Vulpe1 project protocol

Basal liver mineral levels and iron homeostasis in 18 diverse mouse strains   (2010)

McLachlan S, Vulpe CD
With: Lee SM, Steele TM, Hawthorne PL, Zapala MA, Eskin E, Schork NJ, Anderson GJ




Vulpe1_Protocol

Project protocol — Contents

Workflow and sampling
Equipment
Reagents, supplies, and solutions
Procedures
Definitions and calculations
Data
References



Workflow and sampling

Workflow

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)

Equipment

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)

Procedures

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.

Data collected by investigator

• Liver copper (Cu), iron (Fe), and zinc (Zn) contents (dry weight)

• Serum diferric transferrin as a percent of total transferrin

Definitions

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).



References

    Constantine CC, Anderson GJ, Vulpe CD, McLaren CE, Bahlo M, Yeap HL, Gertig DM, Osborne NJ, Bertalli NA, Beckman KB, Chen V, Matak P, McKie AT, Delatycki MB, Olynyk JK, English DR, Southey MC, Giles GG, Hopper JL, Allen KJ, Gurrin LC. A novel association between a SNP in CYBRD1 and serum ferritin levels in a cohort study of HFE hereditary haemochromatosis. Br J Haematol. 2009 Oct;147(1):140-9. Epub 2009 Aug 10. PubMed 19673882   FullText

    Frazer DM, Wilkins SJ, Becker EM, Murphy TL, Vulpe CD, McKie AT, Anderson GJ. A rapid decrease in the expression of DMT1 and Dcytb but not Ireg1 or hephaestin explains the mucosal block phenomenon of iron absorption. Gut. 2003 Mar;52(3):340-6. PubMed 12584213   FullText

    McLaren CE, Barton JC, Eckfeldt JH, McLaren GD, Acton RT, Adams PC, Henkin LF, Gordeuk VR, Vulpe CD, Harris EL, Harrison BW, Reiss JA, Snively BM. Heritability of serum iron measures in the hemochromatosis and iron overload screening (HEIRS) family study. Am J Hematol. 2010 Feb;85(2):101-5. PubMed 20095037