Isoprenoid quantitation in human brain tissue: a validated HPLC-fluorescence detection method for endogenous farnesyl- (FPP) and geranylgeranylpyrophosphate (GGPP).
Farnesyl- and geranylgeranylpyrophosphate (FPP and GGPP) are isoprenoid intermediates in the mevalonate pathway. They play a crucial role in cell survival, growth and differentiation due to their attachment (isoprenylation) to small GTPases (Ras, Rho, etc.). Isoprenoid formation seems to be tightly regulated within the mevalonate pathway and its perturbation has been linked to certain diseases (e.g., cancer, Alzheimer's disease), but tissue levels are unknown. It is therefore of the utmost importance to quantify these isoprenoids in diseased tissue or in tissue after drug administration. The current work describes an isolation procedure utilizing a combination of Extrelut(R) liquid/liquid and reversed-phase solid-phase extraction (SPE) for homogenized human frontal cortex tissue. In addition, after a careful validation of an HPLC-fluorescence method, this assay allowed the determination of nanomolar concentrations of endogenous FPP and GGPP levels (4.5 and 10.6 ng/mg protein, respectively) in human brain tissue. The method is selective, precise (<15% RSD), accurate (<15% relative error) and sensitive over a linear range of 10-400 ng/mL for FPP and 50-1000 ng/mL for GGPP according to the current FDA criteria for bioanalytical method validation. Overall, this new method introduces the ability to simultaneously quantify FPP and GGPP in human brain tissue, and is potentially applicable to several other tissues and species.
Hooff
Gero P
GP
Department of Pharmacology, Biocenter Niederursel, University of Frankfurt, Max-von-Laue-Str. 9, 60438, Frankfurt, Germany.
Volmer
Dietrich A
DA
Wood
W Gibson
WG
Müller
Walter E
WE
Eckert
Gunter P
GP
D001921:Brain / Q000378:metabolism; D001923:Brain Chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis* / Q000737:chemistry*; D006801:Humans; D015394:Molecular Structure; D013729:Terpenes / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism
D005456:Fluorescent Dyes; D013729:Terpenes