Application of covalent affinity chromatography with thiol-disulphide interchange for determination of environmental exposition to heavy metals based on the quantitative determination of Zn-thionein from physiological human fluids by indirect method based on analysis of metal contents.

Intoxication with heavy metals results in numerous poisonings and diseases. They disturb metabolism of the system, are the source of cancer, degeneration changes and others. As a result of kidney damage the urine of people exposed to heavy metals contains different low molecular weight proteins, oligopeptides and amino acids, indicating pathological changes. One of the proteins is a very specific metallopolythiopolypeptide--metallothionein (MT). Based on earlier investigations, a very good correlations has been found between the contents of metallothionein in urine and plasma and the concentration of heavy metals in the blood, urine, kidneys, liver and brain and general in level of exposition to heavy metals. The aim of our investigations was to carry out quantitative isolation of Zn-thionein (Zn-Th), in order to determine the level of exposition to heavy metals. For Zn-Th protein isolation by covalent affinity chromatography with thiol-disulphide interchange (CAC-TDI) was applied, which is a modern technique of separation of a high affinity, good repeatability and reproducibility, allowing specific isolation of the thiol-proteins CAC-TDI gel was used as a solid-phase extraction (SPE) support for preconcentration of Zn-Th protein and Zn bonded with Zn-Th from water, rine, plasma and breast milk samples. The investigations showed unfavourable effect of the support on separation of thiol proteins and good correlation between the concentration of MTs protein added to water, plasma and urine and the concentration of protein indirectly determined via atomic absorption spectrometric (AAS) method, by preconcentration on SPE support metals formerly bound with MT protein and absorbed on CAC-TDI gel and calculated from metals concentration. The present paper is a continuation of earlier experiments on quantitation of Hg-thionein and Cd-thionein in physiological fluids and homogenates.