Re-evaluation of thin layer chromatography as an alternative method for the quantification of prostaglandins from rat Kupffer cells.
In contrast to conventionally used immunoassays, thin layer chromatography (TLC)--by prelabeling of cells with radioactive arachidonic acid (AA)--allows to differentiate between cellularly built and added prostanoids and thus to investigate feedback effects of prostanoids on their own release. PGD2, TXB2 and PGE2 released from zymosan-stimulated Kupffer cells were separated with distinct RF-values, corresponding to those of the pure substances. Quantification of PGD2 and PGE2 gave comparable results with TLC and immunoassays, but measurement in the presence of added prostanoids was only possible with TLC. Moreover TLC was superior to immunoassays in having a longer linear range while being comparably sensitive. Cellularly built TXB2 in its radioactively labeled form was not detectable by TLC. Inhibition of TXB2 release by externally added AA or technical artifacts were excluded, suggesting that the cellular AA-pools used for prostaglandin and thromboxane synthesis differ in their accessibility for added AA. Thus, TLC is a simple, sensitive and precise method for the quantification of cellularly built prostaglandins but not of thromboxane even in the presence of added prostanoids.
Pestel
Sabine
S
Institute of Biochemistry and Molecular Biology, Georg-August University Goettingen, Humboldtallee 23, D-37073 Goettingen, Germany. s.pestel@bc.boehringer-ingelheim.com
Jungermann
Kurt
K
Schieferdecker
Henrike L
HL
D000818:Animals; D002469:Cell Separation; D002855:Chromatography, Thin Layer / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D007728:Kupffer Cells / Q000737:chemistry* / Q000166:cytology*; D008297:Male; D011453:Prostaglandins / Q000032:analysis* / Q000302:isolation & purification; D051381:Rats; D017208:Rats, Wistar
D011453:Prostaglandins