A non-radioactive method for measuring Cu uptake in HepG2 cells

At present, all data on Cu uptake and metabolism have been derived from radioactive uptake experiments. These experiments are limited by the availability of the radioactive isotopes Cu-64 or Cu-67, and their short half-life (12.5 and 62 h, respectively). In this paper, we investigate an alternative method to study the uptake of Cu with natural isotopes in HepG2 cells, a liver cell line used extensively to study Cu metabolism. In nature, Cu occurs as two stable isotopes, Cu-63 and Cu-65 (Cu-63/Cu-65 = 2.23). This ratio can be measured accurately using inductively coupled plasma mass spectrometry (ICP-MS). In initial experiments, we attempted to measure the time course of Cu uptake using Cu-65. The change in the Cu-63/Cu-65 ratio, however, was too small to allow measurement of Cu uptake by the cells. To overcome this difficulty, the natural Cu-63/Cu-65 ratio in HepG2 cells was altered using long-term incubation with Cu-63. This had a significant effect on Cu concentration in HepG2 cells, changing it from 81.9 +/- 9.46 pmol mu g DNA(-1) (week 1) to 155 +/- 8.63 pmol mu g DNA(-1) (week 2) and stabilising at 171 +/- 4.82 pmol mu g DNA(-1) (week 3). After three weeks of culture with 2 mu M Cu-63 the Cu-63/Cu-65 changed from 2.18 +/- 0.05 to 15.3 +/- 1.01. Cu uptake was then investigated as before using Cu-65. Uptake was linear over 60 min, temperature dependent and consistent with previous kinetics data. These observations suggest that stable isotope ICP-MS provides an alternative technique for the study of Cu uptake by HepG2 cells. (c) 2005 Elsevier Inc. All rights reserved.