Quantitating Iron Transport Across the Mouse Placenta In Vivo Using Nonradioactive Iron Isotopes

Summary

This article demonstrates how to prepare and administer transferrin-bound nonradioactive isotopic iron for studies of iron transport in mouse pregnancy. The approach for quantifying isotopic iron in fetoplacental compartments is also described.

Abstract

Iron is essential for maternal and fetal health during pregnancy, with approximately 1 g of iron needed in humans to sustain a healthy pregnancy. Fetal iron endowment is entirely dependent on iron transfer across the placenta, and perturbations of this transfer can lead to adverse pregnancy outcomes. In mice, measurement of iron fluxes across the placenta traditionally relied on radioactive iron isotopes, a highly sensitive but burdensome approach. Stable iron isotopes (⁵⁷Fe and ⁵⁸Fe) offer a nonradioactive alternative for use in human pregnancy studies.

Under physiological conditions, transferrin-bound iron is the predominant form of iron taken up by the placenta. Thus, ⁵⁸Fe-transferrin was prepared and injected intravenously in pregnant dams to directly assess placental iron transport and bypass maternal intestinal iron absorption as a confounding variable. Isotopic iron was quantitated in the placenta and mouse embryonic tissues by inductively coupled plasma mass spectrometry (ICP-MS). These methods can also be employed in other animal model systems of physiology or disease to quantify in vivo iron dynamics.

Introduction

Iron is critical for various metabolic processes, including growth and development, energy production, and oxygen transport¹. Maintenance of iron homeostasis is a dynamic, coordinated process. Iron is absorbed from food in the duodenum and transported around the body in the circulation bound to the iron transport protein transferrin (Tf). It is utilized by every cell for enzymatic processes, incorporated into hemoglobin in nascent erythrocytes, and recycled from aged erythrocytes by macrophages. Iron is stored in the liver when in excess and lost from the body through hemorrhage or cell sloughing. The amount of iron in circulation is the result....

Protocol

All animal protocols and experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California Los Angeles.

1. Preparation of ⁵⁸Fe-Tf

NOTE: The protocol uses ⁵⁸Fe; however, an identical protocol can be used for ⁵⁷Fe. Either isotope can be used and disposed of as a standard iron chemical without additional precautions.

1. Dissolve ⁵⁸Fe in 12 N HCl at 50 µL of HCl/mg of ⁵⁸Fe.
   1. Add HCl to the metal in the glass vial supplied by the vendor, and replace the cap loo....

Results

An earlier study using stable iron isotopes to measure iron transport demonstrated that maternal iron deficiency resulted in the downregulation of the placenta iron exporter, FPN⁴. FPN is the only known mammalian iron exporter, and the absence of FPN during development results in embryonic death before E9.5²⁹. To determine whether the observed decrease in FPN expression translated functionally to decreased placental iron transport, ⁵⁸Fe-Tf was injected intravenou.......

Discussion

Iron is important for many biological processes, and its movement and distribution within the body are highly dynamic and regulated. Stable iron isotopes provide a consistent and convenient alternative to radioactive isotopes for the assessment of the dynamics of iron homeostasis. A critical step in the protocol is keeping track of all the tissue weights and volumes. Iron is an element and therefore cannot be synthesized nor broken down. Thus, if all weights and volumes are carefully logged, all the iron within the syste.......

Materials

Name	Company	Catalog Number	Comments
58Fe-iron metal	Trace Sciences International	Fe-58
Amicon ultra-15 centrifugal filter, 30 kDa cutoff	Millipore Sigma	UFC903024
Centrifuge tubes, 15 mL	Fisher Scientific	14-959-49B
Centrifuge tubes, 50 mL	Millipore Sigma	CLS430829
Centrifuge, Sorvall Legend Micro 17 Microcentrifuge	Fisher Scientific	75002432
Centrifuge, Sorvall Legend RT
Delicate task wipers	Fisher Scientific	06-666
Diet: iron-deficient (4 ppm iron)	Envigo Teklad	TD.80396
Diet: standard chow (185 ppm iron)	PicoLab	5053
Dissecting scissor with 30 mm cutting edge	VWR	25870-002
Forceps 4-1/2 inch length	McKesson	157-469
HEPES	Fisher Scientific	BP310-500
Homogenizer, Bio-Gen PRO200	PROScientific	01-01200
Human apo-transferrin (apo-Tf)	Celliance	4452-01	no longer available, alternative: Millipore 616419
Hydrochloric acid (HCl)	Fisher Scientific	A144S-500
Hydrogen peroxide (H2O2), 35 wt.% solution in water	Cole-Parmer	EW-88216-36
Insulin Syringes, BD Lo-Dose U-100	Fisher Scientific	14-826-79
Isoflurane	VETone	502017
Isoflurane vaporizor	Summit Anesthesia Solutions
Metal heat block	Fisher Scientific
Micro centrifuge tube with flat screw-cap	VWR	16466-064
Microcentrifuge tubes 1.5 mL low-retention	Fisher Scientific	02-681-320
Microcentrifuge tubes 2.0 mL low-retention	Fisher Scientific	02-681-321
Millex-GP syringe filter unit, 0.22 µm, polyethersulfone, 33 mm, gamma-sterilized	Millipore Sigma	SLGP033RS
Nitrilotriacetic acid (NTA)	Sigma	72560-100G
Needle 25 G x 5/8 in. hypodermic general use	Fisher Scientific	14-826AA
pH Strips, plastic pH5.0-9.0	Fisher Scientific	13-640-519
Razor blades 0.22 mm	VWR	55411-050
Scale (g)	Mettler Toledo	PB1502-S
Scale (mg)	Mettler Toledo	Balance XS204
Sodium bicarbonate (NaHCO3)	Sigma	S5761-500G
Sodium chloride (NaCl)	Fisher Scientific	S671-3
Sodium hydroxide (NaOH)	Fisher Scientific	SS266-1
Sterile syringe, slip tip (1 mL)	Fisher Scientific	309659
Trichloroacetic acid (TCA)	Fisher Scientific	A322-500
Software
ImageLab	Bio-Rad
SigmaPlot	Systat