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Title: THE USE OF AFFINITY CONCENTRATORS FOR ON-LINE PRE-CONCENTRATION OF METALLOTHIONEIN PRIOR TO ISOFORM SEPARATION BY CAPILLARY ZONE ELECTROPHORESIS

Author
item BEATTIE JOHN H - THE ROWETT RESEARCH INST.
item SELF RON - UNIV E. ANGLIA, NORWICH
item Richards, Mark

Submitted to: Proceedings of the British Electrophoresis Society
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/20/1994
Publication Date: N/A
Citation: N/A

Interpretive Summary: Trace elements such as zinc and copper are required to support growth and development in all animal species, including humans. There has been a great deal of interest in the scientific community in developing indicators of zinc and copper status. One such status indicator is a zinc- and copper-binding protein called metallothionein (MT). Although MT has been actively studied for over thirty years, there continues to be a need for new rapid and sensitive methods to assay levels of MT. Although there are a number of techniques to measure MT levels in tissues and physiological fluids, none currently exist that offer a quantitative analysis of all MT molecular forms (called isoforms) in fluids such as serum or urine where MT levels are very low. Immunoassay methods are only partially effective in this type of analysis, although they do possess the requisite sensitivity and selectivity. The purpose of this work was to develop a means to concentrate MT present in tissue samples prior to analysis of the protein by capillary zone electrophoresis. By utilizing a device called an analyte concentrator it was possible to extend the detection limits for MT 700 fold. This work constitutes a first step toward analysis of serum and urine samples for MT by capillary electrophoresis and thus opens the possibility for developing a less invasive method to assess trace element status of animals and humans by way of MT isoform analysis.

Technical Abstract: UV absorbance is a convenient detection method for monitoring a wide variety of different components separated by capillary electrophoresis (CE). However, a major disadvantage of UV detection is its low sensitivity and this is a major factor limiting the application of CE as an analytical technique. In order to improve sensitivity, several methods of on-line sample preconcentration by electro-focusing, including stacking and isotachophoresis, have been investigated and the potential of on-line solid phase affinity preconcentrators for this purpose have also been demonstrated. We have developed methods for the separation of isoforms of metallothionein (MT), a low Mr metal-binding proteiny. While a variety of modifications to the capillary surface and electrolyte chemistry have been used to resolve many MT isoforms in liver samples, there are few available alternatives to UV detection and thus sensitivity limits the application of these methods to samples with low MT levels, such as urine or plasma. In this study we have investigated the use of a C18 concentrator which was fitted to the capillary inlet to extend the detection limits for MT isoform analysis. Samples were pressure loaded onto the concentrator for up to 5 min., eluted using 33% acetonitrile and then subjected to electrophoresis in borate or phosphate buffer. The purified isoforms MT-1 and MT-2 from rabbit liver were best resolved using an acidic acetonitrile eluent and an electrolyte of 30 mM phosphate buffer pH 7. Sensitivity enhancement was >700-fold and the relationship between loading time and isoform absorbance peak area was linear when only a small proportion of concentrator capacity was used. Analysis of sheep liver extracts demonstrated that both sensitivity and specificity were enhanced.