Name: quantitating iron transport across the mouse placenta in vivo using nonradioactive iron isotopes
Uploaded: 2022-05-10T14:00:00
Description: Watch this Scientific Journal Video about Quantitating Iron Transport Across the Mouse Placenta In Vivo Using Nonradioactive Iron Isotopes at JoVE.com

The authors acknowledge the use of the ICP-MS facility within the UC Center for Environmental Implications of Nanotechnology in CNSI at UCLA for their assistance with optimizing the protocol for 58Fe measurements. The study was supported by the NIH National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (K01DK127004, to VS) and NIH National Institute of Child Health and Human Development (NICHD) (R01HD096863, to EN).

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.
<ol>
	<li>Dissolve 58Fe in 1...

<ol>
	<li>Ganz, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+iron+homeostasis.">Systemic iron homeostasis.</a> Physiological Reviews. 93 (4), 1721-1741 (2013).</li><li>Aschemeyer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-function+analysis+of+ferroportin+defines+the+binding+site+and+an+alternative+mechanism+of+action+of+hepcidin.">Structure-function analysis of ferroportin defines the binding site and an alternative mechanism of action of hepcidin.</a> Blood. 131 (8), 899-910 (2018).</li><li>Sangkhae, V., Nemeth, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+iron+homeostatic+hormone+hepcidin.">Regulation of the iron homeostatic hormone hepcidin.</a> Advances in Nutrition. 8 (1), 126-136 (2017).</li><li>Sangkhae, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+maternal+iron+status+on+placental+and+fetal+iron+homeostasis.">Effects of maternal iron status on placental and fetal iron homeostasis.</a> Journal of Clinical Investigation. 130 (2), 625-640 (2020).</li><li>Whittaker, P. G., Lind, T., Williams, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+absorption+during+normal+human+pregnancy:+a+study+using+stable+isotopes.">Iron absorption during normal human pregnancy: a study using stable isotopes.</a> British Journal of Nutrition. 65 (3), 457-463 (1991).</li><li>Whittaker, P. G., Barrett, J. F., Lind, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+erythrocyte+incorporation+of+absorbed+non-haem+iron+in+pregnant+women.">The erythrocyte incorporation of absorbed non-haem iron in pregnant women.</a> British Journal of Nutrition. 86 (3), 323-329 (2001).</li><li>O'Brien, K. O., Zavaleta, N., Abrams, S. A., Caulfield, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+iron+status+influences+iron+transfer+to+the+fetus+during+the+third+trimester+of+pregnancy.">Maternal iron status influences iron transfer to the fetus during the third trimester of pregnancy.</a> American Journal of Clinical Nutrition. 77 (4), 924-930 (2003).</li><li>Young, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+hepcidin+is+associated+with+placental+transfer+of+iron+derived+from+dietary+heme+and+nonheme+sources.">Maternal hepcidin is associated with placental transfer of iron derived from dietary heme and nonheme sources.</a> Journal of Nutrition. 142 (1), 33-39 (2012).</li><li>Delaney, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+absorption+during+pregnancy+is+underestimated+when+iron+utilization+by+the+placenta+and+fetus+is+ignored.">Iron absorption during pregnancy is underestimated when iron utilization by the placenta and fetus is ignored.</a> American Journal of Clinical Nutrition. 112 (3), 576-585 (2020).</li><li>Klatt, K. C., Smith, E. R., Barberio, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+a+more+stable+understanding+of+pregnancy+micronutrient+metabolism.">Toward a more stable understanding of pregnancy micronutrient metabolism.</a> American Journal of Physiology-Endocrinology Metabolism. 321 (2), 260-263 (2021).</li><li>Fisher, A. L., Nemeth, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+homeostasis+during+pregnancy.">Iron homeostasis during pregnancy.</a> American Journal of Clinical Nutrition. 106, 1567-1574 (2017).</li><li>van Santen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+iron+regulatory+hormone+hepcidin+is+decreased+in+pregnancy:+a+prospective+longitudinal+study.">The iron regulatory hormone hepcidin is decreased in pregnancy: a prospective longitudinal study.</a> Clinical Chemistry and Laboratory Medicine. 51 (7), 1395-1401 (2013).</li><li>Millard, K. N., Frazer, D. M., Wilkins, S. J., Anderson, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+expression+of+intestinal+iron+transport+and+hepatic+regulatory+molecules+explain+the+enhanced+iron+absorption+associated+with+pregnancy+in+the+rat.">Changes in the expression of intestinal iron transport and hepatic regulatory molecules explain the enhanced iron absorption associated with pregnancy in the rat.</a> Gut. 53 (5), 655-660 (2004).</li><li>Bothwell, T. H., Pribilla, W. F., Mebust, W., Finch, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+metabolism+in+the+pregnant+rabbit;+iron+transport+across+the+placenta.">Iron metabolism in the pregnant rabbit; iron transport across the placenta.</a> American Journal of Physiology. 193 (3), 615-622 (1958).</li><li>Dyer, N. C., Brill, A. B., Raye, J., Gutberlet, R., Stahlman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal-fetal+exchange+of+59+Fe:+radiation+dosimetry+and+biokinetics+in+human+and+sheep+studies.">Maternal-fetal exchange of 59 Fe: radiation dosimetry and biokinetics in human and sheep studies.</a> Radiation Research. 53 (3), 488-495 (1973).</li><li>Contractor, S. F., Eaton, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+transferrin+in+iron+transport+between+maternal+and+fetal+circulations+of+a+perfused+lobule+of+human+placenta.">Role of transferrin in iron transport between maternal and fetal circulations of a perfused lobule of human placenta.</a> Cell Biochemistry &amp; Function. 4 (1), 69-74 (1986).</li><li>Baker, E., Morgan, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+transferrin+in+placental+iron+transfer+in+the+rabbit.">The role of transferrin in placental iron transfer in the rabbit.</a> Quartly Jounrnal of Experimental Physiolology and Cognate Medical Sciences. 54 (2), 173-186 (1969).</li><li>Fleming, R. E., Feng, Q., Britton, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knockout+mouse+models+of+iron+homeostasis.">Knockout mouse models of iron homeostasis.</a> Annual Review of Nutrition. 31, 117-137 (2011).</li><li>Soares, M. J., Varberg, K. M., Iqbal, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemochorial+placentation:+development,+function,+and+adaptations.">Hemochorial placentation: development, function, and adaptations.</a> Biology of Reproduction. 99 (1), 196-211 (2018).</li><li>Jones, H. N., Powell, T. L., Jansson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+placental+nutrient+transport--a+review.">Regulation of placental nutrient transport--a review.</a> Placenta. 28 (8-9), 763-774 (2007).</li><li>Rossant, J., Cross, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Placental+development:+lessons+from+mouse+mutants.">Placental development: lessons from mouse mutants.</a> Nature Reviews Genetics. 2 (7), 538-548 (2001).</li><li>Takata, K., Kasahara, T., Kasahara, M., Ezaki, O., Hirano, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunolocalization+of+glucose+transporter+GLUT1+in+the+rat+placental+barrier:+possible+role+of+GLUT1+and+the+gap+junction+in+the+transport+of+glucose+across+the+placental+barrier.">Immunolocalization of glucose transporter GLUT1 in the rat placental barrier: possible role of GLUT1 and the gap junction in the transport of glucose across the placental barrier.</a> Cell and Tissue Research. 276 (3), 411-418 (1994).</li><li>Shin, B. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunolocalization+of+GLUT1+and+connexin+26+in+the+rat+placenta.">Immunolocalization of GLUT1 and connexin 26 in the rat placenta.</a> Cell and Tissue Research. 285 (1), 83-89 (1996).</li><li>Bastin, J., Drakesmith, H., Rees, M., Sargent, I., Townsend, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localisation+of+proteins+of+iron+metabolism+in+the+human+placenta+and+liver.">Localisation of proteins of iron metabolism in the human placenta and liver.</a> British Journal of Haematology. 134 (5), 532-543 (2006).</li><li>Klausner, R. D., Ashwell, G., van Renswoude, J., Harford, J. B., Bridges, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+of+apotransferrin+to+K562+cells:+explanation+of+the+transferrin+cycle.">Binding of apotransferrin to K562 cells: explanation of the transferrin cycle.</a> Proceedings of the National Academy of Sciences of the United States of America. 80 (8), 2263-2266 (1983).</li><li>Tsunoo, H., Sussman, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+transferrin+binding+and+specificity+of+the+placental+transferrin+receptor.">Characterization of transferrin binding and specificity of the placental transferrin receptor.</a> Archives of Biochemistry and Biophysics. 225 (1), 42-54 (1983).</li><li>Sangkhae, V., Nemeth, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Placental+iron+transport:+The+mechanism+and+regulatory+circuits.">Placental iron transport: The mechanism and regulatory circuits.</a> Free Radical Biology and Medicine. 133, 254-261 (2019).</li><li>McCarthy, R. C., Kosman, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+analysis+of+iron+accumulation+by+endothelial+cells+of+the+BBB.">Mechanistic analysis of iron accumulation by endothelial cells of the BBB.</a> Biometals. 25 (4), 665-675 (2012).</li><li>Donovan, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+iron+exporter+ferroportin/Slc40a1+is+essential+for+iron+homeostasis.">The iron exporter ferroportin/Slc40a1 is essential for iron homeostasis.</a> Cell Metabolism. 1 (3), 191-200 (2005).</li><li>Stefanova, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+hepcidin+and+its+agonist+mediate+resistance+to+selected+infections+by+clearing+non-transferrin-bound+iron.">Endogenous hepcidin and its agonist mediate resistance to selected infections by clearing non-transferrin-bound iron.</a> Blood. 130 (3), 245-257 (2017).</li><li>Ramos, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+distinct+pathways+of+hepcidin+regulation+by+acute+and+chronic+iron+loading+in+mice.">Evidence for distinct pathways of hepcidin regulation by acute and chronic iron loading in mice.</a> Hepatology. 53 (4), 1333-1341 (2011).</li><li>Kulandavelu, S., Qu, D., Adamson, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+function+in+mice+during+normal+pregnancy+and+in+the+absence+of+endothelial+NO+synthase.">Cardiovascular function in mice during normal pregnancy and in the absence of endothelial NO synthase.</a> Hypertension. 47 (6), 1175-1182 (2006).</li><li>Lu, C. C., Matsumoto, N., Iijima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Placental+transfer+and+body+distribution+of+nickel+chloride+in+pregnant+mice.">Placental transfer and body distribution of nickel chloride in pregnant mice.</a> Toxicology and Applied Pharmacology. 59 (3), 409-413 (1981).</li><li>Gunshin, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slc11a2+is+required+for+intestinal+iron+absorption+and+erythropoiesis+but+dispensable+in+placenta+and+liver.">Slc11a2 is required for intestinal iron absorption and erythropoiesis but dispensable in placenta and liver.</a> Journal of Clinical Investigation. 115 (5), 1258-1266 (2005).</li></ol>

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...

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 pregnancy5,6,7,8,9. 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 gestation5,6, 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 levels8,9. 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 rodent4,13 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 unidirectional14,15 and that diferric transferrin is a major source of iron for the placenta and fetus16,17. 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 layers22,23. 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>58Fe-iron metal</td>
			<td>Trace Sciences International</td>
			<td>Fe-58</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon ultra-15 centrifugal filter, 30 kDa cutoff</td>
			<td>Millipore Sigma</td>
			<td>UFC903024</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes, 15 mL</td>
			<td>Fisher Scientific</td>
			<td>14-959-49B</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes, 50 mL</td>
			<td>Millipore Sigma</td>
			<td>CLS430829</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge, Sorvall Legend Micro 17 Microcentrifuge</td>
			<td>Fisher Scientific</td>
			<td>75002432</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge, Sorvall Legend RT</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Delicate task wipers</td>
			<td>Fisher Scientific</td>
			<td>06-666</td>
			<td></td>
		</tr>
		<tr>
			<td>Diet: iron-deficient (4 ppm iron)</td>
			<td>Envigo Teklad</td>
			<td>TD.80396</td>
			<td></td>
		</tr>
		<tr>
			<td>Diet: standard chow (185 ppm iron)</td>
			<td>PicoLab</td>
			<td>5053</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scissor with 30 mm cutting edge</td>
			<td>VWR</td>
			<td>25870-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps 4-1/2 inch length</td>
			<td>McKesson</td>
			<td>157-469</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher Scientific</td>
			<td>BP310-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Homogenizer, Bio-Gen PRO200</td>
			<td>PROScientific</td>
			<td>01-01200</td>
			<td></td>
		</tr>
		<tr>
			<td>Human apo-transferrin (apo-Tf)</td>
			<td>Celliance</td>
			<td>4452-01</td>
			<td>no longer available, alternative: Millipore 616419</td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCl)</td>
			<td>Fisher Scientific</td>
			<td>A144S-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide (H2O2), 35 wt.% solution in water</td>
			<td>Cole-Parmer</td>
			<td>EW-88216-36</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin Syringes, BD Lo-Dose U-100</td>
			<td>Fisher Scientific</td>
			<td>14-826-79</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>VETone</td>
			<td>502017</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane vaporizor</td>
			<td>Summit Anesthesia Solutions</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Metal heat block</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro centrifuge tube with flat screw-cap</td>
			<td>VWR</td>
			<td>16466-064</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes 1.5 mL low-retention</td>
			<td>Fisher Scientific</td>
			<td>02-681-320</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes 2.0 mL low-retention</td>
			<td>Fisher Scientific</td>
			<td>02-681-321</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex-GP syringe filter unit, 0.22 &#181;m, polyethersulfone, 33 mm, gamma-sterilized</td>
			<td>Millipore Sigma</td>
			<td>SLGP033RS</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrilotriacetic acid (NTA)</td>
			<td>Sigma</td>
			<td>72560-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle 25 G x 5/8 in. hypodermic general use</td>
			<td>Fisher Scientific</td>
			<td>14-826AA</td>
			<td></td>
		</tr>
		<tr>
			<td>pH Strips, plastic pH5.0-9.0</td>
			<td>Fisher Scientific</td>
			<td>13-640-519</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blades 0.22 mm</td>
			<td>VWR</td>
			<td>55411-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Scale (g)</td>
			<td>Mettler Toledo</td>
			<td>PB1502-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Scale (mg)</td>
			<td>Mettler Toledo</td>
			<td>Balance XS204</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate (NaHCO3)</td>
			<td>Sigma</td>
			<td>S5761-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Fisher Scientific</td>
			<td>S671-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Fisher Scientific</td>
			<td>SS266-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile syringe, slip tip (1 mL)</td>
			<td>Fisher Scientific</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloroacetic acid (TCA)</td>
			<td>Fisher Scientific</td>
			<td>A322-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageLab</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SigmaPlot</td>
			<td>Systat</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitating iron transport across the mouse placenta in vivo using nonradioactive iron isotopes

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.

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...

Watch this Scientific Journal Video about Quantitating Iron Transport Across the Mouse Placenta In Vivo Using Nonradioactive Iron Isotopes at JoVE.com

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

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.

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

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 ...

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.

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