inhibition of Thr172 dephosphorylation by still unidentified
AMPK-specific protein phosphatase(s) [4]. Once activated,
AMPK concomitantly inhibits ATP-consuming anabolic processes
and promotes ATP-generating catabolic pathways via direct phos-
phorylation of multiple downstream effectors, leading to restora-
tion of cellular energy balance [5].
Since subtle changes in cellular energy status would directly
affect AMPK activity, accurate determination of adenine nucleotide
concentrations is essential in the framework of studies investigating
the pleiotropic role of the kinase in cell and tissue functions. Several
methods have been reported for measuring cell and/or tissue ATP,
ADP, and AMP levels, including luciferase-based bioluminescence
assays and ultraviolet (UV)-based high-performance liquid chro-
matography (HPLC) [6–8]. HPLC has the advantage of high
sensitivity and allows the separation and simultaneous quantifica-
tion of a wide range of nucleotides. Furthermore, taking into
account that HPLC systems are among the most common appara-
tus available in laboratory technical platforms, the cost per sample
remains generally lower for large sample size in comparison with
single-nucleotide commercial assay kits.
In this chapter, we describe a step-by-step protocol for rapid,
highly sensitive, reproducible, and simultaneous determination of
ATP, ADP, and AMP concentrations in cell or tissue samples by
reversed-phase HPLC. Of note, the accurate determination of
adenine nucleotide levels will also depend upon the extraction
procedure used. We therefore include a description of such impor-
tant step that was successfully applied in various cells and tissues
[9–13].2 Materials
Prepare all solutions using ultrapure water (mQ water; sensitivity
18 MΩ-cm at 25C) and analytical grade reagents. Prepare and
store all reagents at room temperature (unless otherwise indicated).2.1 Equipment 1. Polytron homogenizer.
- HPLC system (DIONEX UltiMate 3000, Thermo Scientific).
- SUPELCOSIL™reversed-phase column (LC-18-DB, Sigma-
Aldrich).
2.2 Extraction
Buffers
- Perchloric acid/EDTA solution: 10% (v/v) HClO 4 ,25mM
EDTA. Add ~70 ml of mQ water to a glass beaker containing a
magnet. Add 14.3 ml of 70% perchloric acid. Weigh 930.6 mg
of EDTA disodium salt dihydrate and transfer to the glass
beaker. Mix gently until complete dissolution. Make up to
100 ml, transfer in a glass bottle, and store at 4C.
230 Noemı ́Garcı ́a-Tardo ́n and Bruno Guigas