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INSTRUMENTATION II BIOCHEMICAL APPLICATIONS IN MIGRAINE RESEARCH Alfred Nji Fonteh, PhD.

INSTRUMENTATION II BIOCHEMICAL APPLICATIONS IN MIGRAINE RESEARCH Alfred Nji Fonteh, PhD. HMRI- Molecular Neurology Program 99 N El Molino Avenue Pasadena, CA 91101. OBJECTIVES. Overview of hypotheses Important molecules in migraine research

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INSTRUMENTATION II BIOCHEMICAL APPLICATIONS IN MIGRAINE RESEARCH Alfred Nji Fonteh, PhD.

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  1. INSTRUMENTATION II BIOCHEMICAL APPLICATIONS IN MIGRAINE RESEARCH Alfred Nji Fonteh, PhD. HMRI- Molecular Neurology Program 99 N El Molino Avenue Pasadena, CA 91101

  2. OBJECTIVES • Overview of hypotheses • Important molecules in migraine research • Methods for measuring fatty acids and eicosanoids • Research data • Methods for measuring steroid hormones • Research data • Methods for measuring protein and peptides • Research data • Proposed pathway from discovery to the consumer

  3. A) COMPOSITIONAL ANALYSES • DNA • PROTEINS • LIPIDS • CARBOHYDRATES • ELEMENTS • B) MECHANISM BASED ANALYSES • VASOACTIVITY (peptides, fatty acids, hormones, NT) • INFLAMMATION (prostanoids, peptides, cytokines) • PAIN (receptors, fatty acid amides, peptides) • OXIDATION (fatty acids, proteins) • ION CHANNELS (receptors, elements, NT)

  4. Free Fatty Acids • LC-APCI-ECNICI-MS/MS • Add IS to CSF • Extract steroids • Derivatize to PFBr • Run on C8 LC using methanol gradient • Detect ions using ECNI-MS/MS

  5. Total Fatty Acids Add IS EXTRACT LIPIDS (Base hydrolysis for total) SPE DERIVATIZE PFBr + DIPE NICI-GC/MS LC-NICI/APCI-MS Analyze Data

  6. PUFA levels in CSF 60 0.5 3 0.8 50 0.4 2 0.6 40 0.3 30 0.4 0.2 20 1 0.2 0.1 10 0 0.0 0.0 0 EPA/DGLA/LA AA/LNA -Ist Bar DHA -2nd Bar Migraineur (+HA) Control Migraineur (-HA) Migraineur (+HA) Control Migraineur (-HA) Fatty acids (nmol/nmol lipid phosphorus) Total FA OA/SA Control Migraineur (-HA) Migraineur (+HA) Control Migraineur (-HA) Migraineur (+HA)

  7. ANALYTICAL METHODS IN EICOSANOID RESEARCH A r a c h i d o n i c A c i d 1 5 H E T E 1 5 - k e t o - 1 3 , 1 4 - d i h y d r o - P G F G C / M S , H P L C / M S / M S E L I S A , G C / M S , H P L C / M S / M S 2 a G C / M S , H P L C / M S / M S 1 2 H E T E P G G E L I S A , G C / M S , H P L C / M S / M S P G F 2 2 a E L I S A , G C / M S , H P L C / M S / M S P G H 2 P G I 5 - H E T E T X A 2 H P L C / M S / M S 2 G C / M S , H P L C / M S / M S P G E 2 T X B E L I S A , G C / M S , H P L C / M S / M S 2 L T A 6 - k e t o - P G F 4 1 a E L I S A , G C / M S , H P L C / M S / M S E L I S A , G C / M S P G D 2 P G A L T C4 2 E L I S A , G C / M S , H P L C / M S / M S G C / M S , H P L C / M S / M S E L I S A , H P L C / M S / M S L T B 2 , 3 - d i n o r - 6 k e t o - P G F 4 1 a E L I S A , G C / M S , H P L C / M S / M S P G J G C / M S 2 E L I S A , G C / M S , H P L C / M S / M S 1 5 - k e t o - d i h y d r o - P G E L T D 2 4 G C / M S , H P L C / M S / M S H P L C / M S / M S 2 , 3 - d i n o r - T X B 2 E L I S A , G C / M S L T E 4 H P L C / M S / M S

  8. PDB 1PGE heme ibuprofen analog membrane binding domain Prostaglandin H Synthase 2

  9. Add IS EXTRACT LIPIDS (add 4-5 vols of ethanol) SPE NICI-GC/MS + ANALYSIS OF DATA • EICOSANOID MEASUREMENT • EIA/RIA/ELISA • BIOASSAYS • TLC/HPLC • NICI-GC/MS • DERIVATIZE (methoxyime-pentafluorobenzylester trimethylsilyl esters) • 1) Methoxylamine (MOX for ketone group) • 2) 20 % pentafluorobenzyl bromide (PFBr) and 20 % diisopropylethylamine • (DIPE) in acetonitrile for carboxyle groups) • 3) N,O-bis(trimethyl-silyl)-trifluoroacetamide (BSTFA) for hydroxyl groups

  10. TOTAL ION CHROMATOGRAM

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