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

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

Research output: Contribution to journalArticle

18 Citations (Scopus)

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

Cite this

A non-radioactive method for measuring Cu uptake in HepG2 cells. / Fosset, C ; McGaw, B A ; Reid, M D; McArdle, H J.

In: Journal of Inorganic Biochemistry, Vol. 99, No. 5, 05.2005, p. 1018-1022.

Research output: Contribution to journalArticle

Fosset, C ; McGaw, B A ; Reid, M D ; McArdle, H J. / A non-radioactive method for measuring Cu uptake in HepG2 cells. In: Journal of Inorganic Biochemistry. 2005 ; Vol. 99, No. 5. pp. 1018-1022.
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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.",
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AU - McGaw, B A

AU - Reid, M D

AU - McArdle, H J

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N2 - 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.

AB - 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.

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KW - Cu uptake

KW - HepG2

KW - isotope dilution

KW - plasma-membrane vesicles

KW - copper uptake

KW - trophoblast cells

KW - rat hepatocytes

KW - phen green

KW - iron pool

KW - metabolism

KW - transport

KW - ceruloplasmin

KW - disease

U2 - 10.1016/j.jinorgbio.2005.01.005

DO - 10.1016/j.jinorgbio.2005.01.005

M3 - Article

VL - 99

SP - 1018

EP - 1022

JO - Journal of Inorganic Biochemistry

JF - Journal of Inorganic Biochemistry

SN - 0162-0134

IS - 5

ER -