Isoprenoid quantitation in human brain tissue: a validated HPLC-fluorescence detection method for endogenous farnesyl- (FPP) and geranylgeranylpyrophosphate (GGPP).
Isoprenoid quantitation in human brain tissue: a validated HPLC-fluorescence detection method for endogenous farnesyl- (FPP) and geranylgeranylpyrophosphate (GGPP).
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DOI：
10.1007/s00216-008-2306-3
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发表时间：
2008-10
影响因子：
4.3
通讯作者：
Eckert, Gunter P.
中科院分区：
文献类型：
--
作者：
Hooff, Gero P.;Volmer, Dietrich A.;Wood, W. Gibson;Mueller, Walter E.;Eckert, Gunter P.
关键词：
法呢基焦磷酸和香叶基香叶基焦磷酸（FPP和GGPP）是甲羟戊酸途径中的类异戊二烯中间体。它们通过与小GTP酶（Ras、Rho等）的附着（异戊二烯化）在细胞存活、生长和分化中起关键作用。类异戊二烯的形成似乎在甲羟戊酸途径内受到严格调节，并且其扰动与某些疾病有关（例如，癌症，阿尔茨海默病），但组织水平未知。因此，在患病组织中或给药后在组织中定量这些类异戊二烯是极其重要的。目前的工作描述了一种分离程序，利用Extremut ®液/液和反相固相萃取（SPE）的组合，用于均质化的人额叶皮层组织。此外，在仔细验证HPLC-荧光方法后，该测定法允许测定人脑组织中内源性FPP和GGPP水平（分别为4.5和10.6 ng/mg蛋白质）的纳摩尔浓度。该方法具有选择性、精密度（< 15% RSD）、准确度（< 15%相对误差）和灵敏度，根据现行FDA生物分析方法验证标准，FPP的线性范围为10 - 400 ng/mL，GGPP的线性范围为50 - 1000 ng/mL。总的来说，这种新方法引入了同时定量人脑组织中FPP和GGPP的能力，具有应用于其他几种组织和物种的前景。
Farnesyl- and geranylgeranylpyrophosphate (FPP and GGPP) are isoprenoid intermediates in the mevalonate pathway. They play a crucial role in cell survival, growth and differentiation by 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 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® 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 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 with the prospect for application to several other tissues and species.
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影响因子：
2
作者：
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通讯作者：
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