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Methods of Measurement and Detection in Neurochemistry

Methods of Measurement and Detection in Neurochemistry. Neels van der Schyf, B.Pharm., M.S., Ph.D. and Paul R. Carlier, Ph.D. (pcarlier@vt.edu, 231-9219) February 25 , 2010 http://filebox.vt.edu/users/pcarlier/PRCNeurochemistry10.ppt. Objectives.

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Methods of Measurement and Detection in Neurochemistry

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  1. Methods of Measurement and Detection in Neurochemistry Neels van der Schyf, B.Pharm., M.S., Ph.D. and Paul R. Carlier, Ph.D. (pcarlier@vt.edu, 231-9219) February 25, 2010 http://filebox.vt.edu/users/pcarlier/PRCNeurochemistry10.ppt

  2. Objectives • Introduction to selected methods of measurement and detection in neurochemistry; • Overview of analytical methodology for the detection and measurement of brain chemicals in vitro, in vivo, ex vivo and in situ; • Overview of methods for the localization of particular neurochemicals in the brain in vivo and ex vivo.

  3. So, what can one measure? ?

  4. Biogenic amines • Catecholamines and their metabolites • 5-Hydroxytryptamine (serotonin) • Trace amines • -phenylethylamine • m- and p-tyramine • phenylethanolamine • m- and p-octopamine • Tryptamine • Histamine

  5. Some catecholamines and their O-methylated amine metabolites (and enzymes responsible)

  6. Enzymes involved in catecholamine metabolism • Tyrosine hydroxylase (TH) • Aromatic L-amino acid decarboxylase (AADC) • Dopamine-b-hydroxylase (DBH) • Phenylethanolamine N-methyltransferase (PNMT) • Monoamine oxidase (MAO) • Catechol-O-methyltrasferase (COMT)

  7. Serotonin (5-hydroxytryptamine, 5-HT)

  8. Structures of some trace amines: A) -phenylethylamine; B) phenylethanolamine; C) m-tyramine; D) p-tyramine; E) m-octopamine; F) p-octopamine and g) tryptamine

  9. Histamine

  10. Basic Methods &Applied Technologies

  11. 1. Chromatography B • Principles of chromatography A

  12. Gas chromatography (GC) • Introduction

  13. Flame Ionization Detector (FID) Nitrogen Phosphorous Detector (NPD) Electron Capture Detector (ECD) Thermal Conductivity Detector (TCD) Flame Photometric Detector (FPD) Atomic Emission Detector (AED) Photoionization Detector (PID) Electrical Conductivity Detector (ELCD) Mass Spectrometer (MS) Detectors for GC

  14. Flame Ionization Detector (FID) Electron Capture Detector (ECD) The ECD is extremely sensitive to molecules containing highly electronegative functional groups such as halogens, peroxides, quinones, and nitro groups Most widely used and generally applicable detectors for gas chromatography

  15. Mass Selective Detector (MS) (see also Mass Spectrometry) Mass spectrometry is based upon the ionization of solute molecules in the ion source and the separation of the ions generated on the basis of their mass/charge ratio by an analyzer unit.

  16. increases volatility reduces polarity improves extraction efficiency from aqueous media introduces functional groups sensitive to specific detection, eg. Silylation Acylation Alkylation Esterification Derivatization for GC (1) Trimethylsilylating agents

  17. Trimethylsilylation of phenol Derivatization for GC (2, cont.) N-2,6-dinitro-4-trifluoromethylphenyl, O-trimethylsilyl derivatives of DA and NE

  18. Acylation reaction with trifluoroacetic anhydride Acylation reaction with N-fluoroacyl-imidazole Derivatization for GC (3, cont.) • Typical acylation reaction Products formed when DA and NE are reacted with trifluoroacetic anhydride under anhydrous conditions

  19. Commonly used alkylating reagents Alkylation of phenol with diazomethane Derivatization for GC (4, cont.)

  20. Esterification (first choice for derivatization of acids) Derivatization for GC (5, cont.) A typical esterification reaction

  21. Advantages Inexpensive Several amines at once Separation of positional isomers (meta vs. para) Limitations Advantages/limitations of GC • Thermolability • Lack of specificity • Interference from tissue, • solvents, reagents

  22. Gas chromatography/Mass spectrometry (GC-MS) • Selected ion monitoring (SIM) • MS is adjusted so that one or more ions characteristic of the compound under investigation are monitored continuously as an aliquot of the sample from the GC/MS interface outlet enters the ion source. • Ionization techniques • EI (electron ionization) • PCI (positive chemical ionization) • NCI (negative chemical ionization)

  23. Gas chromatography/Mass spectrometry (GC-MS) (Remember Mass Selective Detector?)

  24. Neurotoxic urinary acids in rats found by GC/MS

  25. High-performance liquid chromatography (HPLC) Typical HPLC column

  26. Detection in HPLC • UV • Fluorescence • Electrochemical

  27. Detection limits for catecholamines with UV, fluorimetric and electrochemical detection using HPLC Substance Detection limit (ng) UV Fluorimetric Electrochemical NE 5 2 0.015 L-DOPA 10 5 0.10 E 5 2 0.15 DA 5 5 0.20

  28. Neurotoxins in the brains of baboons and urine of humans, found with HPLC

  29. In vivo Microdialysis

  30. In vivo Microdialysis setup

  31. In vivo microdialysis in rat brain

  32. LC/MS interface!

  33. PRC200: A Triple Reuptake Inhibitor • Promising antidepressant drug with activity at three transporters Liang, Shaw, Boules, Briody, Robinson, Oliveros, Blazar, Williams, Zhang, Carlier, Richelson* J. Pharm. Exp. Therap.2008, 327, 573-583. http://dx.doi.org/10.1124/jpet.108.143610

  34. PRC200 Microdialysis Experiments • Behavioral models (forced swim test, tail suspension test) consistent with known reuptake inhibtion-based antidepressants • In vivo microdialysis coupled with HPLC/EC detection • Rats cannulated in the medial prefrontal cortex (mPFC) or nucleus accumbens NA) • experiments carried out 3-5 days following surgery • Assess extracellular NE, 5-HT, DA, DOPAC, HVA, 5-HIAA, levels following administration of drug or saline

  35. medial Prefontal Cortex (mPFC) NE 5-HT HVA DOPAC

  36. medial Prefontal Cortex (mPFC) • PRC200 significantly increases NE and 5-HT in the mPFC; decrease 5-HIAA (5-HT intracellular metabolite). • No change in DA: mPFC is devoid of DA transporters. Slight reduction in DOPAC? 5-HIAA

  37. Nucleus accumbens (NA) DA 5-HT DOPAC HVA

  38. Nucleus accumbens (NA) 5-HIAA • PRC200 significantly increases DA and 5-HT in the core of the NA. • Large reduction in intracellular metabolites of DA (DOPAC and HVA) and 5-HT (5-HIAA) • No change in NE seen: core of NA is devoid of NE transporters

  39. 2. Radioenzymatic assay (REA) REA is an assay of the catalytic activity of an enzyme based on the use of a radioactive substrate, i.e. an enzyme is used to change a substrate chemically into a radiolabeled product that can be measured by scintillation, primarily: Catecholamine + *3H-S-adenosylmethionine *3H-product Gamma counter and beta scintillation counter setup

  40. Materials that can be measured and enzymatic activity that can be assessed by REA Catecholamines NE E DA NEGlu EGlu DAGlu MeDA MeNE IsoproterenolIsoetharine N,N, dipropylDA Dobutamine 2 MeDA Non catecholamines Octopamine Phenylethylamine Phenylethanolamine Phenylpropanolamine Enzymes DOPA decarboxylase DBH PNMT COMT COMT assay for catecholamines PNMT assay for NE

  41. Distribution of very-high affinity binding sites of [11C]Ro15-4513 in monkey brain visualized by PET. ACg; anterior cingulate SM; medial septum Acc; nucleus accumbens Amy; amygdala Hipp; hippocampus Ins; insula 3. Positron Emission Tomography (PET) can also use 13N, 15O, 18F http://en.wikipedia.org/wiki/Positron_emission_tomography

  42. PET in neurodegenerative diseases

  43. Brain MAO and cigarette smoking (PET study)

  44. Localization of a novel subtype of prostacyclin receptor in the rat brain using typical in vitro autoradiography from 15R-[3H]tolyl-isocarbacyclin (a stable and potent agonist for a novel CNS-type of prostacyclin receptor) in rat brain sections. Abbreviations in the figures define specific regions of the brain. 4. Autoradiography advantage: can use beta-emitters like 3H or 125I; for RNA transcripts probes are normally labeled with 32P 33P,or 35S.Note: gamma-emitters can also be used. http://www.lab.anhb.uwa.edu.au/hb313/main_pages/timetable/lectures/autoradiography.htm

  45. 5. Single Photon Emission Computed Tomography (SPECT) http://www.physics.ubc.ca/~mirg/home/tutorial/intro.html

  46. SPECT study of DA transporter (DAT) in brain of baboons with [123I] -CIT need gamma-emitters: can also use 99mTc, 111In

  47. SPECT used for DA detection in PD A dynamic sequence of 5-min transverse [123I]IBZM images taken at the level of the striatum, showing the initial uptake of the tracer throughout the brain, followed by washout in brain regions with no DA receptors. After about an hour, the distribution of activity represents the concentration of DA receptors, particularly in the striatum.

  48. SPECT study & DA binding in baboon brain with [123I]IBZM

  49. 6. Magnetic Resonance

  50. 6.1 Magnetic Resonance Imaging (MRI) High resolution imaging, post-pallidotomy, to define lesion sites. http://www.neuroguide.com/gregg.html

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