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 of the balance between the consumption and the supply of iron, the latter being tightly regulated by the hepatic hormone hepcidin (HAMP), the central regulator of iron homeostasis¹. Hepcidin functions to limit iron bioavailability in blood by occluding or inducing ubiquitination and degrading the iron exporter ferroportin (FPN)². Reduction in functional FPN leads to decreased dietary iron absorption, iron sequestration in the liver, and decreased iron recycling from macrophages¹.

Hepcidin is regulated by iron status, inflammation, erythropoietic drive, and pregnancy (reviewed in ³). Given that iron homeostasis is highly dynamic, it is important to understand and measure the total iron pool and iron distribution and turnover. Animal studies traditionally relied on radioactive iron isotopes, a highly sensitive yet burdensome approach to measure iron dynamics. However, in more recent studies, including the study presented here⁴, nonradioactive, stable iron isotopes (⁵⁸Fe) are utilized to measure iron transport during pregnancy^5,6,7,8,9. Stable isotopes are valuable tools for studying nutrient metabolism (reviewed in ¹⁰). The use of stable iron isotopes in human studies demonstrated that i) iron absorption increases toward the end of gestation^5,6, ii) transfer of dietary iron to the fetus is dependent on maternal iron status⁷, iii) maternally ingested heme iron is more readily incorporated by the fetus than nonheme iron⁸, and iv) iron transfer to the fetus is negatively correlated with maternal hepcidin levels^8,9. These experiments measured iron isotopes in sera or their incorporation into RBCs; however, measurement of iron incorporated into RBCs alone may underestimate true iron absorption⁹. In the current study, both heme and nonheme iron are measured in tissues.

During pregnancy, iron is required to support the expansion of maternal red blood cell volume and for transfer across the placenta to support the growth and development of the fetus¹¹. Fetal iron endowment is wholly dependent on iron transport across the placenta. During human¹² and rodent^4,13 pregnancy, hepcidin levels dramatically decrease, increasing plasma iron availability for transfer to the fetus.

The fundamentals of placental iron transport were initially characterized in the 1950s-70s using radioactive tracers (⁵⁹Fe and ⁵⁵Fe). These studies determined that iron transport across the placenta is unidirectional^14,15 and that diferric transferrin is a major source of iron for the placenta and fetus^16,17. The current understanding of placental iron transport is more complete, although some key iron transporters and regulatory mechanisms remain unknown. Mouse models have been essential for understanding iron regulation and transport¹⁸ because the key transporters and mechanisms are remarkably similar. Both human and mouse placentae are hemochorial, that is, maternal blood is in direct contact with the fetal chorion¹⁹. However, there are some notable structural differences.

The syncytiotrophoblast is the placental cell layer that separates the maternal and fetal circulation and actively transports iron and other nutrients²⁰. In humans, the syncytiotrophoblast is a single layer of fused cells. In contrast, the mouse placenta consists of two syncytiotrophoblast layers²¹, Syn-I and Syn-II. However, gap junctions at the interface of Syn-I and Syn-II allow the diffusion of nutrients between layers^22,23. Thus, these layers function as a single syncytial layer similar to the human syncytiotrophoblast. Additional similarities and differences between human and mouse placentae are reviewed by Rossant and Cross²¹. Placental iron transport is triggered by the binding of iron-Tf from maternal blood to the transferrin receptor (TfR1) localized on the apical side of the syncytiotrophoblast²⁴. This interaction induces iron-Tf/TfR1 internalization via clathrin-mediated endocytosis²⁵. Iron is then released from Tf in the acidic endosome²⁶, reduced to ferrous iron by an undetermined ferrireductase, and exported from the endosome to the cytoplasm by a yet-to-be determined transporter. How iron is chaperoned within the syncytiotrophoblast also remains to be described. Iron is eventually transported to the fetal side by the iron exporter, FPN, localized on the basal or fetal-facing surface of the syncytiotrophoblast (reviewed in²⁷).

To understand how physiological and pathological regulation of TfR1, FPN, and hepcidin affects placental iron transport, stable iron isotopes were utilized to quantitate iron transport from the maternal circulation to the placenta and embryo in vivo⁴. This paper presents the methods for preparing and administering isotopic iron-transferrin to pregnant mice, processing of tissues for ICP-MS, and calculating iron concentrations in tissues. The use of stable iron isotopes in vivo can be adapted to investigate iron regulation and distribution in different animal models to investigate physiologic and pathologic iron regulation.

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 loosely. To dissolve the iron, warm the ⁵⁸Fe/HCl solution to 60 °C for 1 h. If still not dissolved, leave the solution overnight at room temperature in the fume hood to dissolve.
      NOTE: Dissolved ⁵⁸Fe/HCl solution is yellowish-orange in color.
      Fe₃O_4(s) + 8HCl_(aq) → Fe^(II)Cl_2(aq) + 2Fe^(III)Cl_3(aq) + 4H₂O
2. Oxidize any remaining Fe^(II)Cl₂ to generate the Fe^(III)Cl₃ solution.
   1. Warm up the ⁵⁸Fe/HCl solution to 60 °C with the cap off to facilitate oxidation.
   2. Add 1 µL of 35% H₂O₂ per 50 µL of ⁵⁸Fe/HCl solution to further facilitate oxidation.
      Fe^(II)Cl_2(aq) + O₂ + 4HCl → 4Fe^(III)Cl_3(aq) + 2H₂O
3. Prepare the ferric chloride (⁵⁸Fe^(III)Cl₃) solution.
   1. Leave the ferric chloride solution in the hood at 60 °C with the cap off to evaporate the sample.
      NOTE: Evaporation may take between one and several days.
   2. Reconstitute ⁵⁸Fe^(III)Cl₃ to 100 mM with ultrapure H₂O, and calculate the amount of ultrapure H₂O required based on the initial metal weight used in step 1.1 (molecular weight of ⁵⁸Fe^(III)Cl₃ is 162.2).
4. Prepare ⁵⁸Fe^(III)-nitrilotriacetate (NTA) by incubating ⁵⁸Fe^(III)Cl₃ with NTA at a 1:5 molar ratio in the presence of 20 mM NaHCO₃.
   1. Prepare 500 mM NTA in 1 N NaOH.
   2. Prepare 5x transferrin-loading buffer (0.5 M HEPES, pH 7.5; 0.75 M NaCl).
   3. Prepare 1 M NaHCO₃ in ultrapure H₂O.
   4. To a 15 mL conical tube, add 150 µL of 100 mM ⁵⁸Fe^(III)Cl₃ solution (from step 1.3.2), 150 µL of 500 mM NTA prepared in 1 N NaOH, 480 µL of ultrapure H₂O, 200 µL of 5x transferrin loading buffer, and 20 µL of 1 M NaHCO₃ solution.
   5. Incubate the mixture for 5 min at room temperature.
5. Load apo-Tf with ⁵⁸Fe^(III)-NTA to form ⁵⁸Fe-Tf.
   NOTE: This protocol was adapted from McCarthy and Kosman²⁸.
   1. Dissolve 500 mg of apo-Tf in 4 mL of 1x Tf-loading buffer.
   2. To the 15 mL conical tube in step 1.4.4 containing 1 mL of the ⁵⁸Fe^(III)-NTA solution, add 4 mL of apo-Tf solution.
      NOTE: This is a 3:1 molar ratio of ⁵⁸Fe-NTA with apo-Tf. Each Tf contains 2 Fe binding sites; excess ⁵⁸Fe-NTA was added to ensure that Tf was fully loaded.
   3. To allow maximal loading of ⁵⁸Fe-NTA onto apo-Tf, check that the solution is at pH 7.5, and adjust the pH, if necessary, with NaHCO₃ or HCl.
   4. Incubate for 2.5 h at room temperature.
6. Remove excess unbound ⁵⁸Fe^(III)-NTA and released NTA.
   1. Transfer the ⁵⁸Fe-Tf solution to a molecular weight cutoff column (30 kDa cutoff) and centrifuge at 2,500 × g for 15 min at room temperature.
   2. Wash the column with 10 mL of 1x transferrin-loading buffer and centrifuge at 2,500 × g for 15 min at room temperature. Repeat the wash and centrifugation, perform a saline wash with 10 mL of saline, and centrifuge at 2,500 × g for 15 min at room temperature.
7. Calculate the concentration of ⁵⁸Fe-Tf.
   NOTE: Due to the addition of excess ⁵⁸Fe in step 1.5.2, assume that all transferrin is diferric. As 500 mg of apo-Tf was used, ~500 mg ⁵⁸Fe-Tf was produced in step 1.5.4.
   1. Measure the volume recovered from centrifugation after the saline wash in step 1.6.2.
   2. Divide 500 mg by the volume recovered to determine the concentration (in mg/mL) of the ⁵⁸Fe-Tf solution.
8. Sterilize the ⁵⁸Fe-Tf solution using a 0.22 µm syringe filter; store at 4 °C until ready to use.
   NOTE: ⁵⁸Fe-Tf solution was used between 1 to 4 weeks post preparation.

2. Set up timed mouse pregnancies

1. Use 6- to 8-week-old female mice. Place animals on a low-iron diet (4 ppm iron) or standard chow (185 ppm iron) for 2 weeks prior to mating and maintain animals on the respective diets throughout pregnancy.
2. Option 01: Confirm pregnancy by weight gain at E7.5.
   1. Set up multiple breeding cages. For each cage, combine 2 females with 1 male overnight; the following day when animals are separated is considered embryonic day (E)0.5. Weigh females at E7.5 to determine if pregnant. Mate males again with females that did not gain weight.
      NOTE: In WT C57BL/6, a weight gain of 1 g at E7.5 is a good indicator of pregnancy. This method ensures that implantation occurred within a specific 16 h timeframe, allowing for synchronous treatment of all animals that became pregnant during the same mating period.
3. Option 02: Confirm pregnancy by plug checks.
   1. Combine 2 females with 1 male and perform daily plug checks to determine if copulation has occurred.
      NOTE: This method may result in staggered pregnancies, and the presence of a plug does not guarantee pregnancy.

3. Administer ⁵⁸Fe-Tf intravenously to E17.5 pregnant mice

1. Prepare ⁵⁸Fe-Tf from step 1.8 for injection.
   1. Prepare ⁵⁸Fe-Tf solution at 35 mg/mL in saline; inject 100 µL per mouse.
   2. Fill an insulin syringe with 100 µL of the ⁵⁸Fe-Tf solution.
      NOTE: Each dose contains 3.5 mg of human ⁵⁸Fe-Tf (5 µg of ⁵⁸Fe).
2. Anesthetize a pregnant mouse using isoflurane.
   1. Use an isoflurane regulator with a chamber.
   2. Use the following settings: 5% isoflurane, 2 L/mL of O₂, 2 min.
   3. Confirm the mouse is anesthetized by looking for lack of response to a toe pinch.
   4. Apply eye lubricant to the surface of the eye and place the mouse on a heating pad.
3. Slowly and carefully inject the ⁵⁸Fe-Tf solution into the retro-orbital sinus.
4. Allow the mouse to recover from anesthesia; do not leave the animal unattended until it has regained sufficient consciousness to maintain sternal recumbency.
5. Six hours post injection, euthanize E17.5 pregnant females by isoflurane overdose.
   1. Perform a cardiac puncture to exsanguinate the mouse as a form of secondary euthanasia.
   2. Pin the feet down with needles for stabilization.
6. Collect the placentae and embryo livers.
   1. Using sterile forceps and dissection scissors, carefully remove the uterus from the pregnant mouse. Cut off a placental fetal-placental unit, which comprises a single fetus and placenta in the amniotic sac surrounded by a portion of the uterus.
   2. Carefully cut through the uterus and amniotic sac without disturbing the fetus and placenta.
   3. Peel back the amniotic sac and remove the fetus and placenta.
   4. Cut the umbilical cord.
   5. Blot the fetus and placenta on a clean task wipe to remove the excess amniotic fluid.
   6. Record the weights of the whole placentae.
   7. Cut each placenta in half with a razor blade, place each half in a 2.0 mL tube, and snap-freeze in liquid nitrogen.
      NOTE: Because ⁵⁸Fe does not require special handling precautions and disposal, one-half of the placentae can be used for ⁵⁸Fe measurement and the other half for any other analyses, including quantitation of transferrin receptor (TFR1) and ferroportin (FPN) expression by western blotting and qPCR.
   8. To collect embryo livers, sacrifice the embryo: use a razor blade to rapidly decapitate the embryo.
      NOTE: At E17.5, all embryos in the uterus must be euthanized individually, even if they are not used in the study.
   9. Pin down the embryo for stabilization, leaving the abdomen exposed.
   10. Using dissection scissors, make a small incision where the umbilical cord was attached, insert one end of the dissection scissors into the incision, and perform a median plane cut toward the coronal plane about ¼ inch. Then, perform transverse plane cuts to expose the fetal liver.
   11. Use forceps to remove the fetal liver.
   12. Record the weights of the whole embryo livers.
   13. Place the whole embryo livers in 2 mL tubes and snap-freeze them in liquid nitrogen.
       NOTE: Alternatively, only a portion of the embryo liver can be used for ⁵⁸Fe measurement if additional analyses are desired. Using 2.0 mL tubes allows for better tissue homogenization than 1.5 mL tubes.
7. Store the tissues indefinitely at -80 °C.

4. Process tissues for quantitative iron analysis by ICP-MS

1. Process the placentae and fetal livers for the quantitation of nonheme iron.
   1. Thaw placental halves and whole fetal livers, and weigh placental halves (see step 3.6.12 for recording fetal liver weights).
   2. Add 400 µL of protein precipitation solution (0.53 N HCl, 5.3% TCA).
   3. Homogenize the tissue using an electric homogenizer.
   4. Incubate the samples at 100 °C for 1 h.
   5. Cool the samples in room temperature water for 2 min.
   6. Open the caps to release pressure, then close the tubes again.
   7. Centrifuge at 17,000 × g for 10 min at room temperature to pellet tissue debris.
   8. Carefully transfer the supernatant to a new labeled tube.
   9. Send samples off for ICP-MS analysis.
2. Process the placentae and fetal livers for the quantitation of heme-iron.
   NOTE: Following extraction of nonheme iron in step 1, the iron remaining in the pellet is predominantly heme.
   1. Record the weight of each pellet from step 4.1.7.
   2. Digest the pellets in 10 mL of concentrated 70% HNO₃ supplemented with 1 mL of 30% H₂O₂
      NOTE: Consult with the ICP-MS core or center to optimize the volume of HNO₃ for specific studies; the volume will partly be dependent on sample weight.
   3. Heat the samples to 200 °C for 15 min.
   4. Send the samples off for ICP-MS analysis.
      NOTE: If distinguishing between heme and nonheme iron sources is not required and only total iron is measured, whole tissue can be digested in HNO₃ as the first step.

5. Data analysis

NOTE: Data from ICP-MS has been provided as ⁵⁶Fe and ⁵⁸Fe concentrations in ng/mL or mg, ppb (Table 1). ⁵⁶Fe is the most abundant iron isotope in nature, and its measurement reflects iron accumulation in the placenta/embryo over the entire pregnancy, whereas ⁵⁸Fe measurement reflects iron that was transferred during 6 h after injection.

1. Subtract the natural abundance of ⁵⁸Fe (0.28% of total Fe) from the measured ⁵⁸Fe values.
2. Calculate total nonheme ⁵⁸Fe.
   1. Calculate embryo liver total nonheme iron (ng) by first multiplying the iron concentration (ng/mL) calculated in step 5.1 by the volume (mL) during initial processing in step 4.1.2 to estimate total ⁵⁸Fe.
   2. Calculate the amount of iron in the whole placenta by taking the total weight of the placenta measured in step 3.6.6 and dividing it by the weight of the placenta processed in step 4.1.1. Multiply this value by the total nonheme iron (ng) calculated in step 5.2.1 to obtain the total nonheme ⁵⁸Fe content of the placenta.
3. Calculate total heme ⁵⁸Fe.
   1. Calculate total heme ⁵⁸Fe by first multiplying the iron concentration (ng/mg) calculated in step 5.1 by the weight of the pellet (in mg) measured in step 4.2.1.
   2. Then, divide the total weight of the placenta measured in step 3.5.1 by the weight of the placenta pellet measured in step 4.2.1. Multiply this value by the total heme iron (ng) calculated in step 5.3.1 to obtain total heme ⁵⁸Fe content of the placenta.
4. Sum the calculated nonheme and heme ⁵⁸Fe values to determine the total iron content for each tissue.

Figure 1: Visual summary of steps in the protocol. (A) Preparation of ⁵⁸Fe-transferrin. (B) In vivo administration of ⁵⁸Fe-transferrin. (C) Tissue collection and storage. (D) Processing of the placenta and embryo liver for quantitation of metal species by ICP-MS. Abbreviations: Fe = iron; NTA = nitrilotriacetic acid; Tf = transferrin; PPS = protein precipitation solution; Sup = supernatant; TCA = trichloroacetic acid; ICP-MS = inductively coupled plasma mass spectrometry. Please click here to view a larger version of this figure.

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

List of materials used in this article

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