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. PGD 2,...

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Bibliographic Details
Published in:Prostaglandins & other lipid mediators 2005, Vol.75 (1), p.123-139
Main Authors: Pestel, Sabine, Jungermann, Kurt, Schieferdecker, Henrike L.
Format: Article
Language:English
Subjects:
Animals
Arachidonic acid
Cell Separation
Chromatography, Thin Layer - methods
Eicosanoids
Enzyme-linked immuno sorbent assay (ELISA)
Enzyme-Linked Immunosorbent Assay
High performance liquid chromatography (HPLC)
Kupffer Cells - chemistry
Kupffer Cells - cytology
Male
Prostaglandins - analysis
Prostaglandins - isolation & purification
Radioimmunoassay (RIA)
Rat
Rats
Rats, Wistar
Thin layer chromatography (TLC)
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Summary: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. PGD 2, TXB 2 and PGE 2 released from zymosan-stimulated Kupffer cells were separated with distinct R F-values, corresponding to those of the pure substances. Quantification of PGD 2 and PGE 2 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 TXB 2 in its radioactively labeled form was not detectable by TLC. Inhibition of TXB 2 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.
ISSN:1098-8823
DOI:10.1016/j.prostaglandins.2004.11.001