Extraction of dolichyl phosphate and its quantitation by straight-phase high-performance liquid chromatography

Abstract

A procedure for the rapid quantitation of dolichyl phosphate by high-performance liquid chromatography on silica is described. The compound elutes as a single peak at 6 ml in excellent yield. The method employs isocratic elution and requires no column treatment between runs; the limit of sensitivity is in the nanogram range. Dolichyl-11-phosphate, which elutes at 7 ml, can be used as an internal standard, thereby eliminating the requirement for preparation of [3H]dolichyl phosphate. The procedure was used in development of a facile assay for free dolichyl phosphate in rat liver. For the assay of total dolichyl phosphate (free and chemically bound), it was found that when rat liver is first saponified and then extracted with ethyl ether, the amount of dolichyl phosphate present in the ether extract is significantly greater than the sum of the amounts found in extracts derived by treating the tissue first with chloroform/methanol (2/1) and then with chloroform/ethanol/water (10/10/3). Using these new procedures, the level of total dolichyl phosphate in rat liver was found to be 14.7 +/- 3.5 micrograms g-1 wet wt (n = 28). Levels in six other organs are also reported.