A rapid procedure for the separation and analysis of metabolites of the sterol and dolichol pathways.
We have previously shown that of several methods examined, the maximum yield of dolichyl phosphate (Dol-P) from rat liver was achieved by saponification of the tissue and subsequent extraction with diethyl ether (R.K. Keller et al. (1985) Anal. Biochem. 147, 166-173). In the present report, we have developed a rapid procedure using non-toxic solvents which resolves the ether extract on a C18 cartridge column into four major fractions: (1) fatty acids; (2) squalene and sterols; (3) dolichol; (4) Dol-P. The utility of the new procedure was demonstrated by preparing the four fractions from liver slices which had been incubated with [3H]acetate. HPLC analysis of the sterol, dolichol and Dol-P fractions yielded well resolved elution profiles, thereby allowing determination of radioactivity incorporated into the major isoprenoids and their metabolites.
Keller
R K
RK
Department of Biochemistry and Molecular Biology, University of South Florida, College of Medicine, Tampa 33612.
Nellis
S W
SW
Cuadrado-Simonet
L
L
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004288:Dolichol Phosphates / Q000302:isolation & purification / Q000378:metabolism; D004286:Dolichols / Q000302:isolation & purification* / Q000378:metabolism; D066298:In Vitro Techniques; D008099:Liver / Q000378:metabolism; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D012997:Solvents; D013261:Sterols / Q000302:isolation & purification* / Q000378:metabolism
D004288:Dolichol Phosphates; D004286:Dolichols; D012997:Solvents; D013261:Sterols