure 3-5. Nitr ite was observed in all ga nglia. In particular, in 72

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buccal and cerebral ganglia NADPHd results showed that high NOS activities were observed (Moroz 2006), detection of nitrite and nitrate pr ovided additional evidence that NO may play a role in activating the feeding mechanism in Aplysia californica. Figure 3-5. Nitrite and nitrate concentrati ons of hemolymph and central ganglia in Aplysia californica. (Bars are concentrations SD, n = 17 for hemolymph and n = 6 for each ganglia measurements) The intracellular concentration of nitrite in ga nglia was detected in the range of 0.6-2.7mM, but it was not observed in the hemolymph. In our study, nitrate concentration in Aplysia hemolymph was approximately 1.4mM. In contrast it was reported that the concentration in hemolymph of Pleurobranchaea californica and Lymnaea stagnalis were 1.83mM and 32M, respectively (Cruz et al. 1997). N itric oxide in oxygen-containi ng, abiotic solutions barely produces NO3; however, in biological systems the pres ence of heme proteins and other oxidants facilitates further rapid oxidation of NO2 to NO3. The absence of nitrite in hemolymph was also reported in vertebrate samples such as urine and plasma, due to the presence of oxyhemoglobin (Meulemans & Delsenne 1994). 73

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74 Conclusion The data clearly demonstrates the ability to measure nitrite and nitrate from biological samples with sensitivity suffici ent for single cell analysis. In Aplysia californica, nitrate anion accumulation was observed in hemolymph and in cen tral ganglia, but it was widely distributed throughout the animals bodies. In contrast, the nitrite anion was located in specific areas. The next step will utilize this sepa ration system to tackle the analysis of basal animals with CE coupled with contactless conductivity detection. Th e ratio of nitrite to nitrate will be used for evaluating NO production and will provide strong evidence for levels of actual NOS activity.

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CHAPTER 4 NITRIC OXIDE (NO) SINGNALIN G IN TRICHOPLAX ADHAERENS Introduction Nitric oxide (NO) is a widespread intracellu lar and intercellular signaling molecule in vertebrates and invertebrates with a variety of functions in the ner vous, cardiovascular and immune systems (Cristino et al. 2008 Garthwaite & Boulton 1995, D'Atri et al. 2009). It is generated from L-arginine and molecular oxygen by the enzyme NO synthase (NOS), which occurs in mammals in at least three distinct isoforms: neuronal, i nducible and endothelial (Ignarro 2000). In recent years, a growing body of evidence has implicated NO-signaling in various organisms throughout the phylogenetic scal e, notably invertebrates, where it has been shown to play important roles in a vari ety of functions (Cristino et al. 2008). Trichoplax adhaerens is an enigmatic disk-like an imal consisting of only four morphologically identifiable cell types arranged into 3 layers surface, middle and lower (Schierwater et al. 2009a). The animal lacks anterior-pos terior polarity, but shows distinct dorsal-ventral surfaces through its intriguing righting behavior, and it ha s gland cells with digestive function in the ventral epithelium. In the absence of su fficient morphological characters, its phylogenetic placement has long been controversial, and recent molecular data have not been able to resolve the issue (Mil ler & Ball 2008). The recently released genome and transcriptome information reveal several genes coding for tr ansmitter synthesis enzymes and neuroendocrinelike signaling molecules (Srivast ava et al. 2008). Thus the st udy of placozoans may provide insights into the early evolution of the nervous system. Speci fically, three isof orms of nitric oxide (NO) synthases were identified in Trichoplax, as well as receptor components homologous to all major neurotransmitter systems in bilate rians, including mammals. However, there is no direct evidence for the presence of NO-related metabolites and neurotransmitters. 75

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Detection and quantification of NO are especi ally complex tasks, because of the small active concentrations, high reactivity with biogeni c free radicals, metal coordinating molecules, and other biogenic-active species in vivo (Ignarro et al. 1993). These biological factors result in a short lifetime and complex analytical signature for this molecule. Currently, there are two main analytical methods, direct and indirect, to measure NO activity in nitrergic neurons under biological conditions. Direct NO detection methods include the following: chemiluminescence assay, spectrophotometry, fluorometry, electroc hemical techniques, and electron paramagnetic resonance (EPR) spectroscopy (Ye et al. 2008). However, the direct NO assay techniques suffer from sensitivity or specificity problems in biological systems. In contrast, indirect methods mainly use NADPH-diaphorase (NADPH-d) histochemistry, in-situ hybridization, or detection of NOs oxidation products (nitrite and nitrate) or its co-product (L-citrulline) (Cristino et al. 2008). It was demonstrated that neuronal concentr ations of nitrite/nitr ate could reflect NOS activity in the rat brain tissue (Salter et al. 1996). Thus, indirect me thods are preferred for measuring concentrations of NOS-related metabolites, and provide good evidence for NOS activity. Capillary electrophoresis (CE) is an effici ent, ultra-small-volume separation method suitable for the determination of charged species in an aqueous media. It has been widely used for the analysis of amino acids and derivatives (Zhu et al. 2005). Compared to other detection modes, laser-induced fluorescence (LIF) detect ion in CE exhibits the best performance characteristics, limits of detection, and linearity (Boudko 2007). Along with L-arginine and citrulline, general am ino-acid profiling of Trichoplax was investigated. As most analytes are not fluorescent, they are commonly derivatized usin g fluorogenic reagents that either are not fluorescent at the excitation wavelength prior to reaction or have nonfluorescent hydrolysis 76

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products. o-Phthalaldehyde (OPA) was chosen as the de rivatization reagent in this work because it is a fluorogenic reagent and reacts with analytes within a few seconds. For nitrite and nitrate detecti on, contactless conductivity dete ction (CCD) was used, due to the excellent sensitivity to small ions. In CCD, the impedance is measured as a function of the cell capacitance value, which depends on multiple factors, including the dielectric (conductivity) profile of the environment and the geometry of the cell and electrodes (Zemann 2003). The major challenge of CCD in the determination of n itrite and nitrate in biological samples is the high concentration of Cl. In vivo, the concentration of Cl is 2-3 orders of magnitude higher than NO2 or NO3 and its electrophoretic mobility is close to that of NO2 and NO3 (Stratford 1999). To reduce chloride interference and improve NO2 detection, a sample cleanup procedure was developed using a home-made solid-phase mi croextraction (SPME) cartridge filled with Dionex OnGuard-Ag cationic exchange resin (Boudko et al. 2002). To establish that NOS enzymatic activity is responsible for producing the Arg/Cit ratio and nitrite measured in Trichoplax, the whole animal was incubated in NOS inhibitor, primarily, NGnitro-l-arginine methyl ester (L-NAME). In addition, another NOS inhibitor, L-N6-(1iminoethyl)-lysine (L-NIL), showed very effective inhibition (Bodnarova et al. 2005, Hansel et al. 2003, Legrand et al. 2009). Methods and Materials Chemicals and Reagents All chemicals for buffers were purchased from Sigma-Aldrich, and standard amino acids were purchased from Fluka. Ultrapure Milli-Q water (Milli-Q filtration system, Millipore, Bedford, MA) was used for all buffers, stan dard solutions, and sample preparations. 77

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Animal Culture Trichoplax adhaerens were cultured in glass dishes containing red sea water at room temperature until use. The red sea water was replaced every 30 days. NOS Inhibitor Incubation After the animals were isolated from the culture medium, they were placed in a 0.5mL PCR tube and incubated with certain concentratio ns of NOS inhibitors for 30 minutes at room temperature, followed by washing wi th artificial sea water. Then all the water was removed and 1uL of Milli Q water was dropped onto the animal, and the tube was stored at -80oC until use. Amino Acids Microanalysis using CE with LIF The CE coupled with the ZETALIF detector (P icometrics, France) was used for the assay of amino acids. In this work a helium-cadmium laser (325nm) from Melles Griot, Inc. (Omnichrome Series56, Carlsbad, CA) was used as the excitation source. Before the photomultiplier tube (PMT), the fluorescence was both wavelength filtered and spatially filtered using a machined 3-mm pinhole. All instrument ation, counting, and high-voltage CE power supply were controlled us ing DAx 7.3 software. All solutions were prepared with ultrapur e Milli-Q water to minimize the presence of impurities. Borate buffer (30mM, pH 9.5) was used for sample preparation. All solutions were filtered using 0.2m filters to remove particulates. The buffers were degassed by ultrasonication for 10 min to minimize the chance of bubble formation. A 75mM OPA / -mercaptoethanol (ME) stock solution was prepared by dissolving 10mg of OPA in 100 L of methanol and mixing with 1mL of 30mM borate and 10 L of -ME. Stock solutions (10mM) of amino acids and neurotransmitters were prepared by dissolving each compound in the borate buffer. OPA and ME were stored in a refrigerator, and fresh solutions were prepared weekly. 78

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All experiments were conducte d using a 75cm length of 50m I.D. 360m O.D. fused silica capillary (Polymicro Technologies, AZ). A 30mM borat e/ 30mM sodium dodecyl sulfate (SDS) electrolyte (adjusted to pH 10.0 with Na OH) was used as the separation buffer for amino acid analysis. Pre-column deri vatization method was used. A 1 L of OPA was incubated in a 0.5mL PCR tube. The total volume of sample, OPA, and internal standard inside the tube was 20 L. For separation steps, the capillary inner-wall was successively washed with 1M NaOH, Milli Q water, and the separation buffer by applyi ng pressure (1900mbar) to the inlet vial. Then the sample was loaded using electrokinetic injection (8kV for 12s ). The separation was performed under a stable 20kV voltage at 20 C. Nitrite/Nitrate Microanalysis using CE with Contactless Conductivity Capillary electrophoresis coupled with a Tr aceDec contactless conductivity detector (Strasshof, Austria) was used for th e assay of nitrite and nitrate in Trichoplax. In order to reduce Clin a sample, OnGuard II Ag (DIONEX Corp ., Sunnyvale, CA) was purchased. Since small sample volumes (20L) are used, custom-built cartridges were used for sample clean-up using a solid phase extraction technique with a minor modification of th e study described in Chapter 3. In brief, 4~5mg of the resin was back loaded in a 10L filterpipette tip, and the micro-cartridge was washed with 1mL of ultrapure water usi ng a 3mL disposable syringe. The pre-washed cartridge was put into a 200L pipette tip to avoid surface contamination during further centrifugation. Extra water remaining in the ca rtridge was removed by centrifugation at 1000rpm for 30 seconds. Then, the assembly was inserted in to 0.5mL PCR tube and a final diluted sample was loaded into the preconditioned cartridge followed by a centrifugation at 1000rpm for 30 seconds, causing the sample to pass through the silver resin. In order to quantitate any potential 79

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sample loss, the custom-made chloride cartridge was tested for sample recovery of both nitrite and nitrate. All experiments were conducte d using a 75cm length of 50m I.D. 360m O.D. fused silica capillary (Polymicro Technologies, AZ) with an insu lated outlet con ductivity cell. Arginine/borate electrolyte was used for a sepa ration buffer with tetradecyltrimethylammonium hydroxide (TTAOH) added as an EOF modifi er. The modifier was prepared from tetradecyltrimethylammonium bromide (TTABr) by an OnGuard-II A cartridge (DIONEX Corp., CA) treated with 1M NaOH. For separation st eps, the capillary inner-wall was successively washed with 1M NaOH, ultrapure water, and the separation buffer (25mM Arg, 81mM Boric acid, and 0.5mM TTAOH, pH 9.0) by applying pr essure (1900mbar) to the inlet vial. Since nitrite and nitrate concentrations were very low in diluted samples, capillary isotachophoresis (CITP), a sample stackin g method, was employed. The leading solution was introduced into the capillary by pressure injection (25mbar for 12s), and then a neuronal sample was loaded using electrokinetic injection (-5kV for 12s). The separation was performed under a stable -15kV voltage at 20 C. Behavior Tests For each pharmacological treatment we obser ved the behavior for 30 minutes without adding chemicals to the seawater (pre-treatment), with chemicals (treatment), and after removing chemicals (post-treatment). A cube of agarogel containing glycine was ma de according to the following procedure. A 1% agarose solution in 10mL Milli Q water was prepared and the solution was brought boiling in a microwave oven to dissolve the agarose. The solution was cooled to room temperature with 80

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gentle stirring. A desired concen tration of glycine was mixed w ith the solution and the mixture was put into a refrigerator. Data Analysis Once an electropherogram was acquired, peak s were assigned based on the electrophoretic mobility of each analyte, and the assignmen ts were confirmed by spiking corresponding standards into the sample. Fivepoint calibration curves (peak area vs. concentration) of analytes were constructed for quantificati on using standard solutions. Other data analysis procedures are described in Chapter 2. Results and Discussion Amino acid analysis by CE-LIF In this study, NO-related me tabolites and potential low molecular weight signaling molecules were identified. First, a series of control tests was performed by injecting Milli Q water and separation buffer, and the peak areas we re subtracted for the animal sample studies (Figure 4-1A). In addition, fresh sea water and Trichoplax culture medium were tested for further control tests (Figure 4-1B). The electropherograms showed th at no significant peaks were observed, but a small amount of glutamate was de tected in the culture medium, probably from the animal food. The eleven compounds were separated clearly (Figure 4-1B) and the components of the Trichoplax sample were identified by relative migration times compared with an internal standard. Several interesting molecu les were identified as shown in Figure 4-1C. Previously it was thought that Trichoplax has no nervous system, and there have been no reports of neurotransmitters. However, for the first tim e, several neurotransmitters were found in the animal with concentrations from 56uM for GAB A to 2.6mM for glycine (Figure 4-1D). In particular, it was interesting that arginine was detected, as well as citrulline, a precursor and co81

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product of nitric oxide, both with relatively high concentrations, 0.35mM for arginine and 0.5mM for citrulline. Figure 4-1. Electropherograms and concentration profiling of Trichoplax adhaerens. Samples were loaded using electrokine tic injection (8kV for 12s), and then analyzed under a stable 20kV voltage at 20oC in 50m I.D. and 360m O.D. capillary with 30mM borate/30mM SDS, pH 10.0. A) Electrophe rograms of Milli Q water and separation buffer. B) Electropherograms of red sea water, Trichoplax culture medium, and standard solutions (1uM). C) Electropherograms of Trichoplax and Trichoplax spiked with standards. D) Concentration profile of Trichoplax (n=5). Peaks: Arginine (Arg), Aspartate (Asp), Citrulline (Cit), Gamma -aminobutyric acid (GABA), Glycine (Gly), Glutamate (Glu), internal standard (i.s.), Phenylalanine (Phe), Serine (Ser), Serotonin (5-HT), Taurine (Tau), and Tryptophan (Trp) Studies of NOS inhibition were conducted with three different types of inhibitors. Nitric oxide is synthesized from a precursor, L-argini ne, with assistance of NOS enzymes, and a co82

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product, L-citrulline, is also produced. While D-NAME was used for ineffective (no inhibition) form, L-NAME and L-NIL served as effective in hibitors. It was expect ed that arginine-tocitrulline ratio would increase after Trichoplax was incubated in either L-NAME or L-NIL, but no change was expected for D-NAME treatment. In Figure 4-2, the arginine-to-citrulline ratio increased by two-fold in case of L-NIL, but there was only a small increase with L-NAME, indicating L-NIL effectively inhibite d NOS enzyme, but L-NAME did not. Figure 4-2. Electropherograms and Arg-to-Cit ratios of Trichoplax adhaerens upon treatment with NOS inhibitors. Samples were loaded using electrokinetic injection (8kV for 12s), and then analyzed under a stable 20kV voltage at 20oC in 50m I.D. and 360m O.D. capillary with 30mM borate/30mM SD S, pH 10.0. A) Electropherograms of Trichoplax incubated with D-NAME (500 M), L-NAME (500M), and L-NIL (1mM) for 30min at room temperature. B) Arg-to-Cit ratio of Trichoplax after treatment with NOS inhibitors. n=5 Nitrite and Nitrate Analysis by CE-Conductivity Trichoplax NO metabolites levels were monitored and concentrations were derived from an in vitro calibration curve prepared from standa rd solutions of nitrate and nitrite at various concentrations (10nM-500 M). With the regression equations, the LOD of nitrate was determined to be 13.3nM for nitrite and 32.4nM for nitrate. These LODs were sufficient to quantify nitrite and nitrate in Trichoplax. 83

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Series of control tests were pe rformed to see if there were any small molecules that might interfere with peak identifications. Water, D-NAME, and L-NIL controls were first tested and no nitrite was observed. However, chloride and nitrate were present, because NOS inhibitor chemicals contain chloride, and nitrate is a comm on impurity in most of ch emicals (Figure 4-3A). Trichoplax by itself and Trichoplax incubated with NOS inhibitors were then analyzed. An effective NOS inhibitor should cause the nitrite level to be lower th an in the animal treated with an ineffective inhibitor. In the control Trichoplax, nitrite was detected and it was about 1.5mM, but after incubated with NOS inhi bitors, no nitrite was observed. It was previously anticipated that nitrite would be present after D-NAME incubation, because it is kno wn not to inhibit NOS activity well. Figure 4-3. Electropherogr ams of controls and Trichoplax upon NOS inhibitors. Separation was conducted in 75cm length of 50m I. D. and 360m O.D. capillary with 84

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arginine/borate buffer, pH 9.0. All samples were loaded using electrokinetic injection (-1kV for 12s), and then analyzed under a stable -15kV voltage at 20oC. A) Electropherograms of Milli Q water, D-NAM E, and L-NIL. B) Electropherograms of Trichoplax only, and Trichoplax incubated with D-NAME (500uM), L-NAME (500uM), and L-NIL (1mM). C) Nitrite and nitrate concentration profiling after NOS inhibition. n=5 Locomotory phases in Trichoplax A single Trichoplax was placed into a Petri-dish and movements were observed for one hour with one picture obtained ever y 10 seconds. In Figure 4-4C red trace indicates the path that Trichoplax took during 60 minutes of observation and the camera s hows clear activ e changes of surface. Trichoplax clearly indicates explor atory phases during which Trichoplax moves quickly (red arrow) followed by phases of almost complete inactivity (Figure 4-4E). Figure 4-4. Trichoplax behavioral analysis (Control). A) Image of Trichoplax in experimental arena. B) Subtracted image of Trichoplax in experimental arena. C) Trace analysis of 85

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Trichoplax behavior. D) Analysis of surface ar ea movement. A periodicity of these activity bursts is visible. E) Trace analysis of Trichoplax (Heyland et al. 2008) NO as a modulator of locomotion Addition of 8-Bromoguanosine 3,5-cyclic monophosphate (8-Bromo-cAMP), NO-donor, 6-(2-Hydroxy-1-methyl-2-nitro sohydrazino)-N-methyl-1-hexanamine (NOC9) and 8-BromocGMP all lead to an increase of the detectab le surface area and activity (data not shown). The NO donor NOC9 resulted in a relatively fast reco very (i.e < 30 minutes) from the treatment while both 8-bromo-cAMP and cGMP treatment resulted in no recovery. These results are consistent with the metabolic degradati on of these compounds in vertebrates. Figure 4-5. Trichoplax behavior analysis (NO modulators). A) 8Bromo-cAMP. B) NO donor (NOC9). C) 8-Bromo-cGMP. Video files were analyzed and the area of each individual was measured for each frame. Th ese values were averaged for all three individuals and plotted as a function of time (upper panel) Average activities of the entire time period were calculated for each individual and compared between pre-, 86

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experiment and postusing th e average activity for all thre e individuals (Heyland et al. 2008) Glycine as a chemoattractant in Trichoplax We tested the response of Trichoplax individuals in response to a local glycine source and compared it to the control. Specifically we pr epared agar doped with 1mM glycine and placed a small block into the experimental arena. The control treatment was ag ar doped with seawater. We then placed one Trichoplax in the arena and filmed its move ment over 30 minutes using time lapse photography. In all three trials, the Trichoplax moved towards the glycine source. Figure 4-6. Trichoplax behavior analysis (Glycine). A) A representative example of the movement experiment. B) Distance data as a function of time for all three individuals tested. The path was quantified by calculati ng the distance between the glycine source and the individual (d1) relative to the di stance between the control source and the individual (d2). Data were normalized by calculating the difference between d1 and d2. Scale bar 2mm (Heyland et al. 2008) The extensive genome analysis of Trichoplax is progressing, and a considerable amount of important information has been published (Srivastava et al. 2008, Schierwater et al. 2009b). In our study, several interesting sign aling molecules were found; for example, glycine was the most abundant amino acid and iGlyRs have b een shown to be highly expressed in Tricholax. DOPA decarboxylase and DBH-like monooxygenase, which are involved in dopamine, noradrenaline 87

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and adrenaline synthesis in adrenergic cells, and are present, as well as putative vesicular amine transporters which are used for neurotransmitte r uptake (Srivastava et al. 2008). However, DA was not found in our study, but it may be obser ved with LOD improvement. Schuchert showed that, although Trichoplax has no nervous system, it has behavioral responses to environmental stimuli, and sensitivity to the neuropeptide RF amide (Schuchert 1993). In our study, the animals were attracted to the glycine, but not to the control. An important aspect of the current studies is the ability to perform direct whole animal measurements of endogenous concentr ations of NO-related metabolites. Trichoplax samples have the concentrations of nitr ites (0.15.03mM), nitrates (2 .26.43mM), Cit (0.50.05mM), and Arg (0.35.01mM). Such a situation is e xplained by functionally active NOS consuming Arg, converting it into Cit and NO, which subseque ntly undergoes oxidation to nitrite and nitrate. Biological roles of NO in marine invertebrates are related to feeding, defense, learning, metamorphosis, and swimming (Moroz et al. 2000, Fiore et al. 2004, Katzoff et al. 2002, Leise et al. 2004, Moroz et al. 2004). The NO/cGMP signa ling pathway is considered to play an important role in the swimming pattern of Aglantha (Moroz et al. 2004). In our study, the NO donor NOC9 aided in a relatively fast recovery while both 8-br omo-cAMP and cGMP treatment resulted in no recovery. This means that cAMP or cGMP may not be able to modulate Trichoplax locomotion, but NO is needed to initiate the signaling pathway. The current work represents the first studies of enzyme activity in Trichoplax via both Arg/Cit and nitrite/nitrate measurements. Moreover, knowledge of the endogenous concentrations of argi nine and citrulline is important in the designing pharmacologica l tests using competitive NOS inhibitors. Conclusion The measurement of the oxidation products of NO-related metabolites us ing CE is a useful method for examining small-volume animals and co mplements other technique s presently in use. 88

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89 These findings underscore the importance of employi ng state-of-the-art microchemical analytical techniques in conjunction with traditiona l physiological, histoc hemical, and molecular techniques in order to confirm NOS activity.

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CHAPTER 5 USING CE FOR METABOLOMIC PROF ILING OF THE BASAL ANIMALS: CTENOPHORES, CNIDARIANS, PLACOZOA, AND SPONGES Introduction While bilateria comprise a well-defined phyloge netic group, both the branching order of the lower animal phyla, including porifera, plac ozoa, cnidarians and ctenophores, and their relationships to bilateria are stil l controversial (Claus 2008, Philippe et al. 2009). In terms of morphology, Trichoplax and sponges were placed into the gr oup of simplest animals, but a largescale phylogenomic analysis recently put ctenophor es into the basal anim al group (Miller & Ball 2008). Immunohistochemical and physiological tests have been conducted in cnidarians, sponges and ctenophores (Kass-Simon & Pierobon 2007, Cr istino et al. 2008, Pang & Martindale 2008a, Pang & Martindale 2008b, Ramoino et al. 2007), but few direct chemical analyses, for compounds like amino acids and signaling molecule s, have been performed in many marine invertebrate species. Thus, analysis of the nitr ic oxide related metabolites and potential signaling molecules in the biological samples may provide insight into the nervous systems of marine animals. While the primary objective of this study invol ved the identification and characterization of key potential signaling molecules of low molecular weight and their metabolites, our effort was also focused on the analysis of D-Aspartic acid (D-Asp) and D-Glut amic acid (D-Glu). In particular, D-Asp is found in the central nervous systems of a variety of animals, including mammals (Sakai et al. 1998, D'Aniello 2007) a nd mollusks (Miao et al. 2006, Song et al. 2006). In addition, D-Glu naturally presents in ma ny microbes (Glavas & Tanne r 2001), as well as in plants and animals (Corrigan 1969, Kera et al. 1996). Kera et al. reported that D-Glu may play a role in Aplysia central nervous systems (Quan & Liu 2003). 90

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To accomplish this goal, capilla ry electrophoresis (CE) was us ed, because of its efficient and ultra-small-volume separation abilities for the separation and analysis of charged species. As described in Chapter 2, CE has been widely used for the analysis of amino acids and derivatives (Zhu et al. 2005, Boudko 2007, Poinsot et al. 2008) In addition to L-arginine and citrulline determinations, general amino-acid profiling of the basal animals was performed. As most analytes are not fluorescent, th ey are commonly derivatized usi ng fluorogenic reagents that either are not fluorescent prior to reacti on or have nonfluorescen t hydrolysis products. oPhthalaldehyde (OPA) was chosen as the deriva tization reagent in this work, because it is a fluorogenic reagent which reacts with analytes within a few seconds In addition, for nitrite and nitrate detection, contactless conductivity detection (CCD) wa s used, due to the excellent sensitivity to small ions. In this study, a number of pot ential signaling molecules were found, including glycine, GABA, glutamate, and aspartate, with concentrat ions up to the millimolar level. In particular, serotonin (5-HT) was detected in the tentacle area of Sarsia, one of cnidarians. Also, D-Glu was identified in most of ctenophor es, and D-Asp was quantified in Sarsia, Trichoplax, and Sycon coactum. Methods and Materials Chemicals and Reagents All chemicals for buffers were purchased from Sigma-Aldrich, and standard amino acids were purchased from Fluka. Ultrapure Milli-Q water (Milli-Q filtration system, Millipore, Bedford, MA) was used for all buffers, stan dard solutions, and sample preparations. Sample Preparation Samples of Trichoplax adhaerens were cultured in glass dish es containing red sea water at room temperature by the courtesy of Jim Nether ton. The red sea water was refreshed every 30 91

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days. Each Trichoplax was collected from the culture dish by a glass pipette and transferred into a 0.5mL PCR tube under a stereomicroscope. The culture medium was removed and the animal was covered with 1 L Milli-Q water, followed by storage at -80 oC until use. Ctenophores (Pleurobrachia, Beroe, Bolinopsis, and Mnemiopsis) and cnidarians (Aglantha, Sarsia, Aequorea, and Phialidium) were collected at the University of Washington Friday Harbor Laboratories (FHL), San Ju an Island, WA. Speci fic body parts of both ctenophores and cnidarians were dissected under a stereomicroscope using a scissor and tweezers. The samples were stored in a PCR tube containing Milli-Q water at -80 oC until use. All the works were performed by the scientist in the FHL. Sponges (Sycon coactum and Aphrocallistes vastus) were collected using the manipulator arm of the remote operated vehicle ROPOS (Rem ote Operated Platform for Ocean Science; ropos.com) at San Jose Islets, Barkley Sound, Canada. The pieces were cut and stored in a 0.5mL PCR tube at -80oC until use. Amino Acids Microanalysis using CE with LIF The CE coupled with the ZETALIF detector (P icometrics, France) was used for the assay of amino acids. In this work a helium cadmium laser (325nm) from Melles Griot, Inc. (Omnichrome Series56, Carlsbad, CA) was used as the excitation source. Before the photomultiplier tube (PMT), the fluorescence was the wavelength filtered. All instrumentation, counting, and high-voltage CE power supply were controlled using DAx 7.3 software. Borate buffer (30mM, pH 9.5) was used for sample preparation, and all solutions were filtered using 0.2m filters to remove particulates. The buffers were degassed by ultrasonication for 10 min to minimize the chance of bubble formation. A 75mM OPA / -mercaptoethanol ( ME) stock solution was prepared by dissolving 10mg of OPA in 100 L of methanol and mixing with 1mL of 30mM borate and 10 L of -ME. Stock solutions (10mM) of amino acids and 92

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neurotransmitters were prepared by dissolving each compound in the borate buffer. OPA and ME were stored in a refrigerator, and fresh solutions were prepared weekly. All experiments were conducte d using a 75cm length of 50m I.D. 360m O.D. fused silica capillary (Polymicro Technologies, AZ). A 30mM borat e/ 30mM sodium dodecyl sulfate (SDS) electrolyte (adjusted to pH 10.0 with Na OH) was used as the separation buffer for amino acid analysis. For pre-column derivatization method, 1 L of OPA was incubated at 20oC for 6 minutes in a 0.5mL PCR tube with 18 L sample and 1 L internal standard. For separation steps, the capillary inner-wall was successively wash ed with 1M NaOH, Milli Q water, and the separation buffer by applying pressure (1900mbar) to the inlet vial. Then the sample was loaded using electrokinetic inje ction (8kV for 12s). The separati on was performed under a stable 20kV voltage at 20 C. Nitrite/Nitrate Microanalysis using CE with Contactless Conductivity Capillary electrophoresis coupled with a Tr aceDec contactless conductivity detector (Strasshof, Austria) was used for the assay of nitr ite and nitrate in the basal animals. In order to reduce Clin a sample, OnGuard II Ag (DIONEX Corp., Sunnyvale, CA) was purchased. Since small sample volumes (20L) were analyzed, custom-built cartridges were used for sample clean-up as described in Chapter 3. In order to quantitate any potential sample loss, the custommade chloride cartridge was tested for samp le recovery of both nitrite and nitrate. The buffer preparation, injection procedure, and separation are described in Chapter 4. Data Analysis After an electropherogram was acquired, peak s were assigned based on the electrophoretic mobility of each analyte, and the assignmen ts were confirmed by spiking corresponding standards into the sample. Fivepoint calibration curves (peak area vs. concentration) of analytes 93

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were constructed for quantificati on using standard solutions. Other data analysis procedures are described in Chapter 2. Results and Discussion Neurotransmitters and Their Metabolites in Basal Animals Figure 5-1. Electropherograms of basal animals. A) Sponges. B) Ctenophores. C) Cnidarians and placozoa. Samples were loaded using electr okinetic injection (8kV for 12s) and then analyzed under a stable 20kV voltage at 20oC in 50m I.D. and 360m O.D. capillary with 30mM borate/30mM SDS, pH 10.0. Arrows: black (5-HTP) and red (Tyr) 94

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Two sponges, Aphrocallistes vastus (class Hexactinellida), ca lled glass sponge, and Sycon coactum (class Calcarea ), were analyzed. The electropherograms in figure 5-1A of representative samples of A. vastus show high concentrations of GABA an d taurine, while high concentrations of glycine and taurin e were detected in S. coactum. Interestingly, both animals have 5-HTP, which is a direct precursor of serotonin, but it was not detected in these analyses. Sponges have a complex intercellular communication system, a nd the possible mechanisms of a coordinating center involve internal spreading of the chemical signal in the extracellular ma trix, cell motility, and cell-cell and cell-substratum inter actions (Ramoino et al. 2007, Ellwanger et al. 2007). Neurotransmitters (e.g., epinephrine, norenephrine, and serotonin), as well as the neurotransmitter-related enzyme monoaminoxidase, have been found in myocyte-like cells of Sycon ciliatum (class Calcarea) (Lentz 1966). Also, cells containing a gene encoding a putative metabotropic glutamate/GABA receptor have been observed in the sponge Geodia cydonium (class Demospongiae), and pharmacological data supported the existence of such a receptor by demonstrating that it can be activated by L-Glu (Perovic et al. 1999). Four different animals were investigated, including Pleurobrachia, Mnemiopsis, Bolinopsis, and Beroe, as well as their specific body re gions (Figure 5-1B). Metabolite concentrations are presented in Table 5-1. In most of these ctenophores, high concentrations of taurine and citrulline were detected. This is a particularly interesting result for Pleurobrachia since it was reported that NOS was localized ne ar the mouth and gut areas (Claus 2008). A bundle of axon-like processes arch es over the epithelial cells in the aboral sense organ of Pleurobrachia and Mnemiopsis for regulating swimming behavior of these animals (Tamm & Tamm 2002). The ctenophores are recognized as an important gr oup for understanding the early evolution of key anatomical characters, such as body symmetries, the mesoderm, and the 95

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neurosensory system (Jager et al. 2008). Recently, a large-scale phylogenomic analysis of ctenophores placed them at the bo ttom of the metazoans, but a fe w data are available on their metabolites (Miller & Ball 2008). In our work, we investigated four different species: Aglantha, Sarsia, Aequorea, and Phialidium (Figure 5-1C). Glycine was high in most of these animals, and it was interesting that 5-HT was identified in Sarsia. It has been reported that 5-HT pl ays an important role in an early stage of metamorphosis in Phialidium gregarium (McCauley 1997) and in Eudendrium racemosum (Zega et al. 2007). In Aglantha digitale, the nitric oxide/cyclic guanosine monophosphate (NO/cGMP) signaling pathway modul ates the rhythmic swimming associated with feeding, possibly by means of putative nitrergic sensory neur ons in the tentacles (Moroz et al. 2004). A considerable amount of data has s hown the accumulation of neurotransmitters, such as acetylcholine, GABA, and glutamate, in cnidarians (Kass-Simon & Pierobon 2007), but more animal groups still need to be analyzed. Glu and Asp Enantiomer Analysis in Basal Animals D-Aspartic acid (D-Asp), an endogenous amino acid present in vertebrates and invertebrates, plays an important role in the neuroendocrine system, as well as in the development of the nervous system (D'Aniello 2007). However, previously no direct chemical data on the enantiomers in the basal animals have been available. Therefore, D enantiomers were analyzed in Trichoplax, sponges, ctenophores, and cnidaria ns using a chiral selector, -CD. Although it was surprising to iden tify D-Glu and D-Asp in these samples, detection was, nonetheless, confirmed by spiking with each standard (Figure 5-2). The quantification data are presented in Table 5-2. D-Asp ha s been found near the brain (Song et al. 2008), endocrine (D'Aniello et al. 2000), retina (Lee et al. 1999), and nervous tissues (Spinelli et al. 2006) in the various animals. It is considered that D-Asp ma y aid in the construction of neuronal networks. 96

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Figure 5-2. Electropherograms of Gl u and Asp enantiomers in the basal animals. A) Placozoa. B) Sponges. C) Ctenophores. D) Cnidarians. Peaks: 1) L-Glu, 2) D-Gl u, 3) D-Asp, and 4) L-Asp. Samples were loaded using elec trokinetic injection ( 8kV for 12s) and then analyzed under a stable 20kV voltage at 20oC in 50m I.D. and 360m O.D. capillary with 15mM borate and 10mM -CD, pH 10.0 Nitrite and Nitrate Assay in the Basal Animals In sponges, the nitrite concentrations in a millimolar range were observed in Sarcia and Beroe (Figure 5-3B,C). There was a report that si gnificant amounts of NO were detected in the homogenates of the mouth area from Aplysia californica, previously shown to be NO-positive, and in individual NOS-containing buccal neurons from the freshwater snail, Lymnaea stagnalis (Kim et al. 2006). Nitrite and nitr ate are useful indicators of nitr ic oxide activity and have been 97

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used for this purpose in various an imals (Boudko 2007). The presence of Ca2+-dependent, heatstress-activated nitric oxide s ynthase (NOS) activity was demonstrated previously in dendritic sponge cells (Giovine et al. 2001). Regulation of nitric oxide production by the NOS and control by the natural inhibitor asymmetric dimethyl arginine (ADMA) were also observed in S. domuncula (Muller et al. 2006). Furthermore, in Aglantha, NO serves as a modulator to affect the swimming pattern (Moroz et al. 2004). Therefore, Sarcia, aequorea, and Phialidium should be carefully investigated to determine the role of NO in the swimming motion. Figure 5-3. Nitrite and nitrate el ectropherograms of the basal anim als. A) Sponges, B) Cnidarians and placozoa, C) Ctenophores, D) Concentra tion profiles of nitrite and nitrate (n=3). Note: whole body (WB), aboral organ (AO) 98

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99 Conclusion Four different basal animal groups, including placozoa, ctenophora, cn idaria, and porifera, were analyzed by the CE-LIF system to identif y and quantify potential signaling molecules. In particular, nitric oxide-related me tabolites (Arg, Cit, nitrite, and nitrate) were observed in the mouth region of Sarsia and Beroe, suggesting that NO acts on m odulate the feeding mechanism. We found that D-Asp may play im portant roles in placozoa, pori fera, and cnidaria, and that ctenophores may use D-Glu as a signaling molecule The current system reliably operated for a large-scale animal analysis, although some impr ovements in LODs and sample preparation methods can still be made to minimize errors.

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Table 5-1. Metabolite concentrations in basal animals. Species Region Cit Ser 5-HTP Tyr GAB A Gly Tau Phe Trp Glu Asp Arg 5-HT Beroe AOa 25.0.6 2.6.3 0.22.01 2.25 0.18 0.61.07 1.97.06 2.21.13 9.94.83 3.78.2 1.88.15 3.53.26 Mouth 38.7.1 4.46.45 0.43.04 7.47.90 2.49.21 4.88 0.38 5.29.37 9.45.49 6. 74.58 9.93.37 5.31.31 Comb 24.8.06 2.91.17 0.91.08 6.53.38 0.89.08 3.51 0.3 4.76.22 11.2.42 4. 40.28 1.50.07 6.12.42 Dots 22.1.73 2.03.05 2.71. 17 7.85.54 0.58.01 11. 2.3 9.14.18 4.69.17 3. 08.01 10.8.25 9.42.72 Mnemiopsis AOa 136 23.8 3.1 24 83 2.4 30 10 26 Mouth 2040 205 261 89 715 360 149 320 346 163 126 Lobe 277 37 4.4 46 121 4.3 13 56 23 41 Ctenrow 81 43 7.3 447 76 187 7 113 62 30 78 Bolinopsis WBa 165 28 6.5 730 27 25 34 14 17 Pleurobrachia Comb 5.99.1 1.26.1 0.34.02 0.91.07 23.7. 5 33.7 1.63.07 5.49.2 1.58.08 27.4.3 3.05.3 Bodywall 2.82.1 0.75.08 0.23 0.02 0.28.03 30.5.6 2.86 1.50 4.17.2 2.30.07 1.58.1 4.06 AOa 8.81.3 1.23.1 1.48.1 6. 84.7 22.8.5 10.9.04 1.73.04 10.4.7 3.49.01 31.4.2 4.70 Mouth 15.7.5 1.57.1 0.42.04 4.24.3 20.6.7 4.07. 3 3.75.3 6.39.6 2.08 0.2 3.32.1 6.60.4 Stomach 44.0.2 4.95.3 2.81. 2 15.6.1 64.3.2 12.1 0.8 17.7.9 25.4.6 7. 40.6 13.4.0 38.9.0 Tentacle 24.5.9 3.16.2 0.77. 08 1.38.07 20.4.6 1.40 0.1 1.94.2 8.20.4 6. 66.6 12.0.4 4.55.2 Trichoplax WBa 1650 446 62 3520 82 177 1210 622 900 A. vastus WBa 60 57 72 421 156 1120 34.4 40.2 24 52 48 S. coactum WBa 49 18 20 13 2490 82 4 12 12 13 10 5.4 Phialidium WBa 27 11 8 1840 17 12 7 Sarsia WBa 8.4 9 10.8 2.9 381 47 2 2.2 5.6 18 4.8 28 Tentacle 18.7 16 0.57.04 3.8 508 83 16 4.2 40 6 18 19 Mouth 201 156 236 39 1700 522 56 102 164 55 50 Aequorea WBa 73 87 28 82 116 340 34 69 22 17 Aglantha WBa 57 23 23 2370 37 29 18 107 38 108 a. WB (whole body), AO (aboral organ) Results are in MSD, n=3-5 100

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Table 5-2. Land D Glu and Asp concentrations in basal animals Specie s Region L-Glu D-Glu D-Asp L-Asp Beroe AOa 25.51.9 2.480.5 9.740.8 Mouth 9.541.9 0.670.1 6.411.6 Coms b Dots 14.73.1 1.590.09 7.531.3 Mnemiopsis AOa 3917 2817 Mout h Lobe 8037 2 3315 40236 4122 Ctenrow 3712 454 Bolinopsis WBa 9412 41 4118 Pleurobrachia Combs 27.12.03 9.660.71 Bodywall 10.13.39 10.25.39 AOa 6.540.61 0.41 7.450.32 Mouth 12.31.36 1.39 11.20.85 Stomach Tentacle 36.83.27 3.140.37 16.91.04 11.70.6 5 157069 2.720.32 9.820.57 2640154 Trichoplax WBa 138071 A. vastus WBa 8048 12459 S. coactum WBa 5823 8640 7422 Phialidium WBa 3910 261 Sarsia WBa 214 240.5 Tentacle 416 260.4 Mouth 25521 9263 Aequorea WBa 10137 426 Aglantha WBa 19416 3924 4923a. WB (whole body), AO (aboral organ) Results are in MSD, n=3-5 101

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CHAPTER 6 COMPARATIVE ANALYSIS OF MULLUSCA: SQUID, NAUTILUS, AND APLYSIA CALIFORNICA Introduction The cephalopods, comprising the squid, octopus and cuttlefish, have arguably the most advanced nervous systems among the invertebrate s and are certainly the most sophisticated systems within the phylum Mollusca (Williamson & Chrachri 2004). In particular, cephalopod nervous systems have served successfully for decades in efforts to understand the basic biology of normal nerve function, from the flow of ion cu rrents in the nerve impulse to the motor systems of axon transport (Grant et al. 2006). In addition, Aplysia californica (Phylum: Mollusca) has been useful for cell and molecular biological stud ies of behavior, learning, and memory, because of the accessibility of individual ganglia and specific neurons within these ganglia and the ability to identify individual nerve cells that play roles in specific behaviors (Moroz et al. 2006). Since the structure and origin of th e giant fiber system of the squid Loligo were described, a vast literature on the physiology, behavior, and biochemistry of the giant fiber system has been published (Grant et al. 2006, Giuditta et al. 2008). The jet propulsion locomotory behavior depends upon the giant axons that innervate the muscles of the man tle. The diameter of the giant axon can be up to 1mm, and its length may exte nd several centimeters depending on the species and size of the animal. About 3-10 L of pure axoplasm, uncontamin ated by sheath or glial cells, can be collected from a single giant axon, depe nding on size, making it po ssible to collect up to 30-100 L of axoplasm from one squid (2 axons per squid) (Grant et al. 2006). There have been several reports about the glutam ate as a signaling molecule in Sepioteuthis sepioidea (Garcia & Villegas 1995, Garcia 1996) and in Loligo (Lieberman & Sanzenbacher 1992). The only other extant group with in the class cephalopod is the Nautilus, which consists of 5 species. They have retained the heavy extern al protective shell but have relatively simple 102

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nervous systems, presumably reflecting the ancestral, more primitive form (Williamson & Chrachri 2004). Unlike other cephalopods, Nautilus is long-lived, slow growing, and largely sedentary, scavenging for its food rather than actively hunting prey (Crook & Basil 2008). It lives predominately in deep (>300m), cold waters surrounding the coral reef s of the Indo-Pacific, and in minimal light penetration habitat. Nautilus relies mostly on olfaction and touch to locate food sources (Shigeno et al. 2008). The apparent similarity between living Nautilus and ancestral cephalopods suggests that the Nautilus may provide important insights into the evolution of complexity in invertebrate nervous systems (Crook & Basil 2008). Catecholamines (e.g., noradrenaline, adrenalin and dopa mine) and serotonin (5-HT) we re identified by HPLC with electrochemical detection and by immunohistoc hemistry, respectively, in the central cardiovascular system of Nautilus pompilius Linne (Springer et al. 2005). The nerve fibers of the shell-producing organs (m antle and siphuncle) in Nautilus pompilius contain the neurotransmitter acetylcholine, catecholamines and Phe-Met-Phe-Arg-NH2 (FMFR-amide) (Westermann et al. 2002). The importance of Aplysia californica as a reductionist model for studies in molecular neurobiology, electrophysiology, learning, and memory has steadily increased over the past three decades (Capo et al. 2009, Kandel 2001, Moroz et al. 2006). This marine algavore inhabits intertidal and sublittora l zones along the Pacific coast of th e United States and Mexico, where it lays large benthic egg masses (Capo et al. 2009). Eggs hatch afte r 7-10 days, releasing planktotrophic veliger larvae, whic h have been reported to remain in the plankton for at least 35 days, and then pass through metamorphosis, juvenile, and adult Aplysia stages (Kriegstein et al. 1974). Aplysia ganglia and individual neurons were analyzed by assaying the biosynthetic enzyme which decarboxylates DOPA and 5-HTP to form dopamine or 5-HT, respectively 103

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(Weinreich et al. 1972). Embryonic cells containing catecholamines, Phe-Met-Arg-Phe-NH2 (FMRF-amide), and 5-HT were iden tified by immunohistochemistry in Aplysia (Dickinson et al. 2000). However, there have been few direct chemical analyses of signaling molecules in the giant axon of Squid, in the specific body parts of Nautilus (e.g., neurons, muscles, retina cells, and cerebral cord), or in the chem osensory and embryonic cells of Aplysia californica. In order to address this issue, L-arginine and citrulline determinations, as well as general amino-acid profiling of the basal animals were performed. Capillary electrophoresis (CE) was used because of its efficient and ultra-small-volume capabili ties for the separation and analysis of charged species. As described in Chapter 2, CE has been widely used for the analysis of amino acids and derivatives (Zhu et al. 2005, Boudko 2007, Poinso t et al. 2008). As most analytes are not fluorescent, they are commonly derivatized usin g fluorogenic reagents that either are not fluorescent prior to reac tion or have nonfluoresce nt hydrolysis products. o-Phthalaldehyde (OPA) was chosen as the derivatization reagent in this work, because it is a fluorogenic reagent which reacts with analytes with in a few seconds. In addition, fo r nitrite and nitr ate detection, contactless conductivity detection (C CD) was used, due to the excellent sensitivity to small ions. Methods and Materials Chemicals and Reagents All chemicals for buffers were purchased from Sigma-Aldrich, and standard amino acids were purchased from Fluka. Ultrapure Milli-Q water (Milli-Q filtration system, Millipore, Bedford, MA) was used for all buffers, stan dard solutions, and sample preparations. Sample Preparation Freshly caught squid (Loligo pealei), were obtained at the Mari ne Biological Laboratory (Woods Hole, MA, USA), and the giant axon (0.9 mm in diameter and 1.5cm in length) was 104

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surgically removed, pl aced in artificial sea water and cleaned. An axoplasm was extruded from the axon, quickly frozen and stored at -80oC until processed. Nautilus (phylum: cephalopoda) was obtaine d from a commer cial supplier (Sea Dwelling CreaturesTM, Los Angeles, CA, USA) Specific body parts of Nautilus were dissected under a stereomicroscope using scissors and tweezers by Dr Leonid Moroz. The samples were stored in PCR tubes containing M illi-Q water at -80 oC until use. Aplysia californica were obtained from either the Aplysia Research Facility (Miami, FL) or Marinus Scientific (Long Beach, CA), depending on the animal size (100-400g). The egg strand (cordon) was obtained from the Miami facility and inspected under a dissecting microscope for collection of second and third day stages. Chemos ensory cells were also obtained under the microscope. All individual samples were st ored in separate 0.5mL PCR tubes at -80oC until use. Amino Acids Microanalysis using CE with LIF A CE coupled with the ZETALIF detector (Picom etrics, France) was used for the assay of amino acids. In this work a helium cadmium laser (325nm) from Melles Griot, Inc. (Omnichrome Series56, Carlsbad, CA) was used as the excitation source. Before the photomultiplier tube (PMT), the fluorescence was wavelength filtered. All instrumentation, counting, and high-voltage CE power supply were controlled using DAx 7.3 software. All solutions were prepared with ultrapur e Milli-Q water to minimize the presence of impurities. Borate buffer (30mM, pH 9.5) was used for sample preparation. All solutions were filtered using 0.2 m filters to remove particulates. The buffers were degassed by ultrasonication for 10min to minimize the chance of bubble formation. A 75mM OPA / -mercaptoethanol (ME) stock solution was prepared by dissolving 10mg of OPA in 100 L of methanol and mixing with 1mL of 30mM borate and 10 L of -ME. Stock solutions (10mM) of amino acids and 105

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neurotransmitters were prepared by dissolving each compound in the borate buffer. OPA and ME were stored in a refrigerator, and fresh solutions were prepared weekly. All experiments were conducte d using a 75cm length of 50m I.D. 360m O.D. fused silica capillary (Polymicro T echnologies, AZ). A 30mM borate/30mM sodium dodecyl sulfate (SDS) electrolyte (adjusted to pH 10.0 with Na OH) was used as the separation buffer for amino acid analysis. In pre-column derivatization, 1 L of OPA was incubated in a 0.5mL PCR tube with 18L of sample, and 1L internal standard (total volume = 20 L). For separation steps, the capillary inner-wall was successively washed with 1M NaOH, Milli Q water, and the separation buffer by applying pressure (1900mbar) to the inle t vial. Then the sample was loaded using electrokinetic injection (8kV for 12s). The sepa ration was performed under a stable 20kV voltage at 20 C. Nitrite/Nitrate Microanalysis using CE with Contactless Conductivity Capillary electrophoresis coupled with a Tr aceDec contactless conductivity detector (Strasshof, Austria) was used for th e assay of nitrite and nitrate in Trichoplax. In order to decrease the concentration of Clin a sample, OnGuard II Ag (DIONEX Corp., Sunnyvale, CA) was purchased. Since small sample volumes (20L) were involved, custom-built cartridges were prepared as described in Chapter 3. The buffer preparation, injection procedure, and separation are described in Chapter 4. Data Analysis After an electropherogram was acquired, peak s were assigned based on the electrophoretic mobility of each analyte, and the assignmen ts were confirmed by spiking corresponding standards into the sample. Fivepoint calibration curves (peak area vs. concentration) of analytes 106

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were constructed for quantificati on using standard solutions. Other data analysis procedures are described in Chapter 2. Results and Discussion Squid Axoplasm Analysis Figure 6-1. Electropherograms a nd concentration profiling of Squid. A) Standards and Squid axoplasm samples. B,C) Concentration prof ile (n=4). Samples were loaded using electrokinetic injection (8kV for 12s), and then analyzed under a stable 20kV voltage at 20oC in 50m I.D. and 360m O.D. capill ary with 30mM borate/30mM SDS, pH 10.0 In the squid axoplasm, high concentrations of Gly, Glu, and Asp were observed, and for the first time 5-HT and GABA were quantitated (Figure 6-1). It was demons trated that L-Glu and 5-HT are endogenous in the nerv es innervating squid chromatophores (pigment-containing and light-reflecting cells) and that the radial mu scles contain receptors for both substances (Messenger et al. 1997). These results suggest that L-Glu is an excitatory transmitter at squid chromatophore muscles, and in contrast 5-HT acts to relax the muscles. The jet propulsion locomotory behavior of squid depends upon the signaling evoked by a system of giant neurons beginning with two large neurons in the brain (Grant et al. 20 06). The neurons integrate most 107

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sensory input to transmit excitatory impulses by L-Glu to the dorsal stellate ganglia and giant axons that innervate the muscles of the mantle. Figure 6-2. Nitrite and nitr ate electropherograms and c oncentration profile. A) Squid axoplasm. B) Concentration profiles of Squid axoplasm (n=3) Furthermore, Arg and Cit were present in high concentration (Figure 6-1) as well as nitrite and nitrate (Figure 6-2) It was demonstrated that NO is an integral part of the complex mechanisms implicated in the initiation and maintenance of the symbiont infection of the light organ of the Hawaiian bobtail squid, Euprymna scolopes, by symbiotic Vibrio fischeri cells (Davidson et al. 2004). Indeed, it was shown that NO was released into the mucus secreted by the light organ, where the symbiotic bacteria aggregated before migrating into the final sites of colonization (Palumbo 2005). In earlier work on the Sepia officinalis ink gland cells, activation of the NMDA glutamate receptor caus ed an influx of calcium (Palumbo et al. 2000). The calcium binds to calmodulin and activates NOS to produce NO, which subsequently targets guanylyl cyclase to produce hi gher levels of cGMP. The cG MP activates tyrosinase by phosphorylation through a protein ki nase G, with consequent in crease of melanin formation (Palumbo et al. 2000). cGMP also induces secretion of i nk constituents from mature cells (Fiore et al. 2004). 108

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The results in figure 6-3 indicate that D-Asp was present in the Squid axoplasm. The DAsp may play an important role in the synaptic signaling. It was reported that neither Dnor LAsp was capable of gating a squid glutamate receptor (SqGluR). However when applied alongside glutamate, both isomers slowed significan t glutamate gating of the current, opening the possibility that these substances could act as neuromodulators (Brown et al. 2007). Figure 6-3. Electropherograms a nd concentration profile of Gl u and Asp enantiomers in the Squid axoplasm. A) Standards and Squid. B) Enantiomer concentrations (n=3). Samples were loaded using electrokinetic in jection (8kV for 12s) and then analyzed under a stable 20kV voltage at 20oC in 50m I.D. and 360m O.D. capillary with 15mM borate and 10mM -CD, pH 10.0 Nautilus Analysis The cerebral cord, retina cells, muscle, and neurons of Nautilus were investigated. As shown in figure 6-4, the cerebral cord contains high concentrations of Arg, Cit, and GABA. In the Nautilus heart region, endothelia n itric oxide synthase (eNOS) was identified on endothelial cells, where the heart may be modulated (Springer et al. 2004). In the study of the nerve endings of the mantle and the siphuncle, it was demonstr ated that reactions we re occurring by using antibodies against serotonin a nd the tetrapeptide FMRF-amide, and observing the presence of specific acetylcholinesterase yi elded positive results (Westermann et al. 2002). Additionally, the 109

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HPLC-analyses showed that in the mantle and in the siphuncle the contents of dopamine were 190ng and 160ng per gram of tissue, respectivel y, suggesting that dopamine may be a neurotransmitter. Figure 6-4. Electropherograms a nd concentration profiling of Nautilus. A) Specific body regions. Arrows: red (5-HTP) and black (Gly). B) C oncentration profile (n=4). Samples were loaded using electrokinetic in jection (8kV for 12s), and then analyzed under a stable 20kV voltage at 20oC in 50m I.D. and 360m O.D. capillary with 30mM borate/30mM SDS, pH 10.0 Figure 6-5. Nitrite and nitrate electropherograms and concentration profile. A) Specific body parts of Nautilus. B) Concentration profiles of Nautilus (n=3) 110

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In figure 6-5B, muscle contains the relatively high concentration of nitrite and shows the large Arg-to-Cit ratio, suggesting that nitric oxide acts as a vasodilatator or signaling molecule. It was reported that NO donor S-nitroso-N-acetylpenicillamine (SNAP) caused relaxation from the star fish Asterias rubenstube feet and the apical muscle of the body wall (Melarange & Elphick 2003). Figure 6-6. Electropherograms a nd concentration profile of gl u and asp enantiomers in the Nautilus. A) Nautilus body parts. B) Enantiomer concentrations (n=3). Samples were loaded using electrokinetic in jection (8kV for 12s) and then analyzed under a stable 20kV voltage at 20oC in 50m I.D. and 360m O.D. capillary with 15mM borate and 10mM -CD, pH 10.0 Interestingly, D-Asp was present in the sa mples except the retina cells (Figure 6-6). Previous work by D`Aniello et al showed that in the retina of Sepia officinalis, D-Asp occured at relative high concentrations (2.0-3.0 mol/g tissue) (D'Aniello et al. 2005). When the animal was left in the dark, the concentrations of D-Asp si gnificantly decreased in th e retina; however, when the animals were exposed to the light again, D-As p returned to the previo us levels (D'Aniello 2007). In contrast, in our re sult the retina cells of Nautilus D-Asp was not present possibly because the Nautilus has been considered that the struct ure and visual acuity of the primitive, 111

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lensless eye lends support to the hypo thesis that vision is of limite d use in the deep ocean (Crook & Basil 2008). Aplysia californica Analysis In Aplysia chemosensory cells, relatively high con centrations of Gly, Tau, Glu, and Asp were observed, as well as high levels of Arg and Cit (Figure 6-7A,B). Also, nitrite was present in the millimolar concentration (Figure 6-7C,D). The distribution of putative nitric oxide synthase (NOS)-containing cells in the mollusc Aplysia californica was studied by using NADPHdiaphorase (NADPH-d) histochemistry, and it show ed that chemosensory areas (the mouth area, rhinophores, and tentacles) expressed the most in tense staining, implying a role for NO as a modulator of chemosensory processing (Moroz 2006). A role for the NO-cGMP pathway in mediating chemosensory activati on of feeding in the mollusc Lymnaea stagnalis was suggested by intense NADPH diaphorase staining observed in nerve fibers that project from sensory cells in the lips to the CNS and by the presence in the CNS of a NO-activated guanylyl cyclase (Elphick et al. 1995). Also, behavioral experiments on the L. stagnalis showed that hemoglobin (NO scavenger) prevented feeding and methyl ene blue (inhibitor of guanylyl cyclase) significantly delayed the onset of feeding. Mobley et al (Mobley et al. 2008) used the metabolite profilin g (e.g., Arg, Glu, Asp, Gly, Tau, and Glutathione) technique to determin e whether metabolite profiles can identify cell classes of chemosensory tissues within and acros s different species, incl uding mouse, zebrafish, lobster and squid. For example, the high arginine and low taurine content in lobster statistically separated its olfactory receptor ne uron (ORN) classes from those of other species. High glycine content throughout the zebrafish ol factory epithelium (OE) separa ted most of its cell classes from the other species. Although chemosensory systems across species share many similarities, 112

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the heterogeneous environments, osmotic pressure, and toxicant exposure cr eate differences that are reflected in their metabolite profiles. Figure 6-7. Electropherogram a nd concentration profiles of Aplysia californica chemosensory cells. A, B) Major amino acids profiling by CE-LIF. C, D) Nitrite and nitrate profiling by CE-CCD. (n=3) The embryonic cells (2-3 days after egg-laying) were analyzed to profile amino acids and nitrite and nitrate (Figure 6-8). Previous work by Dickinson et al (Dickinson et al. 2000) showed that Phe-Met-Arg-Phe-NH2 (FMRFamide)-like-immunoreactive (L IR) cells first appeared during the trochophore stage (2.5-4 days after egg-laying), that by the veliger stag e (5-7 days) serotoninLIR cells appeared in the apical organ, a nd that shortly before hatching (8-10 days) catecholamine-containing cells appeared around the mouth and in the foot in the mullusc Aplysia 113

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californica (Dickinson et al. 2000). In addition, in another mollusc Phestilla sibogae cells containing 5-HT, catecholamines, and FMRFamidelike peptides were already present by the earliest veliger stages (5-7 days), but the trochophore stage was not explored because the autofluorescent yolk obscured visualizati on in these earliest embryos (Croll 2006). Figure 6-8. Electropherograms and concen tration profiles of embryonic cells of Aplysia californica. A) Amino acid profiles. 2nd day sample (70-fold dilution) and 3rd day sample (167-fold dilution). B, C) Major am ino acid concentrations in the cells. D) Nitrite and nitrate. E) Concentratio ns of nitrite and nitrate. (n=3) Conclusion The present study shows that th ere are distinct differences in metabolites among mulluscs, including the axoplasm of squid, the CNS of Nau tilus, and chemosensory and embryonic cells of 114

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115 Aplysia californica. Serotonin-containing axoplas m was identified in the giant axon of squid. In neuron and muscle of Nautilus, 5-HT was also present and may play a role as a neuromodulator. Along with the NADPH-d results (Moroz 2006), nitric oxide-metabolites (Arg, Cit, nitrite, and nitrate) provided additional evidence that ch emosensory cells use NO to activate the feeding mechanism in Aplysia californica. These results further can be used to understand the CNS in the mollusca and even to compare with other phyla animals.

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BIOGRAPHICAL SKETCH Dosung Sohn was born in Seoul, Korea in 1974. He completed his undergraduate career at the Inha University in Incheon, Korea and rece ived the masters degree at the same place under the research direction of Wan In Lee graduating in 2001. He continued his education at the University of Florida under Weihong Tan graduating in 2009. 133