Determination of Energy Charge Potential in the C6 Glioma and the HepG-2 Cell Culture

Abstract

The energy charge potential (ECP) is the ratio among adenosine 5'-triphosphate (ATP), -diphosphate (ADP), and -monophosphate (AMP); it reflects the high-energy-bond content in biological systems. Under normal physiological conditions, the value of ECP is between 0.7 and 1.0. In this study, two different methods were used to extract adenosine nucleotides (ATP, ADP, and AMP) for determination of the ECP in the C6 glioma and the HepG-2 human hepatoma cell lines. In the first method, cells were removed from culture plates by trypsinization before the nucleotides were extracted using perchloric acid. Some of the trypsinized cells were used for a cell count, thereby allowing the calculation of an average nucleotide level per cell. In the second method, perchloric acid was added directly to the culture plate immediately after the medium was removed. This method resulted in an instant termination of any enzymatic process that might degrade the nucleotides and alter their levels in cells. Adenosine nucleotides were determined by high-performance liquid chromatography. The direct extraction method yielded a total adenosine nucleotide (TAN) level more than twofold higher than that obtained following trypsinization. For the C6 glioma cells, the ratio of ATP to ADP to AMP in the TAN pool was 0.70:0.15:0.15, as compared to 0.36:0.27:0.37 when cells were extracted following trypsinization. Similarly, for the HepG-2 cells, the ratio was 0.62:0.15:0.23 following direct extraction, as compared to 0.15:0.28:0.57 following trypsinization. Using the results from the direct extraction method, the value of the cellular ECP was 0.78 ± 0.06 for the C6 glioma cells and 0.70 ± 0.06 for the HepG-2 cells. Compared to the ECP values obtained using the trypsinization method (0.50 ± 0.05 and 0.29 ± 0.12, respectively), the ECP values derived using the direct extraction method were significantly greater in both cell lines and were suggestive of the energy status of normal, healthy cells.