Iron Supplementation in Suckling Piglets: How to Correct
`Iron Deficiency Anemia without Affecting Plasma
`Hepcidin Levels
`
`Rafał R. Starzyn´ ski1, Coby M. M. Laarakkers2,3, Harold Tjalsma2,3, Dorine W. Swinkels2,3, Marek Pieszka4,
`Agnieszka Stys´ 1, Michał Mickiewicz5, Paweł Lipin´ ski1*
`
`1 Institute of Genetics and Animal Breeding, Jastrze˛biec, Poland, 2 Department of Laboratory Medicine (LGEM 830), Radboud University Medical Center, Nijmegen, The
`Netherlands, 3 Hepcidinanalysis.com, Nijmegen, The Netherlands, 4 National Research Institute of Animal Production, Balice, Poland, 5 Mifarmex Ltd, Michało´ w-Grabina,
`Poland
`
`Abstract
`
`The aim of the study was to establish an optimized protocol of iron dextran administration to pig neonates, which better
`meets the iron demand for erythropoiesis. Here, we monitored development of red blood cell
`indices, plasma iron
`parameters during a 28-day period after birth (till the weaning), following intramuscular administration of different
`concentrations of iron dextran to suckling piglets. To better assess the iron status we developed a novel mass spectrometry
`assay to quantify pig plasma levels of the iron-regulatory peptide hormone hepcidin-25. This hormone is predominantly
`secreted by the liver and acts as a negative regulator of iron absorption and reutilization. The routinely used protocol with
`high amount of iron resulted in the recovery of piglets from iron deficiency but also in strongly elevated plasma hepcidin-25
`levels. A similar protocol with reduced amounts of iron improved hematological status of piglets to the same level while
`plasma hepcidin-25 levels remained low. These data show that plasma hepcidin-25 levels can guide optimal dosing of iron
`treatment and pave the way for mixed supplementation of piglets starting with intramuscular injection of iron dextran
`followed by dietary supplementation, which could be efficient under condition of very low plasma hepcidin-25 level.
`
`Citation: Starzyn´ ski RR, Laarakkers CMM, Tjalsma H, Swinkels DW, Pieszka M, et al. (2013) Iron Supplementation in Suckling Piglets: How to Correct Iron Deficiency
`Anemia without Affecting Plasma Hepcidin Levels. PLoS ONE 8(5): e64022. doi:10.1371/journal.pone.0064022
`
`Editor: James R. Connor, Penn State Hershey Medical Center, United States of America
`
`Received February 9, 2013; Accepted April 9, 2013; Published May 30, 2013
`Copyright: ß 2013 Starzyn´ ski et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
`unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
`
`Funding: This work was supported by The National Science Centre grant
`2012/05/E/NZ5/02126 (http://www.ncn.gov.pl/) and Mifarmex Ltd (http://mifarmex.
`pl). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
`
`Competing Interests: The authors have the following interests: Coby M.M. Laarakkers, Harold Tjalsma and Dorine W. Swinkels are employees of the Radboud
`University Medical Centre (Nijmegen, The Netherlands) that amongst others offers hepcidin measurements to the scientific, medical and pharmaceutical
`communities through the service unit "hepcidinanalysis.com" on a fee per sample basis. Michal Mickiewicz is employee of Mifarmex Ltd, who partly funded this
`study. There are no further patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on
`sharing data and materials, as detailed online in the guide for authors.
`
`* E-mail: p.lipinski@ighz.pl
`
`Introduction
`
`Iron deficiency is considered to be the most common
`mammalian nutritional deficiency [1] and is most prevalent in
`the neonatal period [2]. Neonatal IDA is particularly frequent and
`severe in pigs, regardless of the breed and the system of piglet
`rearing [3], [4]. Iron scarcity in piglets is the result of interplay of
`several distinct risk factors such as low level of
`iron stores,
`increased iron requirements,
`limited external supply and the
`immaturity of molecular mechanisms of iron absorption [5], [6].
`For decades pigs have been selected for large litter size, high birth
`weight and fast growth, which resulted in greater body blood
`volume, red blood cells (RBC) count, and in consequence,
`in
`increased iron demands. Both hepatic iron reserves and the sow’s
`milk are therefore nowadays not sufficient to meet iron require-
`ments in suckling piglets [3], [4], [7]. Moreover, the molecular
`machinery responsible for iron absorption in newborn piglets is
`not fully developed, and this may explain a reduced responsiveness
`of these animals to oral iron therapy [8], [9]. Consequently, the
`use of parenteral injection of exogenous iron to prevent deepening
`iron deficiency in suckling piglets has been well documented and is
`
`iron
`[12]. Various
`[11],
`obligatory in pig breeding [10],
`supplementation strategies (in terms of time and route of iron
`administration as well as the amount and the form of supplemental
`iron) have been tested and many of them proved to be beneficial in
`correcting IDA in newborn piglets as evaluated by measuring their
`RBC indices and serum iron status [10], [11], [12], [13], [14],
`[15].
`Over the past eleven years major insights have been made into
`the role of hepcidin in the tuning of systemic iron homeostasis
`[16], [17]. Hepcidin is a peptide hormone, mainly secreted by the
`liver, which acts as a negative regulator of iron absorption and its
`reutilization by macrophages of the reticuloendothelial system
`(RES). By binding to ferroportin, the only known iron exporter,
`hepcidin causes ferroportin internalization and degradation, hence
`decreasing the absorption of dietary iron by enterocytes and the
`release of iron from macrophages that have engulfed senescent
`RBCs, into the plasma. Inappropriate low hepcidin levels cause
`the iron overload disorders, whereas increased hepcidin expression
`leads to anemia due to the insufficient intestinal iron absorption
`and iron arrest in the RES [16].
`
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`Considering that hepcidin is up-regulated by high iron
`concentrations in the plasma and the liver [16], we aimed in this
`study to establish an optimal strategy of iron supplementation in
`piglets, which could both improve RBC indices and minimize
`dysfunctional induction of hepcidin expression that could poten-
`tially have an adverse effect on iron utilization.
`The intramuscular injection of large amount of iron dextran to
`suckling piglets is a relatively simple therapy, which in general,
`efficiently corrects iron deficiency anemia (IDA), widely occurring
`in pig neonates, and therefore is routine practice in pig breeding
`[10], [11], [12]. We have recently demonstrated that excessive
`loading of piglets with iron dextran induces hepcidin expression at
`the mRNA level in the liver, and may perturbate the utilization of
`iron released from this compound by blocking ferroportin [10],
`[20]. It has been well established that after injection, iron dextran
`is rapidly taken from plasma and deposited in the reticuloendo-
`thelial system [21]. The elemental iron is then released from the
`complex with polyisomaltose by macrophages (mainly by Kupffer
`cells) and successively re-enters plasma via a ferroportin-dependent
`pathway. Next, the blood flow iron is complexed with transferrin
`(Tf) and transported to the bone marrow to allow hemoglobin
`synthesis. It seems that the optimal iron supplementation in piglets
`should meet two main criteria: 1) the delivery of sufficient amount
`of iron for erythropoiesis; 2) the avoidance of a dysfunctional
`increase in plasma hepcidin levels.
`We have recently reported that by both reducing the amount of
`supplemented iron and modifying the timing of its dosage, we can
`improve the hematological status of piglets while attenuating their
`hepatic hepcidin mRNA levels [10]. Here, we have developed a
`mass spectrometry assay to quantify bioactive hepcidin-25 in piglet
`plasma and assessed different protocols of piglet supplementation
`with iron dextran for their ability to stimulate recovery from
`neonatal IDA without inducing an unfavorable increase in plasma
`hepcidin.
`
`Plasma Hepcidin Level in Iron Supplemented Piglets
`
`Materials and Methods
`
`Ethics Statement
`Use of animals in the experiment and all procedures were
`approved by the Second Local Ethical Committee on Animal
`Testing at the Institute of Pharmacology in Krakow (permission
`no. 975).
`
`Animals, Experimental Design, and Blood Samples
`Collection
`Experiments were conducted at the pig farm Brzezie belonging
`to the National Research Institute of Animal Production (Balice,
`Poland). A total of 27 Polish Landrace 6 Polish Large White
`piglets housed in standard conditions (approx. 70% humidity and
`a temperature of 2262uC in standard cages with straw bedding)
`were used in the experiments. During the 28-day experiment sows
`were allowed to nurse their piglets. The feed (Prestarter, was
`manufactured at the feed mill of the Experimental Station of the
`National Research Institute of Animal Production in Brzezie; iron
`content 0.75 mg Fe/kg feed mixture) was offered to piglets from
`day 7 to day 28. Piglets were taken from 3 litters delivered by 3
`multipara sows. They were allotted to one of following experi-
`mental groups (9 piglets per group) on the basis of balanced body
`weight (b.w.) at birth (Figure 1): A) piglets routinely supplemented
`by intramuscular injection with 150 and 40 mg Fe/kg b.w. on
`days 3 and 21 postpartum, respectively; B) piglets supplemented
`with 37.5 mg Fe/kg b.w. on days 3 and 14 postpartum; C) piglets
`supplemented with 37.5 mg Fe/kg b.w. on day 3 postpartum only.
`Iron was administered to piglets by intramuscular injection in the
`neck in the form of iron dextran (FeDex), a complex of ferric ions
`with low molecular weight dextran (Suiferron, Mifarmex Ltd,
`Michało´w-Grabina, Poland or Ferran 100, Vet-Agro, Lublin,
`Poland). Blood samples for analyses were taken from all piglets on
`days 3, 14, 21, and 28 of life. At the same periods all animals were
`weighed. Blood was drawn by venipuncture of the jugular vein
`(Vena jugularis externa) into tubes coated with heparin or EDTA as
`anticoagulants. Heparinized blood was immediately spun down (at
`
`Figure 1. Experimental design scheme. 3 various protocols of iron dextran administration in piglets are indicated as A, B and C. Iron dextran was
`injected to piglets on days indicated by arrows. Blood samples were collected on days 3, 14, 21 and 28 after birth. When iron dextran injection and
`blood collection fell on the same day, blood was always drawn before iron administration.
`doi:10.1371/journal.pone.0064022.g001
`
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`Plasma Hepcidin Level in Iron Supplemented Piglets
`
`4uC, 2000 rpm, 10 min) to collect plasma. Plasma samples were
`aliquoted and stored at 280uC. EDTA-whole blood was used for
`immediate hematological analyses.
`
`Hematological Analysis
`such as RBC count,
`Red blood cells
`(RBC) parameters
`hemoglobin level (HGB), hematocrit (HCT), mean cell volume
`(MCV), mean corpuscular hemoglobin (MCH), and mean
`corpuscular hemoglobin concentration (MCHC) were determined
`using an automated AVIDIA 2010 analyzer (Siemens, Germany).
`
`Plasma Iron Parameters
`iron binding capacity
`Plasma iron concentration and total
`(TIBC) were determined by colorimetric measurement of the
`absorbance of the iron-chromazurol complex at 630 nm (Alpha
`Diagnostic, Poland). Percent of transferrin (Tf) saturation by iron
`was calculated according to the following formula: TSAT = [-
`plasma iron/TIBC] 6 100.
`
`Plasma Hepcidin-25 Quantification
`Piglet plasma hepcidin-25 measurements were performed by a
`combination of weak cation exchange chromatography and time-
`of-flight mass
`spectrometry (WCX-TOF MS), as described
`
`previously for human plasma samples [17], [18], [19]. Peptide
`spectra were generated on a Microflex LT matrix-enhanced laser
`desorption/ionisation TOF MS platform (BrukerDaltonics). When
`piglet samples were applied, this procedure yielded a peak with
`mass/charge ratio (m/z) of 2750, which corresponds to the
`theoretical mass of pig hepcidin-25 (2749.4 Da), assuming that 4
`intra-molecular disulphide bridges are present as is the case for all
`other known hepcidin molecules (Figure 2). The identity of this
`peak was further confirmed by its specific disappearance from the
`mass spectrum by pre-incubation of piglet plasma samples with
`anti-hepcidin molecules (data not shown). A synthetic human
`hepcidin-25 peptide (Peptide International Inc.) was used as
`internal standard for quantification. Piglet plasma hepcidin-25
`concentrations were expressed as nmol/L (nM). The lower limit of
`detection of this method was 1 nM.
`
`Statistical Analysis
`Data are presented as mean values 6 SD. Statistical analysis of
`results was performed using one-way analysis of variance (one-way
`ANOVA). The significance of differences was verified by Tukey’s
`test using Statgraphics 5.1 program (Manugistics, USA). Statisti-
`cally significant differences between parameters of piglets from
`different groups on a given day of experiment were denoted by
`
`Figure 2. Pig Hepcidin-25 quantification by mass spectrometry. Hepcidin-25 measurements in piglet plasma were performed by peptide
`enrichment through weak cation exchange chromatography coupled to time-of-flight mass spectrometry (WCX-TOF MS). Note that quantification is
`based on the relative intensity of the piglet hepcidin peak with mass/charge ratio (m/z) of 2750 to that of the synthetic internal standard (IS) that is
`spiked in the know concentration of 10 nM to each sample prior to sample work-up. The four spectra illustrate the appearance of hepcidin-25 upon
`34 at days 3 (baseline), 14, 21 and 28.
`iron injection in group A, pig
`doi:10.1371/journal.pone.0064022.g002
`
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`Plasma Hepcidin Level in Iron Supplemented Piglets
`
`different capital and small
`respectively.
`
`Results and Discussion
`
`letters at P#0.01 and P#0.05,
`
`.74A
`
`6 1
`
`.36a
`
`6 1
`
`.62
`
`6 1
`
`.56
`
`6 0
`
`.24A
`
`6 3
`
`.11A
`
`6 2
`
`27.70
`
`28.22
`
`27.98
`
`29.10
`
`13,39
`
`15,21
`
`.88B
`
`6 0
`
`.62ab
`
`6 0
`
`.54
`
`6 0
`
`.47
`
`6 0
`
`.23B
`
`6 1
`
`.63B
`
`6 1
`
`29.62
`
`28.71
`
`27.88
`
`28.16
`
`18,29
`
`18,97
`
`.11B
`
`6 1
`
`.22b
`
`6 1
`
`.04
`
`6 1
`
`.70
`
`6 1
`
`.37B
`
`6 1
`
`.41B
`
`6 1
`
`30,78
`
`29,70
`
`28,94
`
`28,33
`
`19,42
`
`20,16
`
`28
`
`21
`
`14
`
`3
`
`28
`
`21
`
`MCHC(g/dL)
`
`.93A
`
`6 1
`
`.09
`
`6 1
`
`.02aA
`
`6 9
`
`.34aA
`
`6 5
`
`.06
`
`6 5
`
`.34
`
`6 3
`
`17,41
`
`19,30
`
`47.91
`
`53.74
`
`62.09
`
`66.37
`
`.46aA
`
`.62A
`
`6 1
`
`,86
`
`6 0
`
`.60bB
`
`6 4
`
`.86AB
`
`6 5
`
`.01
`
`6 6
`
`.70
`
`6 2
`
`.02aAB
`
`18,43
`
`19,70
`
`61.80
`
`66.14
`
`66.15
`
`69.88
`
`.72B
`
`6 1
`
`,80
`
`6 1
`
`.14bB
`
`6 4
`
`.15bB
`
`6 5
`
`.25
`
`6 6
`
`.39
`
`6 3
`
`20,96
`
`19,37
`
`63.11
`
`67.95
`
`72.46
`
`68.28
`
`.07bB
`
`14
`
`3
`
`28
`
`21
`
`14
`
`3
`
`MCH(pg)
`
`MCV(fL)
`
`To assess the effect of different iron supplementation strategies
`on the iron status and hepcidin levels in piglets, three different
`protocols were employed. The control protocol
`involved two
`injections (split supplementation) of piglets with 150 and 40 mg
`Fe/kg b.w. on day 3 and 21 postpartum, respectively (group A), is
`a routine iron therapy of piglets applied at the farm where the
`study was performed. Our modified supplementation based on the
`protocol
`recently described [10]
`involved two injections of
`37.5 mg Fe/kg b.w. on day 3 and 14 postpartum (group B).
`Piglets from the third group (group C) received 37.5 mg Fe/kg
`b.w. on day 3 only. As shown in Table 1, iron supplementation in
`group C was
`largely insufficient
`to maintain hematological
`parameters and plasma iron status (Figure 3) till the weaning
`(day 28). However, the rationale to include the latter group in this
`study was to determine how long this single injection could
`maintain the appropriate hematological status of piglets. Our
`results clearly show that after day 21,
`the values of all
`hematological parameters (except of RBC count) of piglets from
`group C were significantly lower compared with those form group
`A and B animals. Similarly, iron plasma parameters (Fe plasma
`level, TIBC and Tf saturation) indicated gradually developing iron
`deficiency in group C piglets. In contrast, RBC indices in animals
`from group A and B were in the range of standard piglet
`parameters in this age [22], [23]. Importantly, there were no
`differences in most hematological
`indices between piglets from
`group A and B throughout the experimental period. Significantly
`lower values in piglets from group B were recorded only for MCV
`on day 21 (P#0.01), HCT on day 28 (P#0.01) and MCHC on day
`21 (P#0.05) (Table 1). Similar hematological status of group A
`and B piglets strongly indicates that by applying a modified
`protocol with decreased amounts of
`supplemental
`iron, a
`satisfactory prophylactic effect preventing development of IDA
`in piglets can be achieved till weaning. This observation extends
`our previous results showing the efficacy of split supplementation
`with reduced amounts of iron till day 14 after birth [10].
`It is known that in iron deficiency, a drop of hematological
`parameters is preceded by a decrease in plasma iron level, Tf iron
`saturation and the increase in TIBC values [2]. This scenario was
`perfectly reproduced in group C piglets: decrease in plasma iron
`status recorded on day 14 resulted in a severe deterioration of
`hematological status on day 21. In group B piglets, the plasma iron
`status on day 14 was also found to be at the borderline of iron
`deficiency (note that at that time piglets from both groups received
`the same iron treatment), however, the second injection of iron to
`group B piglets just at that day restored efficiently plasma iron
`level and in consequence increased the values of most RBC
`indices.
`To assess the circulating levels of bioactive hepcidin in the
`circulation of the piglets, we developed and validated a novel mass
`spectrometry-based assay to quantifies hepcidin-25 in piglet
`plasma samples (see Material and Methods section for details).
`As shown in Figure 2, this methodology could detect the peptide
`peak that corresponds to pig hepcidin-25 of (with molecular weight
`of 2750 Dalton)
`in samples from group A piglets following
`injection of the high dose of iron, but not at baseline. In fact, the
`hepcidin-25 concentration in group A piglets showed in general a
`gradual increase throughout the experimental period (Figure 4). In
`contrast, the hepcidin-25 levels in plasma from group B and C
`piglets
`remained just above or below the lower
`limit of
`
`doi:10.1371/journal.pone.0064022.t001
`respectively,betweenparametersofpigletsfromdifferentgroupsonagivendayofexperiment.
`each)describedinthelegendtoFigure1.Hematologicalparametersweredeterminedfor9pigletsfromeachexperimentalgroup.DifferentcapitalandsmalllettersdenotesstatisticallysignificantdifferenceatP#0.01andP#0.05,
`RBC–redbloodcellscount,HGB-hemoglobinlevel,HCT-hematocrit,MCV-meancellvolume,MCH-meancorpuscularhemoglobin,MCHC-meancorpuscularhemoglobinconcentration.A,B,andC–experimentalgroups(n=9
`
`6 1
`
`.55
`
`6 1
`
`7.65
`
`.79
`
`6 0
`
`7.66
`
`.43
`
`6 1
`
`9.28
`
`7.47
`
`.79
`
`6 0
`
`5.94
`
`.75
`
`6 0
`
`5.82
`
`.72
`
`6 0
`
`5.93
`
`.59
`
`6 0
`
`5.17
`
`.80
`
`6 0
`
`5.26
`
`.78
`
`6 0
`
`5.19
`
`.76
`
`6 0
`
`4.85
`
`.59
`
`6 0
`
`4.44
`
`.59
`
`6 0
`
`4.45
`
`.91
`
`6 0
`
`3.98
`
`.41
`
`6 0
`
`3.90
`
`.72
`
`6 0
`
`3.86
`
`A
`
`B
`
`C
`
`14
`
`3
`
`28
`
`21
`
`14
`
`GroupQ3
`(days)
`AgeR
`
`HGB(g/dL)
`
`ParameterRBC(6106/mL)
`
`Table1.Hematologicalparameters(mean6SD)ofpigletssupplementedwithirondextranaccordingtovariousprotocols.
`
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`May 2013 | Volume 8 |
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`Issue 5 | e64022
`
`6 6
`
`.63a
`
`6 6
`
`.34
`
`6 5
`
`.30
`
`6 5
`
`28.41
`
`29.70
`
`30.12
`
`26.27
`
`.40
`
`6 2
`
`.22A
`
`6 2
`
`7.95
`
`.36B
`
`6 1
`
`.13A
`
`6 2
`
`7.92
`
`.31B
`
`6 1
`
`.82
`
`6 1
`
`8.8
`
`.88
`
`6 0
`
`27.18
`
`10.64
`
`9.94
`
`8.15
`
`.09
`
`6 4
`
`.59B
`
`6 1
`
`.41B
`
`6 1
`
`.18
`
`37.39
`
`35.10
`
`32.08
`
`26.22
`
`11.49
`
`10.41
`
`28
`
`21
`
`14
`
`3
`
`28
`
`21
`
`HCT(%)
`
`6 4
`
`.49b
`
`6 4
`
`35.84
`
`34.56
`
`6 5
`
`.94b
`
`6 4
`
`.43
`
`6 1
`
`7.47
`
`.40
`
`6 2
`
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`Plasma Hepcidin Level in Iron Supplemented Piglets
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`Plasma Hepcidin Level in Iron Supplemented Piglets
`
`Figure 3. Plasma iron parameters of piglets supplemented with iron dextran according to various protocols. A; plasma iron level. B;
`total iron binding capacity. C; iron transferrin saturation. Values are expressed as the means 6 S.D. Plasma iron parameters were determined for 9
`piglets from each experimental group. Different capital and small letters denote statistically significant difference at P#0.01 and P#0.05, respectively,
`between parameters of piglets from different groups on a given day of experiment.
`doi:10.1371/journal.pone.0064022.g003
`
`quantification (,1 nM) of our assay throughout the experimental
`period (Figures 4). The fact that we observe very low hepcidin
`plasma concentration measured in all 3 day-old piglets at baseline
`is surprising considering our previous finding of relatively high
`abundance of hepatic hepcidin mRNA levels in piglets of the same
`age, while no large increase in these liver mRNA levels were
`observed upon iron intervention [10]. This discrepancy may relate
`to an unknown hepcidin stimulus during early life in combination
`with immature hepatic hepcidin maturation and secretion
`pathways in these neonates [16] and shows the added value of
`actually assessing the active circulating peptide. On the other
`hand, the absence of circulating hepcidin-25 may relate to a
`relative low sensitivity of the mass spectrometry assay compared to
`mRNA quantification by real-time PCR in plasma from these
`young piglets, although this was not found for mouse bioactive
`hepcidin-25 [24]. Hepcidin concentration measured in plasma
`from group A piglets markedly increased after the first injection of
`high iron dose. Interestingly, in plasma of piglets supplemented
`with 37.5 mg Fe/kg b.w. on day 3 (group B and C), hepcidin-25
`was hardly detectable up to day 14. Moreover, even after the
`second injection of iron dextran to piglets from group B on day 14,
`plasma hepcidin-25 levels continued to be very low till weaning
`(day 28). Notably, our methodology to quantify piglet hepcidin-25
`yielded robust assay characteristics that were similar to those
`published for hepcidin-25 in human plasma samples [17]. For
`
`piglet hepcidin-25, intra-run coefficient of variations (CV) were
`4.4% at 4.1 nM and 12.4% at 1.9 nM (n = 7), and inter-run CVs
`were 6.3% at 3.3 nM and 7.4% at 4.8 nM (n = 8). Dilution
`linearity of the piglet hepcidin-25 assay is shown in Figure S1
`(R2 = 0.995).
`Our results open an interesting field for speculations regarding
`the relationship between iron supplementation, regulation of iron
`metabolism and hematological status. Obviously, injection of high
`amounts of iron rectifies neonatal IDA in pigs as exemplified in
`this study and many others [10], [11], [12], [13]. However, it is
`accompanied by the risk of excessive hepcidin synthesis, which in
`turn may impair both the utilization of
`supplemental
`iron
`deposited in RES macrophages and the absorption of dietary
`iron. Our results clearly show that it is possible to strongly reduce
`the amount of supplemental iron, and still maintain values of RBC
`indices at the proper level without inducing hepcidin-25 expres-
`sion. It is tempting to speculate that after injection of low doses of
`supplemental iron to piglets according to our modified protocol,
`iron is predominantly transferred to the bone marrow, where it
`ensures the correct course of erythropoiesis. In contrast, in piglets
`highly loaded with supplemental iron, iron is partially redistributed
`to hepatocytes, where it induces hepcidin synthesis. Hepatic iron is
`thought to induce hepcidin expression via bone morphogenetic
`protein (BMP) signaling [25], [26]. On the other hand when serum
`iron levels are low, transferrin receptor 1 (TfR1) sequesters HFE
`
`Figure 4. Hepcidin concentration in blood plasma of piglets supplemented with iron dextran according to various protocols. Values
`are expressed as the means 6 S.D. Hepcidin concentration was determined for 5–7 piglets from each group/day. Different capital and small letters
`denotes statistically significant difference at P#0.01 and P#0.05, respectively, between parameters of piglets from different groups on a given day of
`experiment.
`doi:10.1371/journal.pone.0064022.g004
`
`PLOS ONE | www.plosone.org
`
`6
`
`May 2013 | Volume 8 |
`
`Issue 5 | e64022
`
`PGR2020-00009
`Pharmacosmos A/S v. American Regent, Inc.
`Petitioner Ex. 1081 - Page 6
`
`

`

`on hepatocytes, preventing its interaction with transferrin receptor
`2 (TfR2), which is necessary for signaling to hepcidin. As serum
`iron saturation increases, HFE is displaced from its overlapping
`binding site on TfR1 by holo-Tf. HFE is then freed to interact
`with TfR2 and to signal for the increased production of hepcidin
`[27,28]. Accordingly, after the first iron injection group A piglets
`maintain in parallel permanent high plasma iron levels, high Tf
`iron saturation and high hepcidin expression, which is then
`intensified by the second injection of iron dextran on day 21. An
`opposite relationship between iron plasma status and hepcidin
`levels occurs in iron deficient piglets from group C. Piglets from
`group B show an intermediate plasma iron status, which is
`sufficient to fuel erythropoiesis but does not reach a threshold
`indispensable for increasing plasma hepcidin levels.
`Although our studies [10] demonstrate evident benefits of the
`modified protocol of split supplementation of newborn piglets with
`iron dextran, its usefulness in the swine industry may be limited
`because of the necessity for a second iron dextran injection, which
`makes
`this procedure labour-consuming and expensive. Our
`present results provide a molecular background for planning mixt
`parenteral/oral iron supplementation in newborn piglets. It seems
`that early (on day 3) bolus parenteral supplementation with
`reduced amount of iron dextran is indispensable because of the
`extreme iron deficiency in newborn piglets and the high iron
`demand for erythropoiesis. Keeping in mind that the injection to
`piglets of ,40 mg Fe/kg b.w. does not induce hepcidin, we
`hypothesize that the second phase of iron supplementation with
`dietary iron starting on day 7–10 of life is an appropriate way to
`satisfy the iron requirements of piglets more physiologically. We
`have recently demonstrated that in piglets after day 4 postpartum,
`
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`Plasma Hepcidin Level in Iron Supplemented Piglets
`
`the two duodenal iron transporters – DMT1 and ferroportin are
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`
`Supporting Information
`
`Figure S1 Dilution linearity of the piglet hepcidin-25
`assay. A piglet plasma sample containing 8.3 nM (sample
`content 1.00) Hepcidin-25 was diluted 2, 3, 5 and 10 times in
`binding buffer
`(sample content 0.50, 0.33, 0.20 and 0.10,
`respectively) and applied to WCX-TOF MS. The measured
`hepcidin-25 values are indicated in nM. Dilution linearity:
`R2 = 0.995.
`(TIF)
`
`Table S1
`(TIF)
`
`Author Contributions
`
`Conceived and designed the experiments: RRS PL MP. Performed the
`experiments: RRS CMML HT MP AS. Analyzed the data: RRS DWS PL.
`Contributed reagents/materials/analysis
`tools: RRS DWS MP MM.
`Wrote the paper: RRS CMML HT DWS PL.
`
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