Colorimetric Assessment of Deiodinase 1 Activity in Human Liver Microsomes Using the Sandell-Kolthoff Reaction

The assay quantifies DIO1 inhibition using human liver microsomes. It was validated and optimized for robust and standardized handling. This protocol is used in mechanistic toxicology and screening to identify chemicals inhibiting DIO1 and disrupting thyroid hormone homeostasis.

This protocol measures iodide release from RT3 through deiodination via the Sandell-Kolthoff reaction, a practical surrogate readout for DIO1 activity. To begin, obtain an appropriate rectangular transparent container. Add about 250 grams of ion-exchange resin to the container.

Wash the resin with 10%acetic acid and let the resin suspension rest for 10 minutes. Then, slightly tilt the container to aspirate the supernatant. Repeat the wash at least five times in total, or until no more color leaks into the solvent.

After the last wash, add 10%acetic acid, so it makes up approximately 50%of the total volume. Tilt the container so the resin moves to one side. Stabilize the container in the tilted position, and wait about 10 minutes for the phases to separate.

Place a 96 well filter plate on top of a 96 deep well plate. Add 100 microliters of 10%acetic acid into each well of the 96 well filter plate. Cut the tips used for resin casting at a height of about one centimeter to widen the opening at the end.

Use the cut tip to dispense 300 microliters of ion-exchange resin into each well of the 96 well filter plate. Centrifuge the plate assembly with a swing out rotor for microtiter plates at 100 to 200G for one minute to elute the acetic acid into a 96 deep well plate. Check whether the resin is distributed evenly across the plate.

Seal the plate with an impermeable sheet of plastic. Store the plate at four degrees Celsius for a maximum of six months. Prepare iodide dilutions from the iodide source in deionized water.

Add 50 microliters of pure deionized water as a control to a 96 well plate in triplicate. Add 50 microliters of the prepared iodide dilutions to the same plate in triplicate. Add 50 microliters of 40 millimolar cerium solution to each well.

Start the reaction by adding 50 microliters of 25 millimolar arsenite solution. Immediately after adding arsenite, measure the absorbance with a plate reader at 415 nanometers. Then measure the absorbance every minute for 21 minutes.

Prepare the reference item, positive control, negative control, and test item dilution series at the concentration shown. Prepare the substrate mix and a microsome suspension. Place the tubes on ice.

Add 10 microliters of the reference item dilutions, controls, and test item dilutions to the wells according to the plate layout. Add 40 microliters of microsome suspension to each well followed by 50 microliters of freshly prepared substrate mix. Seal the plate with an impermeable sheet of plastic.

Place it on a shaker in an incubator at 37 degrees Celsius and 850 revolutions per minute for two hours. Then place the plate on ice to stop the reaction and leave it there until measurement. Place the ion-exchange resin filled 96 well filter plate on top of a 96 deep well plate.

Add 150 microliters of 10%acetic acid to each well of the filter plate to wet the resin. Centrifuge the plate assembly in a centrifuge with a swing out rotor for microtiter plates at 100 to 200G for one minute to elute the acetic acid into the 96 deep well plate. Replace the used 96 deep well plate with a fresh plate.

Retrieve the incubated 96 well plate from ice. Add 133 microliters of 10%acetic acid to each well. Shake the plate briefly to mix the samples.

Transfer 175 microliters of each sample to the ion-exchange resin filled 96 well filter plate, maintaining the original plate layout. Centrifuge the plate assembly with a swing out rotor for microtiter plates at 100 to 200G for one minute to pass the samples through the resin into the 96 deep well plate. Then remove the ion-exchange resin filled 96 well filter plate.

Transfer 50 microliters of the undiluted sample from the 96 deep well plate into a new 96 well plate. Add 50 microliters of 40 millimolar cerium solution to all wells and start the reaction by adding 50 microliters of 25 millimolar arsenite solution to each well. Immediately after adding arsenite, measure the absorbance with a plate reader at 415 nanometers.

Then measure the absorbance every minute for 21 minutes. The background corrected Sandell-Kolthoff signal increased with increasing microsomal protein. The signal varied across post ion-exchange dilutions.

The one to two dilution yielded the highest signal, while maintaining a sigmoidal response. Comparison of inhibition curves identified five micrograms of microsomal protein per well as the lowest amount producing a sigmoidal response. The reference inhibitor showed a concentration-dependent decrease in iodide release activity, with a sigmoidal inhibition profile.

Tannic acid showed a comparable inhibitory profile with high maximum inhibition. And dibutyl phthalate showed no inhibition across the tested concentration range. These results showed that the assay clearly distinguished inhibitory from non-inhibitory concentration response profiles under standardized conditions.

Maintaining strict uniform handling is a key challenge. A slight variation to timing and handling can significantly affect protocol efficiency. Future studies can adapt the standardized workflow to other sources of deiodinating enzymes, enabling assays for DIO2, DIO3, or dehalogenases.