K. Shinozaki et al., A NEW METHOD OF PURIFICATION AND SENSITIVE BIOASSAY OF PLATELET-ACTIVATING-FACTOR (PAF) IN HUMAN WHOLE-BLOOD, Life sciences, 54(6), 1994, pp. 429-437
Citations number
23
Categorie Soggetti
Biology,"Medicine, Research & Experimental","Pharmacology & Pharmacy
There is no satisfactory assay procedure of PAF in human whole blood i
n terms of sensitivity, reproducibility and simplicity. This is due to
coexisting lipids from plasma and cellular membranes which inhibit me
asurement of PAF in various assay procedures, including bioassay. In t
he present study, an attempt was made to eliminate these interfering l
ipid inhibitors from brood samples. Lipids in human whore blood were e
xtracted according to the method of Bligh and Dyer and the organic lay
er was dried under a stream of nitrogen. Then, the dried organic layer
was dissolved in diethyl-ether and the solution was kept at -20 degre
es C which was then centrifuged. The resulting supernatant was then ap
plied to an anion-exchange column and the PAF fraction was obtained by
step-wise gradient elution. The fraction was further purified by norm
al phase HPLC. Then PAF in the final sample was determined by sensitiv
e bioassay using rabbit platelets containing fibrinogen and epinephrin
e. The recovery rate of PAF throughout this procedure was constant and
satisfactory (37.4+/-9.7%), which was confirmed using [H-3]-PAF. The
lower limit of the present assay was estimated to be 5pg in 1ml of blo
od and it was sensitive enough to detect PAF in brood samples from hea
lthy volunteers and patients with sepsis or liver cirrhosis. Furthermo
re, attempts were made to compare the sensitivity and the recovery of
our method with these of a commercially available radioimmunoassay (RI
A) kit for PAF. However, it was not possible to detect any amount of a
uthentic PAF added to whore blood.