alexa Simple Analysis Method for Metallothionein-1, -2 and -3
ISSN: 2155-9872

Journal of Analytical & Bioanalytical Techniques
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Research Article

Simple Analysis Method for Metallothionein-1, -2 and -3 in the Brain by One-Step Size-Exclusion Column HPLC On-Line Coupling with Inductively Coupled Plasma Mass Spectrometry

Satomi Kameo1,2*, Kunihiko Nakai3, Akira Naganuma4, Hiroshi Koyama2 and Hiroshi Satoh1

1Environmetal Health Sciences, Tohoku University Graduate School of Medicine, Japan

2Department of Public Health, Gunma University Graduate School of Medicine, Japan

3Development and Environmental Medicine, Tohoku University Graduate School of Medicine, Japan

4Laboratory of Molecular and Biochemical Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Japan

*Corresponding Author:
Satomi Kameo
Department of Public Health
Gunma University Graduate School of Medicine
3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan
Tel: +81-(0)27-220-8011
Fax: +81-(0)27-220-8016
E-mail: [email protected]

Received date: October 17, 2014; Accepted date: November 14, 2014; Published date: November 18, 2014

Citation: Kameo S, Nakai K, Naganuma A, Koyama H, Satoh H (2014) Simple Analysis Method for Metallothionein-1, -2 and -3 in the Brain by One-Step Size-Exclusion Column HPLC On-Line Coupling with Inductively Coupled Plasma Mass Spectrometry. J Anal Bioanal Tech 5:224. doi: 10.4172/2155-9872.1000224

Copyright: © 2014 Kameo S, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

 

Abstract

A simple analysis method of metallothionein (MT) isoforms MT-1, MT-2, and MT-3 was developed based on the use of one-step size-exclusion column (SEC) HPLC and on-line coupling with inductively coupled plasma mass spectrometry (ICP-MS). For the elucidation of the functions of MT isoforms in the brain, it is necessary to have a simple method to determine these isoforms. A SEC TSK gel G2000 SWXL PEEK (7.8 mm I.D. × 30 cm) system was used in this study. The HPLC system was connected with a quadrupole ICP-MS. All of the connections were made using PEEK tubing. To induce MTs, cadmium (Cd) chloride was administered to MT-1, 2 null and 129/Sv mice at a dose of 4 mg/kg body weight by i.p. injection. Mice were sacrificed 24 h after treatment under anesthesia. Brains and livers were collected, and all the samples were stored at −80°C until subsequent analyses. Soluble extracts of the livers and brains from 129/Sv mice either treated with cadmium or untreated were analyzed. MT-1, MT-2, and MT-3 were clearly separated by the one-step SEC HPLC-ICP-MS system (monitored at 65Cu, 66Zn, 113Cd and 55Mn for element) using an appropriate buffer (25 mM Tris-12.5 mM HCl containing 20 mM KCl) and an ultrafiltration membrane filter to eliminate high molecular weight proteins over 30,000 MW and Cu, Zn-SOD. Retention times (RTs) of peaks of each isoform were distinguishable; RTs of MT-1, MT-2, and rMT-3 were 8.6, 8.1, and 7.6 min, respectively. MT-1, MT-2, and MT-3 were separated clearly using this system. This system would be a powerful tool for the separation and metal-component analysis of MT isoforms to elucidate further the biological functions of MTs in the brain.

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