ANION EXCHANGE CHROMATOGRAPHIC PROPERTIES OF URINARY METALLOTHIONEIN AND ITS ROLE IN CADMIUM, COPPER AND ZINC EXCRETION IN CADMIUM-POISONED RATS

Abstract

Urine of rats given daily subcutaneous injection of 0.5 mg Cd/kg for 14 weeks was chromatographed on a Sephadex G-75 column. The eluted fractions were analyzed for cadmium, copper and zinc. A cadmium-and copper-containing protein in the metallothionein fraction was further subjected to DEAE Sephadex A-25 chromatography.
In the Sephadex G-75 chromatogram, most parts (about 90%) of the urinary cadmium and copper were distributed in the metallothionein fraction, but no zinc was detected in this fraction. About 90% of the urinary zinc was recovered from nonprotein fractions near the bed volume. The other minor parts of these urinary metals were distributed broadly in protein fractions corresponding to higher molecular weights. In the control animals without cadmium injection, large parts of the urinary zinc and copper were recovered from the non-protein fractions near the bed volume and very small amounts of these metals were distributed in the high molecular-weight protein fractions. A trace amount of copper, but no zinc was detected in the metallothionein fraction.
The DEAE Sephadex A-25 chromatogram showed that cadmium and copper in the metallothionein fraction could be separated into four distinct peaks ac-companied by corresponding peaks in the UV-absorbance at 250 nm. The first two minor peaks were separated at the initial buffer concentration of 0.05 M Tris-HCl; the other two main peaks were separated at buffer concentrations of 0.19 and 0.26 M, respectively. The separation profile of the latter two peaks was similar to that of hepatic metallothionein. These results indicated that most of the urinary cadmium and copper in the cadmium-poisoned rats was associated with metallothionein.