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

C Fosset, B A McGaw, M D Reid, H J McArdle

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Abstract

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.

Original languageEnglish
Pages (from-to)1018-1022
Number of pages5
JournalJournal of Inorganic Biochemistry
Volume99
Issue number5
Early online date10 Jan 2005
DOIs
Publication statusPublished - May 2005

Keywords

  • inductively coupled plasma-mass spectrometry-
  • Cu uptake
  • HepG2
  • isotope dilution
  • plasma-membrane vesicles
  • copper uptake
  • trophoblast cells
  • rat hepatocytes
  • phen green
  • iron pool
  • metabolism
  • transport
  • ceruloplasmin
  • disease

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