Parvez / Naoi / Nagatsu Methods in Neurotransmitter and Neuropeptide Research

1. Auflage 2013
ISBN: 978-1-4832-9022-5
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: 6 - ePub Watermark

E-Book, Englisch, Band Volume 11, 436 Seiten

Reihe: Techniques in the Behavioral and Neural Sciences

This two-part book provides the most advanced biochemical and histochemical analytical methods on neurotransmitters, neuromodulators, and neuropeptides described by international specialists. The biochemical methods include liquid chromatography/electrochemistry, liquid chromatography/fluorescence and chemiluminescence detection, gas chromatography or liquid chromatography with mass spectrometry, and immunoassays.Morphological and physiological methods are also described, including immunohistochemistry, in situ hybridization, in vivo microdialysis, electrophysiological analysis, microwave irradiation, electrophysiological analysis, micro-anatomical dissection, peptide sequence analysis, quantitative autoradiographic analysis, and isolated CNS preparations. The neurotransmitters and neuropeptides include catecholamines, indoleamines, histamine, polyamines, and various neuropeptides, and their related enzymes. The book is the most comprehensive, advanced and versatile volume on the biochemical, morphological and physiological analysis of neurotransmitters and neuropeptides.

Autoren/Hrsg.

Parvez, S. H.

Naoi, M.

Nagatsu, T.

Inhaltsverzeichnis

1;Front Cover;1
2;Methods in Neurotransmitter and Neuropeptide Research;4
3;Copyright Page;5
4;Table of Contents;10
5;Preface;6
6;List of Contributors;8
7;Chapter 1. Multi-electrode detection system for determination of neurotransmitters;18
7.1;1. Introduction;18
7.2;2. Principles of analysis using multi-electrode detection systems;20
7.3;3. HPLC systems using multi-electrode array detection;28
7.4;4. Analysis of biogenic amines and their metabolites;30
7.5;5. Analysis of amino acids;40
7.6;6. Activities of the enzymes related to monoamine metabolism;43
7.7;7. Biochemical and pharmacological applications;47
7.8;Acknowledgements;51
7.9;References;51
8;Chapter 2. Determination of biogenic amines, their metabolites, and other neurochemicals by liquid chromatography /electrochemistry;58
8.1;1 Introduction;58
8.2;2. Principles;61
8.3;3. Instrumentation;65
8.4;4. Tyrosine metabolites;70
8.5;5. Tryptophan metabolites;75
8.6;6. Enzyme activity;77
8.7;7. Amino acids;80
8.8;8. Choline and acetylcholine;82
8.9;9. Microdialysis and column format;85
8.10;10. Review of general applications of LCEC to drugs and other organics;88
8.11;11. Conclusion;90
8.12;References;91
9;Chapter 3. High-performance liquid chromatography-fluorometric and chemiluminometric detection of biogenic amines and other neurotransmitters;100
9.1;1 Introduction;100
9.2;2. Biogenic amines and their metabolites;101
9.3;3. Neurotransmitter amino acids;119
9.4;4. Neuropeptides;123
9.5;References;133
10;Chapter 4. Combined gas chromatography-mass spectrometry for the analysis of central and peripheral biogenic amines;144
10.1;1 Introduction;144
10.2;2. Methods;145
10.3;3. Results and comments;151
10.4;References;163
11;Chapter 5. Immunochemistryand in situ hybridization of catecholaminesynthesizing enzymes and the related neurotransmitters;168
11.1;1 Introduction;168
11.2;2. General histochemical procedures;169
11.3;3. Phylogenetic and ontogenetic studies of catecholamine systems by immunocytochemistry;177
11.4;4. Conclusion;196
11.5;Acknowledgements;196
11.6;References;197
12;Chapter 6. Electron microscopic exploration of synaptic organization;202
12.1;1 Introduction;202
12.2;2. Conventional cytochemical synaptic studies;203
12.3;3. Further cytochemical synaptic studies;204
12.4;4. Quantification of synaptic ultrastructure;209
12.5;5. Synaptic quantification: stereology;216
12.6;6. Computer reconstruction of synapses;223
12.7;Acknowledgements;230
12.8;References;230
13;Chapter 7. In vivo microdialysis of neurotransmitters and their metabolites;236
13.1;1 Introduction;236
13.2;2. Methodology;237
13.3;3. Determination of extracellular neurotransmitter concentrations;245
13.4;4. Criteria for identification of neurotransmitters;252
13.5;5. Problems of microdialysis;258
13.6;5. Concluding remarks;261
13.7;Acknowledgements;261
13.8;References;262
14;Chapter 8. Electrophysiological analysis of sympathetic function;266
14.1;1 Introduction;266
14.2;2. General principles of electrophysiological techniques for the analysis of sympathetic function;267
14.3;3. Application of electrophysiological analysis to the study of pharmacology;278
14.4;4. Conclusions and future perspectives;285
14.5;References;286
15;Chapter 9. Current status of rapid enzyme inactivation through the use of microwave irradiation;290
15.1;1 Introduction;290
15.2;2. Criteria for evaluating an adequate microwave device;291
15.3;3. A recent model of a microwave device;292
15.4;4. Effect of MWR on the concentration of various endogenous neurochemicals;307
15.5;5. Discussion and conclusions;321
15.6;Acknowledgements;322
15.7;References;322
16;Chapter 10. Precise micro- and macro-anatomical dissection of the rat brain and the investigation of neuroendocrine function;328
16.1;1 Introduction;328
16.2;2. Micro-dissection and micropunch;333
16.3;3. Macro-dissection of the rat brain;352
16.4;4. Conclusion;362
16.5;Acknowledgements;362
16.6;References;363
17;Chapter 11. Analysis of enzymes in catecholamine biosynthesis;366
17.1;1 Introduction;366
17.2;2. Assay methods for tyrosine hydroxylase;367
17.3;3. Assay methods for aromatic L-amino acid decarboxylase;377
17.4;4. Assay methods for dopamine ß-hydroxylase;382
17.5;5. Assay methods for phenylethanolamine N-methyltransferase;388
17.6;6. Conclusion;392
17.7;Acknowledgement;392
17.8;References;393
18;Chapter 12. Catechol-O-methyltransferase;398
18.1;1 Introduction;398
18.2;2. Properties of COMT;398
18.3;3. Distribution and purification;401
18.4;4. The cellular distribution of COMT;403
18.5;5. Methods for the measurement of COMT activity;411
18.6;6. Examples of current procedures;415
18.7;7. Conclusion;420
18.8;References;421
19;Subject index;432

Leseproben

Chapter 2

Determination of biogenic amines, their metabolites, and other neurochemicals by liquid chromatography/electrochemistry

Chester T. Duda and Peter T. Kissinger,     Department of Chemistry, Purdue University, W. Lafayette, IN 47907, and Bioanalytical Systems Inc. 2701, Kent Avenue, West Lafayette, IN 47906-1382, USA

Publisher Summary

This chapter focuses on the determination of biogenic amines, their metabolites, and other neurochemicals by liquid chromatography/electrochemistry. Electrochemistry has a distinct advantage compared to most analytical techniques as it involves a direct conversion of chemical information to an electrical signal without any need for intermediate optical or magnetic carriers. For example, all catechol derivatives can be readily oxidized at a graphite electrode to generate the corresponding orthoquinone, two protons, and two electrons. The combination of gas chromatography and mass spectrometry has revolutionized the ability to handle extremely complex mixtures of chemical substance. The direct conversion of chemical information into electricity gives electrochemical measurements a significant advantage when compared with many other analytical techniques. It has been recognized that considerable advantage results from the coupling of liquid chromatography (LC) with electrochemistry (EC). The chapter discusses the application of LCEC to endogenous compounds of neurologic interest.

1 Introduction

A great many problems in biomedical research involve the determination of fewer than 10 individual substances in very complex samples such as biological fluids or tissue homogenates. In many laboratories it has become routine to isolate a few microliters of perfusate from a living animal using the in vivo microdialysis sampling technique. These samples contain thousands of individual compounds and ions which are thought to be irrelevant to the problem at hand. The amount of sample is frequently limited, particularly in experiments with laboratory animals, and it is often necessary to determine amounts of individual compounds in the picomole range and below. To meet these challenges a selective analytical approach is needed, with good detection limits for substances of interest.

A combination of existing technologies can provide the desired instrumentation. For example, the combination of gas chromatography and mass spectrometry (GCMS) has revolutionized our ability to handle extremely complex mixtures of chemical substances. Unfortunately, this technique does not solve all problems equally well. Many nonvolatile and thermally labile metabolites of biomedical interest are not directly suitable for GCMS. In addition, for many laboratories the expense and complexity of the instrumentation rules it out for routine purposes. Since this chapter was first prepared in the early 1980s, powerful GCMS systems have become available at much lower cost. This trend will continue. LCMS is also gaining in capability at lower cost, but is generally insufficient for neurotransmitter studies.

For over twenty years it has been recognized that considerable advantage results from the coupling of liquid chromatography (LC) with electrochemistry (EC) (see for example Krstulovic, 1986; Kissinger, 1989). While more limited in scope, the LCEC system has many parallels with the GCMS system. In both cases a high-resolution separation technique is coupled to a measurement scheme involving the direct conversion of chemical information into electricity. Many of the compounds which cause problems for the gas-phase technique are well suited to the liquid-phase variant. The detection limits achievable with both methodologies are roughly equivalent. While GCMS is far more versatile and has the edge in molecular specificity, LCEC is less expensive and is more convenient to use for many problems. LCEC systems are sufficiently inexpensive that one laboratory will frequently use several instruments with autosamplers to handle a large sample load. The basic components of an LCEC system are depicted in Fig. 1.

Fig. 1 Basic components of an LCEC system. (Reproduced with permission of Bioanalytical Systems, Inc.)

Phenols and indoles have been known for at least 60 years to be electrochemically reactive. Nevertheless, for all practical purposes it was not until the early 1970s that this reactivity was used to advantage by analytical chemists. Professor Ralph Adams and his co-workers at the University of Kansas were the first to recognize that the ease of oxidation of tyrosine and tryptophan metabolites might provide a ‘handle’ for measurement of these substances in brain tissue. Adams was particularly intrigued by the possibility of using implanted microelectrodes to follow the release of neurotransmitters in vivo. While this revolutionary idea must still be considered to be at a very early stage of development, a number of promising results have already been published. Several excellent reviews on in vivo electrochemistry have appeared in recent years (Marsden et al., 1984; Justice et al., 1985; Justice, 1987).

Electrochemistry has a distinct advantage compared to most analytical techniques in that it involves a direct conversion of chemical information to an electrical signal without need for intermediate optical or magnetic carriers. For example, all catechol derivatives can be readily oxidized at a graphite electrode to generate the corresponding orthoquinone, two protons, and two electrons:

Reaction 1

To use this anodic oxidation analytically, it is most convenient to measure the rate at which electrons are transferred across the electrode-solution interface, in other words, the anodic current, . The instantaneous current is directly proportional to the number of molecules coming into contact with the interface per unit time and can therefore be used to determine the concentration of the reactant in the neighboring solution.

One of the principal problems of electrochemistry is that its molecular specificity is inadequate for many purposes. All catechol derivatives in a complex mixture react similarly and generally cannot be distinguished, one from the other, by an electrode. For this reason it is necessary to incorporate a separation step into the electrochemical experiment. Modern reverse-phase or ion-exchange chromatography is ideally suited to this purpose because ionic mobile phases are used (necessary for electrochemical detection). Modern microparticle columns are capable of rapidly separating closely related compounds in a few minutes with relatively little dilution. Minimizing the dilution inherent in chromatography requires a careful selection of the column diameter to match the volume of sample available.

Liquid chromatography has many advantages for the trace determination of polar organic substances. The number of sample manipulations can often be reduced when compared to gas-phase, fluorescence, chemiluminescence, or radioenzymatic methods. The primary disadvantages are (1) the fact that samples must be processed in series for the final quantitation, and (2) that the reliability of the instrumentation (including columns) is not perfect. While the latter problem has been dramatically improved in the last few years there remains considerable room for further progress, particularly with respect to pumps, autosamplers, and columns. LC systems do require maintenance. Like automobiles, they can last a very long time with proper care. An excellent recent book contains many good ideas on how to care for a liquid chromatograph (Dolan and Snyder, 1989).

Because electrochemistry is a surface technique, it is a simple matter to build thin-layer detector cells with microliter volumes. Such cells are capable of monitoring eluted components without distorting the chromatographic separation. The first experiments in this area were carried out in the spring of 1972 (Kissinger et al., 1973) and since that time over two thousand papers have appeared, many of which are dedicated to neurochemical measurements. The physical principles of electrochemistry will be briefly reviewed in the following section.

2 Principles

Electrochemistry is one of the most sensitive tools available to the analytical chemist. The direct conversion of chemical information into electricity gives electrochemical measurements a significant advantage when compared with many other analytical techniques. Recent advances in metal-oxide semiconductors provide an inexpensive yet effective means to measure very low electric currents. MOSFET electronics combined with an appropriate electrode provides a sensitive and reliable approach to the determination of redox-active substances. Reactions at an electrode can be followed at a rate as low as 10-16 equivalents per second! Electrochemists can now make measurements at electrodes with a radius below 1 µm on a time scale of 10-8 s!

In order to effectively utilize such ‘amperometric’ measurements, several points must be considered. First, electrochemistry is a surface technique; to optimize its use for trace analysis one must enhance the ratio of the surface area relative to the volume of the solution, while keeping the latter small. Second, because electrochemistry is a ‘chemical’ as opposed to a...