Step Procedure accomplished Equipment Data Collected 1 Mice are fed purified diet until 8-wk of age - - 2 Mice are euthanized without fasting - - 3 Blood is collected via cardiac puncture - - 4 Serum is collected and stored for later analysis Centrifuge - 5 Liver is harvested and stored for later analysis Freeze-drier - 6 Liver is processed for measuring mineral content Microwave digestor - 7 Iron, copper, and zinc levels in the liver are measured Mass spectrometer iron, copper, and zinc levels 8 Serum is analyzed for diferric transferrin Gel electrophoresis system serum diferric transferrin 9 Total serum transferrin levels are assessed Western blotting system (total serum transferrin)
- 70°C freezer
- Freeze-drying system (Labconco)
- microwave digestor (CEM microwave accelerated reaction system 5, CEM)
- Vista AX CCD inductively coupled plasma-atomic emission spectrometry (ICP-AES, Varian)
- urea polyacrylamide gel electrophoresis
Reagents, supplies, solutions
• AIN93G purified diet containing ~35 ppm iron (Dyets)
• carbon dioxide gas
• 5 mL syringes
• centrifuge tubes
• microcentrifuge tubes
• liquid nitrogen
• suprapure nitric acid
• metal-free water
• polyclonal antibody to human transferrin (1 in 1,000 dilution; Silenus Laboratories, Hawthorn, Australia)
I. Measurement of serum transferrin levels
a. Mice are fed a purified diet containing ~35 ppm iron ad libitum until 8 wk of age before testing begins.
b. To facilitate the collection of blood, mice are euthanized with carbon dioxide gas without fasting.
c. Blood is collected by cardiac puncture using 19G needle and 5 mL syringe.
d. Collected blood samples are then kept at room temperature for at least 30 min and allowed to settle.
e. To collect serum samples, clotted blood samples are centrifuged at 1,000 g for 15 min.
f. Serum samples are then aliquoted into microcentrifuge tubes, snap-frozen in liquid nitrogen, and stored at -70°C for subsequent analysis.
g. To determine serum diferric transferrin levels, urea polyacrylamide gel electrophoresis system is used.
h. To evaluate total transferrin levels in the same serum samples, Western blotting is used with a polyclonal antibody to human transferrin that cross-reacts with the mouse protein.
II. Measurement of liver mineral content
a. Liver tissues are harvested, snap-frozen in liquid nitrogen, and stored at -70°C for subsequent analysis.
b. To test for liver mineral content, liver tissues are, first, vacuum-dried overnight using a freeze-drying system.
c. Dried liver samples are weighed, and then digested in suprapure nitric acid using a microwave digestor (CEM system 5).
d. Digested liver samples are then diluted with metal-free water to give a final nitric acid concentration of ~1 M of nitric acid.
e. In order to measure levels of iron, copper, and zinc in the liver, an inductively coupled plasma-atomic emission spectrometry is used.
f. The metal content of the liver is expressed as micrograms per gram (µg/g) dry weight of tissue. All samples are measured in triplicates.
• Liver copper (Cu), iron (Fe), and zinc (Zn) contents (dry weight)
• Serum diferric transferrin as a percent of total transferrin
transferrin: serum glycoprotein that binds to iron reversibly to control levels of circulating free iron.
diferric transferrin: form of transferrin that reversibly binds two iron ions (the majority of circulating iron).
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