A modified mercury saturation assay for measuring metallothionein

A thorough re-evaluation of assay procedures for a mercury displacement analytical method for metallothionein (MT) described in 1993, was conducted using an analytical standard of rabbit liver MT-II and MT determinations, in liver, kidney, gill and intestine from lake trout ( Salvelinus namaycush). These studies indicated that: (1) loss of MT by adherence to polypropylene material occurs with the dilution series; (2) use of a calibration curve using a rabbit liver MT-II analytical standard, is superior to the dilution series for determining unknown MT concentrations; (3) Hg incubation time requirements are less in 20% TCA than in 10% TCA; and (4) employing both 20% TCA and heat at 95°C for 5 min to denature non MT proteins results in the most accurate MT spike recoveries, or recoveries that did not differ significantly from those closest to 100%, in liver, kidney, gill and intestine. Calibration curves with an average r 2 of 0.9998 for 69 trials over different days, while passing through the origin, provided evidence of negligible non-specific binding of mercury. Use of water or saline for preparing tissue homogenates, produced slight, but significant, differences in MT estimates in the intestine and kidney. Comparisons between the original and the modified methods were made on liver, kidney and gill from control, sham-injected and Cd-treated lake trout. Differences in MT estimates obtained between the two methods depended upon the tissue. MT estimates in liver by the modified method were 9, 13, and 10% higher in control, sham-injected and Cd-treated groups, respectively, than those resulting from the original assay. MT estimates in gill by the modified method, were 3.2-fold, 4.0-fold and 69% higher in control, sham-injected, and Cd-treated groups, respectively. Corresponding values for kidney were 37, 75, and 57% higher. Because percent recoveries of MT-II spikes were virtually 100%, in all four tissues, using the modified method, MT estimates obtained using these modifications were more accurate than those obtained using the procedures of the original assay. Application of the modified assay to fish tissues in a Canadian biomonitoring program for metal mining, demonstrated that in 1 week an experienced technologist can complete 90 MT analyses of liver and kidney and 75 analyses of gill and intestine.