This protocol is significant because it enables researchers to track and quantitate iron distribution in vivo without the need for radioactivity. Since multiple stable iron isotopes can be detected concurrently within the same sample, this technique can be used to understand the uptake, regulation, and distribution of iron from different sources. To begin, add 12 normal hydrochloric acid to the iron-58 in the glass vial supplied by the vendor, and replace the cap loosely.
To dissolve the iron, warm the solution at 60 degrees Celsius for one hour. If still not dissolved, leave the solution overnight at room temperature in the fume hood to dissolve. Then to generate the ferric chloride solution, oxidize any remaining ferrous chloride by warming up the solution to 60 degrees Celsius with the cap off to facilitate oxidation.
Add one microliter of 35%hydrogen peroxide per 50 microliters of the solution to further facilitate oxidation. Leave the ferric chloride solution in the hood at 60 degrees Celsius with the cap off to evaporate the sample. Then reconstitute the ferric chloride to 100 millimoles with ultrapure water.
Calculate the amount of water required based on the initial metal weight. Next, prepare one milliliter of ferric nitrilotriacetate by incubating the prepared ferric chloride solution with nitrilotriacetate at a one-to-five molar ratio in the presence of 20 millimolar sodium bicarbonate for five minutes at room temperature. Then dissolve 500 milligrams of apotransferrin in four milliliters of transfer and loading buffer, and add four milliliters of this apotransferrin solution to the prepared ferric nitrilotriacetate solution in the 15-milliliter tube.
To allow maximal loading of ferric nitrilotriacetate onto apotransferrin, check that the solution is at pH 7.5 and adjust the pH if necessary with sodium bicarbonate, or hydrochloric acid. Incubate the mixture for 2 1/2 hours at room temperature. Next, remove the excess unbound ferric nitrilotriacetate and released nitrilotriacetate by transferring the iron-58 transferrin solution to a molecular weight cutoff column and centrifuging the column.
After centrifugation, wash the column twice by adding 10 milliliters of transferrin loading buffer and centrifuging the column. Then wash the column with 10 milliliters of saline and centrifuge again. Finally, sterilize the iron-58 transferrin solution using a 0.22-micron syringe filter, and store at four degrees Celsius until ready to use.
Prepare a 35 milligram per milliliter iron-58 transferrin solution in saline. Then place an anesthetized pregnant mouse on a heating pad, and slowly and carefully inject the iron-58 transferrin solution into the retro-orbital sinus. Six hours post-injection, euthanize the mouse, and carefully remove the uterus using sterile forceps and dissection scissors.
Cut off a fetal placental unit comprising a single fetus and placenta in the amniotic sack surrounded by a portion of the uterus. Next, carefully cut through the uterus and amniotic sac without disturbing the fetus and placenta. Then peel back the amniotic sac and remove the fetus and placenta.
After cutting the umbilical cord, blot the fetus and placenta on a clean task wipe to remove excess amniotic fluid, and record the weight of the whole placenta. Cut each placenta in half with a razor blade. Place each half in a two milliliter tube, and snap freeze in liquid nitrogen.
To collect the embryo livers, sacrifice the embryo, and pin down the embryo for stabilization leaving the abdomen exposed. Using dissection scissors make a small incision where the umbilical cord was attached. Insert one end of the dissection scissor into the incision and perform a 1/4-inch median plane cut toward the coronal plane.
Then perform transverse plane cuts to expose the fetal liver. Using forceps, remove the fetal liver, and record the weight of the whole embryo liver. Place the whole embryo liver in a two-milliliter tube and snap freeze it in liquid nitrogen.
Store the tissues indefinitely at minus 80 degrees Celsius. To quantify the non-heme iron in the placentae and fetal livers, thaw the placental halves and whole fetal livers. Then weigh the placental halves.
Add 400 microliters of protein precipitation solution to the tissue samples and homogenize the tissue using an electric homogenizer. Incubate the samples at 100 degrees Celsius for one hour. Then cool them at room temperature water for two minutes.
Open the caps to release pressure. Then close the tubes again. After centrifuging the sample to pellet the tissue debris, carefully transfer the supernatant to a new labeled tube for ICP-MS analysis.
Next to quantify the heme iron, record the weight of the pellet obtained after centrifugation. Then digest the pellets in 10 milliliters of concentrated 70%nitric acid, supplemented with one milliliter of 30%hydrogen peroxide. Heat the samples to 200 degrees Celsius for 15 minutes before sending them for ICP-MS analysis.
ICP-MS measurement allowed the detection of two different iron isotopes. The most abundant iron isotope, iron-56, reflects chronic ion changes in tissues. Another isotope, iron-58, reflects acute changes in the distribution of the injected iron.
Iron-56 measurement confirmed that embryo livers from iron-deficient pregnancies had decreased iron stores compared to those in iron-replete pregnancies. Iron-58 measurement confirmed that in iron-deficient pregnancies, less iron was transferred to the embryo liver over six hours than in iron-replete pregnancies. While performing the procedure, remember to record the weight of the tissues.
These weights are necessary to calculate iron concentrations. Since iron-58 does not require special handling precautions and disposal, unprocessed tissue can be used for other analyses, including, but not limited to western blotting, or qPCR.
