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 SpectrometrySatomi Kameo1,2*, Kunihiko Nakai3, Akira Naganuma4, Hiroshi Koyama2 and Hiroshi Satoh1
- *Corresponding Author:
- Satomi Kameo
Department of Public Health
Gunma University Graduate School of Medicine
3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan
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.
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.