Determination of platelet-activating factor by a chemiluminescence method and its application to stimulated guinea pig neutrophils.
A simple, rapid and sensitive chemiluminescence method has been developed to measure platelet-activating factor (PAF). Hydrogen peroxide generated from PAF, upon phospholipase D cleavage, by choline oxidase is determined as chemiluminescence by a luminol-microperoxidase system. The detection limit of PAF by this method is 5 pmol/tube. The method is reproducible with a 5.5% coefficient of variation at 10 pmol of PAF (n = 5). Lipids were extracted from guinea pig neutrophils after stimulation with cytochalasin B and N-formyl-methionyl-leucyl-phenylalanine, and PAF was isolated by high-performance liquid chromatography and determined by chemiluminescence measurements. The amount of PAF detected was 96.1 +/- 39.7 (mean +/- SD, n = 7) pmol/10(8) cells. This highly sensitive method could be useful for the determination of PAF generated under pathophysiological conditions.
Hasegawa
Y
Y
Department of Clinical Chemistry, School of Pharmaceutical Sciences, Toho University, Chiba, Japan.
Kunow
E
E
Shindou
J
J
Yuki
H
H
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005260:Female; D006168:Guinea Pigs; D007202:Indicators and Reagents; D008163:Luminescent Measurements; D008165:Luminol; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D009504:Neutrophils / Q000187:drug effects / Q000502:physiology*; D010743:Phospholipids / Q000302:isolation & purification; D010972:Platelet Activating Factor / Q000031:analogs & derivatives / Q000032:analysis* / Q000302:isolation & purification
D007202:Indicators and Reagents; D010743:Phospholipids; D010972:Platelet Activating Factor; C037145:1-hexadecyl-2-acetyl-glycero-3-phosphocholine; D009240:N-Formylmethionine Leucyl-Phenylalanine; D008165:Luminol