title: HPLC and mass spectrometry analysis of dolichol-phosphates at the cell culture scale.
abstract: Dolichols (Dol) are polyprenol lipids that are essential structural components of eukaryotic membranes. In addition, the phosphorylated derivatives of Dol function as lipid anchors of mono- and oligosaccharide precursors involved in protein glycosylation. The biological importance of Dol phosphates (Dol-P) is illustrated by the severe outcome of human disorders linked to Dol biosynthetic defects, such as Dol-kinase deficiency. For characterization of inherited human diseases and evaluation of therapeutic trials, cultured cells often serve as a sole possible source for experimentation. Limited amounts of cell culture material render the quantitative analysis of Dol a challenging task. Here, we present HPLC- and mass spectrometry-based approaches to analyze and quantitate Dol-P from cultured human cells. The composition of naturally occurring Dol-P and the saturation state of the alpha-isoprene units was identified by negative-ion electrospray ionization mass spectrometry. Furthermore, fluorescently labeled Dol-P were separated by HPLC and quantified by comparison to known amounts of the internal standard polyprenol-P. The effect of pravastatin, a 3-hydroxy-3-methyl-glutaryl coenzyme-A reductase inhibitor, on the formation of Dol-P in HeLa cells was investigated. As expected, this treatment led to a decrease of Dol-P down to 35% of normal levels.
authors.affiliation: Institute of Physiology, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
authors.forename: Micha A
authors.initials: MA
authors.lastname: Haeuptle
authors.forename: Andreas J
authors.initials: AJ
authors.lastname: Hülsmeier
authors.forename: Thierry
authors.initials: T
authors.lastname: Hennet
mesh_terms: D000873:Anthracenes / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004288:Dolichol Phosphates / Q000032:analysis* / Q000737:chemistry; D005419:Flavonoids / Q000032:analysis / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D010636:Phenols / Q000032:analysis / Q000737:chemistry; D059808:Polyphenols; D017035:Pravastatin / Q000494:pharmacology; D012015:Reference Standards
chemical_list: D000873:Anthracenes; D004288:Dolichol Phosphates; D005419:Flavonoids; D010636:Phenols; D059808:Polyphenols; C027347:9-diazomethylanthracene; D017035:Pravastatin