Recent advances in the research concerning chemical mediators of inflammatory and allergic reactions have implicated two profoundly active lipids, viz. platelet activating factor and members of the leukotriene family. These lipids have strikingly similar pharmacological actions and evidence suggest a close biochemical interaction between these substances. While there are assays for the LTs based on biological activity, RIA, UV and HPLC properties, there exists only biological assays for PAF. Detailed studies of AGEPC (acetyl glycerol ether phosphocholine) which has been described as the structure of PAF, have been hampered without a specific, sensitive method of analysis. Such analytical capability is important to more fully evaluate the physiological role for AGEPC in health and disease. We propose to develop physico-chemical methods of quantitative analysis of PAF, its lyso-analogs, alkyl-acyl diglyceride, and acyl, ether phosphatidylcholine based on fast atom bombardment mass spectrometry and GC/MS using stable isotope techniques. These techniques will add to the existing analytical armamentorium to investigate each intermediate in our proposed acetyl ether PC cycle. This cycle is postulated to operate in certain cells leading to the synthesis of prostanoids, leukotrienes and/or PAF upon biological stimulation. We propose to investigate in detail precursor-product relationships between 1-0-hexadecyl-2-arachidonyl-glycerophosphocholine and eicosanoids using stable isotope and radioisotope tracer techniques. The metabolism of PAF, lyso PAF and 1-0-hexadecyl-2-arachidonyl-GPC will be carried out in cells including polymorphonuclear leukocytes, macrophages, monocytes and pulmonary endothelial cells. Basic information as to the turnover of arachidonic acid-containing lipids will be obtained in the cells under study. Furthermore, it is anticipated that potential pools for prostanoids and lipoxygenase products will be identified. Finally, various inhibitors of the biosynthesis of prostaglandins, leukotrienes and phospholipids will be employed to study the biochemical interrelationships between mediator synthesis. It is by understanding the specific biochemical events surrounding PAF and leukotrienes that development of specific pharmacologic agents, antagonists to these mediators, may be possible.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI020774-06
Application #
3130592
Study Section
Biochemistry Study Section (BIO)
Project Start
1984-01-01
Project End
1989-12-31
Budget Start
1989-01-01
Budget End
1989-12-31
Support Year
6
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Colorado Denver
Department
Type
Schools of Medicine
DUNS #
065391526
City
Aurora
State
CO
Country
United States
Zip Code
80045
Maclouf, J; Murphy, R C; Henson, P M (1990) Transcellular biosynthesis of sulfidopeptide leukotrienes during receptor-mediated stimulation of human neutrophil/platelet mixtures. Blood 76:1838-44
Maclouf, J; Murphy, R C; Henson, P M (1989) Platelets and endothelial cells contribute to the production of LTC4 by transcellular metabolism with neutrophils. Adv Prostaglandin Thromboxane Leukot Res 19:259-62
Fradin, A; Zirrolli, J A; Maclouf, J et al. (1989) Platelet-activating factor and leukotriene biosynthesis in whole blood. A model for the study of transcellular arachidonate metabolism. J Immunol 143:3680-5
Murphy, R C; Clay, K L (1987) Measurement of platelet-activating factor by physicochemical technique s. Am Rev Respir Dis 136:207-10
Haroldsen, P E; Voelkel, N F; Henson, J E et al. (1987) Metabolism of platelet-activating factor in isolated perfused rat lung. J Clin Invest 79:1860-7
Chilton, F H; Murphy, R C (1987) Stimulated production and natural occurrence of 1,2-diarachidonoylglycerophosphocholine in human neutrophils. Biochem Biophys Res Commun 145:1126-33
Chilton, F H; Hadley, J S; Murphy, R C (1987) Incorporation of arachidonic acid into 1-acyl-2-lyso-sn-glycero-3-phosphocholine of the human neutrophil. Biochim Biophys Acta 917:48-56
Haroldsen, P E; Clay, K L; Murphy, R C (1987) Quantitation of lyso-platelet activating factor molecular species from human neutrophils by mass spectrometry. J Lipid Res 28:42-9
Haroldsen, P E; Murphy, R C (1987) Analysis of phospholipid molecular species in rat lung as dinitrobenzoate diglycerides by electron capture negative chemical ionization mass spectrometry. Biomed Environ Mass Spectrom 14:573-8
Chilton 3rd, F H; Murphy, R C (1986) Fast atom bombardment analysis of arachidonic acid-containing phosphatidylcholine molecular species. Biomed Environ Mass Spectrom 13:71-6

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