Quantitative analysis of platelet activating factor treated with pentafluorobenzoyl chloride using gas chromatography/negative ion chemical ionization mass spectrometry.
To confirm that platelet activating factor (PAF) plays an important role as a mediator in acute inflammation and allergic reaction, it is necessary to develop a more sensitive and stable method for measuring trace amounts of PAF in various biological samples. For this reason, the authors have adapted gas chromatography/negative ion chemical ionization mass spectrometry (GC/NICI-MS) (developed by Ramesha and Pickett in 1985) by employing an SPB-1 column and isobutane as the reagent gas. Furthermore, with this method the authors attempted an investigation of the time course of hexadecyl- and octadecyl-PAF production and release from human polymorphonuclear leukocytes (PMNs) stimulated by Ca-ionophore A23187. PMNs were obtained from venous blood from a human donor by centrifugation and were stimulated by 5 microM of Ca-ionophore A23187. PAF was purified using a SEP-PAK silica column and thin layer chromatography, and then was hydrolyzed with phospholipase C. The extract was treated with pentafluorobenzoyl chloride. Using 1-O-hexadecyl-2-acetyl (perdeuterated)-sn-phosphocholine) as an internal standard, the following results were obtained: The standard curve for this quantitative analysis was linear with a correlation coefficient of 0.9997 from 1 pg to 200 ng. The authors assumed that quantities as low as a few picograms of PAF could be measured by the method. The production of hexadecyl-PAF in the cell pellet peaked (8.1 +/- 1.34 ng/10(7) cells) at 2 min after stimulation and that in the supernatant peaked (6.3 +/- 0.97 ng/10(7) cells) at approximately 7 min after stimulation. Octadecyl-PAF could not be detected in this experiment.(ABSTRACT TRUNCATED AT 250 WORDS)
Shindo
K
K
First Department of Internal Medicine, Yokohama City University School of Medicine, Japan.
Hashimoto
Y
Y
D001565:Benzoates / Q000494:pharmacology*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D006801:Humans; D010972:Platelet Activating Factor / Q000032:analysis*; D005464:Fluorobenzenes; D005466:Fluorocarbons
D001565:Benzoates; D010972:Platelet Activating Factor; C014737:pentafluorobenzoyl chloride; D005464:Fluorobenzenes; D005466:Fluorocarbons