Measurement of Cadmium-Induced Metallothionein in Urine by ELISA and Prevention of Overestimation Due to Polymerization*

Urinary metallothionein (MT) is a biological marker of cadmium (Cd) exposure and Cd-induced renal dysfunction. The MT is prone to oxidation due to high cysteine content and forms polymers, which can result in overestimation of the protein by immunochemical methods. The objectives of the present study were to develop an enzyme-linked immunosorbent assay (ELISA) for the measurement of MT in urine and to find ways by which the protein could either be preserved in its monomeric form or converted to this form before analysis to avoid overestimation. Urine specimens analyzed were either from rats repeatedly injected with Cd or from individuals chronically exposed to cadmium through their diets. The MT in rat urine remained in the monomeric form if the urine was collected at 4 degrees C but did not if it was collected at room temperature. The MT was also polymerized if the urine was subjected to repeated freezing and thawing. Overestimation of MT in rat urine occurred (as much as 12-fold) if the MT was polymerized. Addition of 5mM mercaptoethanol to freshly collected rat urine retarded MT polymerization, and addition of 50mM mercaptoethanol converted the polymerized MT to its monomeric form. Analysis of MT in frozen human urine samples revealed that if the urines were not treated with mercaptoethanol, the estimates of MT concentration were up to 11-fold higher than in the treated samples. We conclude that the polymerization of MT in rat and human urines is a serious problem and results in overestimation of the protein by ELISA and that this problem could be overcome by the addition of mercaptoethanol to the urine samples prior to analysis.