Species analysis of metallothionein isoforms in human brain cytosols by use of capillary electrophoresis hyphenated to inductively coupled plasma-sector field mass spectrometry

A new approach for the speciation of metallothioneins (MT) in human brain c ytosols is described. The analysis is performed by application of a newly d eveloped coupling of capillary electrophoresis (CE) with inductively couple d plasma-sector field mass spectrometry (ICP-SFMS). Isoforms of metallothio neins are separated from 30-100 muL sample volumes by CE and the elements C u, Zn, Cd, and S are detected by use of ICP-SFMS. The extraction of cytosols is the first step in the analytical procedure. T issue samples from human brain are homogenized in a buffer solution and Sub mitted to ultra-centrifugation. The supernatant is defatted arid the cytoso l pre-treatment is optimized For CE separation by matrix reduction. The buf fer concentration and pH used for capillary electrophoretic separation of m etallothionein from rabbit liver were optimized. CE with ICP-MS detection i s compared to UV detection. In the electropherograms obtained from the cyto sols three peaks can be assigned to MT-1, MT-2, and MT-3. As an additional method, size-exclusion chromatography (SEC) is applied. Fractions from an S EC separation of the cytosol are collected, concentrated, and then injected into the CE. The detection of sulfur by ICP-SFMS (medium resolution mode) and quantifica tion by isotope dilution have also been investigated as a new method for th e quantification of MT isoforms. The analytical procedure developed has been used for the first time in comp arative studies of the distributions of MT-1, MT-2, and MT-3 in brain sampl es taken from patients with Alzheimer's disease and from a control group.