The influence of lactoferrin, orally administered, on systemic iron homeostasis in pregnant women suffering of iron deficiency and iron deficiency anaemia

Available online at www.sciencedirect.com

Biochimie 91 (2009) 44e51 www.elsevier.com/locate/biochi

Review

The influence of lactoferrin, orally administered, on systemic iron homeostasis in pregnant women suffering of iron deficiency and iron deficiency anaemia Rosalba Paesano a, Miriam Pietropaoli b, Sandra Gessani c, Piera Valenti d,* a

Department of Obstetrician and Gynecology, Sapienza, University of Rome, Rome, Italy b Microbo srl Biotechnology Company, Rome, Italy c Istituto Superiore di Sanita’, Department of Cell Biology and Neurosciences, Rome, Italy d Department of Public Health Sciences, Sapienza, University of Rome, Rome, Italy Received 14 March 2008; accepted 6 June 2008 Available online 14 June 2008

Abstract Iron is a fundamental element for humans as it represents an essential component of many proteins and enzymes. However, this element can also be toxic when present in excess because of its ability to generate reactive oxygen species. This dual nature imposes a tight regulation of iron concentration in the body. In humans, systemic iron homeostasis is mainly regulated at the level of intestinal absorption and, until now, no regulated pathways for the excretion of iron have been found. The regulation and maintenance of systemic iron homeostasis is critical to human health. Excessive iron absorption leads to iron-overload in parenchyma, while low iron absorption leads to plasma iron deficiency, which manifests as hypoferremia (iron deficiency, ID) and ID anaemia (IDA). ID and IDA are still a major health problem in pregnant women. To cure ID and IDA, iron supplements are routinely prescribed. The preferred treatment of ID/IDA, consisting in oral administration of iron as ferrous sulphate, often fails to exert significant effects on hypoferremia and may also cause adverse effects. Lactoferrin (Lf), an iron-binding glycoprotein abundantly found in exocrine secretions of mammals, is emerging as an important regulator of systemic iron homeostasis. Recent data suggest that this natural compound, capable of interacting with the most important components of iron homeostasis, may represent a valuable alternative to iron supplements in the prevention and cure of pregnancy-associated ID and IDA. In this review, recent advances in the molecular circuits involved in the complex cellular and systemic iron homeostasis will be summarised. The role of Lf in curing ID and IDA in pregnancy and in the maintenance of iron homeostasis will also be discussed. Understanding these mechanisms will provide the rationale for the development of novel therapeutic alternatives to ferrous sulphate oral administration in the prevention and cure of ID and IDA. Ó 2008 Elsevier Masson SAS. All rights reserved. Keywords: Lactoferrin; Iron; Hypoferremia; Anaemia; Pregnancy

1. Iron homeostasis and disorders Abbreviations: Lf, lactoferrin; ID, iron deficiency; IDA, iron deficiency anaemia; ROS, reactive oxygen species; DMT1, divalent metal transporter 1; Tf, transferrin; TfR, transferrin receptor; DCYTB, duodenal cytochrome B; FPN, ferroportin; IRP, iron regulatory proteins; IRE, iron regulatory elements; STAT, signal transducer and activator of transcription; IL, interleukin. * Corresponding author. Department of Public Health Sciences, Sapienza, University of Rome, Piazzale Aldo Moro, 5, 00185 Rome, Italy. Tel.: þ39 335 540 3965; fax: þ39 06 4991 4626. E-mail address: [email protected] (P. Valenti). 0300-9084/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2008.06.004

Iron is an essential element for living cells, owing to its ability to gain and loss electrons. However, iron can also be toxic when present in excess because of its capacity to don electrons to oxygen, thus causing the generation of reactive oxygen species (ROS), such as superoxide anions and hydroxyl radicals. Hydroxyl radicals are known to cause tissue injury and organ failure by damaging a number of cellular

R. Paesano et al. / Biochimie 91 (2009) 44e51

components, including DNA, proteins and membrane lipids. Accordingly, all organisms have developed strategies that allow acquiring, binding and storing elemental iron in a nontoxic, readily available form. In this respect, lactoferrin (Lf) in the secretions and transferrin (Tf) in the serum, ensure that the proper 1018 M free iron concentration in human fluids is maintained, thus avoiding iron precipitation, ROS induction and microbial colonisation [1,2]. The total body iron, about 3 g in women and 4 g in men, is mainly incorporated as haemic-iron in the haemoglobin, myoglobin and cytochromes (2e2.7 g), and as non-haemic form in various enzymes. Every day 20 mg of iron, derived primarily from lyses of senescent erythrocytes, are utilised for the de novo synthesis of haem. The systemic iron homeostasis is tightly regulated through iron absorption and storage. Absorption of nearly all dietary iron (1e2 mg daily) takes place in the proximal duodenum and includes the following steps: (i) reduction of iron from the ferric state (III) to the ferrous state (II); (ii) apical uptake by enterocytes followed by transcellular trafficking; and (iii) basolateral efflux by the ferrous iron transporter ferroportin (FPN). The fate of iron absorbed by enterocytes can be either storage as ferritin followed by excretion in the faeces when the senescent erythrocytes are sloughed, or plasma transfer. Once in the portal circulation iron binds to transferrin (Tf) and it is transported to sites of use (erythroid precursors) and storage (hepathocytes) [3]. Excessive iron absorption results into iron-overload in parenchymal tissues, while low iron absorption leads to plasma iron deficiency, which manifests as hypoferremia (iron deficiency, ID) and iron deficiency anaemia (IDA). ID is the most common nutritional deficiency, afflicting about two billion of people in the world, identified by the World Health Organization as one of the 10 risk factors for illness, disability and death. In pregnancy, ID and IDA represents a high risk factor for maternal and infant health that includes preterm delivery, foetal growth retardation, low birth weight, and inferior neonatal health. These pregnancy complications are thought to occur as a consequence of an increased iron requirement, related to enhanced blood volume and development of foetaleplacenta unit [4]. 2. Mechanisms supervising cellular and systemic iron homeostasis Iron concentrations and fluxes are regulated at both the cellular and the systemic level. 2.1. Cellular iron homeostasis The great majority of body cells take up iron via binding holo-transferrin (iron-saturated Tf), present in extracellular fluids and plasma, to the Tf receptor 1 and 2 (TfR1 and TfR2), with subsequent endocytosis [5]. TfR1 is expressed at high levels on erythroid precursors but also on virtually all dividing cells, while TfR2 is predominantly expressed in liver cells and binds holo-transferrin at lower affinity than TfR1. Both diferric (Fe2Tf) and monoferric (FeTf) transferrin

45

traffic into endosomes where the low pH strips the iron from the receptor-ligand complex. The TfR1-Tf is cycled back to the plasma membrane, where at neutral pH, the iron-free Tf is released back to plasma and TfR1 becomes available for the next cycle of iron uptake. In the endosomes, the released ferric ions are reduced by a ferrireductase to ferrous ions, which are then transported across the endosomal membrane into cytoplasm via divalent metal transporter 1 (DMT1) [6]. In the cytoplasm, a portion of the iron is stored by cytoplasmic ferritin, a protein composed of 24 subunits endowed with ferroxidase activity, able to harbour up to 4500 iron atoms per molecule as oxy-hydroxide micelles [7]. Cell iron levels modulate the synthesis of ferritin, TfR1 and DMT1 through transcriptional and post-transcriptional mechanisms. Iron regulatory proteins 1 and 2 (IRP-1 and -2) bind to iron regulatory elements (IRE) in mRNAs encoding proteins involved in cellular iron homeostasis [8]. The binding of IRPs has opposite effects on protein synthesis depending on the location of the target IRE: binding of IRPs to 50 IRE mRNAs inhibits translation thus decreasing protein synthesis, while binding to 30 IRE stabilises mRNA, resulting in increased protein synthesis [9e11]. In iron-deficient cells, ferritin concentration decreases through turnover, and TfR1 concentration increases, while in iron-loaded cells ferritin concentration increases and TfR1 synthesis decreases. Therefore, cellular iron homeostasis is maintained via the IRP/IRE system by modifying the production of proteins involved in cellular iron uptake and storage, according to the cytoplasmic iron concentrations. 2.2. Systemic iron homeostasis Differently from cellular iron homeostasis, systemic iron regulation involves complex mechanisms that only recently have started to be characterised. Different cell types possess distinct pathways for iron acquisition, while all cells have only a unique pathway for iron export into blood. As regard to different cell types, the duodenal enterocytes are involved in dietary iron acquisition, hepatocytes are the main iron store, macrophages recycle iron from the lysis of senescent red blood cells, and finally, the placental syncytiotrophoblast is engaged in iron transport from mother to foetus across placenta [8,12]. Hepatocytes, placental cells, and macrophages import the Tfbound iron from circulation using TfRs, while enterocytes can acquire iron present in food as haem or elemental iron. Haem is imported into the intestine by unknown mechanisms, while elemental Fe(III) is converted on the apical part of duodenal cells to Fe(II) by a ferrireductase (duodenal cytochrome B, DCYTB) and transported into the cells by DMT1 [6]. Interestingly, it has been demonstrated that DMT1 is required for intestinal iron and other divalent metals (zinc, manganese, cobalt, cadmium, copper, nickel) transport or iron delivery to erythroid precursors, while it is not crucial for maternal iron transfer across the placenta to the foetus [13]. Iron absorbed by DMT1 enters the cytosol of enterocytes where it can be stored in the ferritin or exported into plasma by the basolateral iron exporter FPN. This protein, the only known cellular iron exporter from tissues into blood, has been found in all cell

46

R. Paesano et al. / Biochimie 91 (2009) 44e51

types that are involved in iron export, including the duodenal cells, hepatocytes, macrophages, and placental cells [14]. Tissue-specific deletion of the FPN gene causes iron accumulation in enterocytes, hepatocytes and above all in macrophages, which require FPN to efficiently recycle iron from lysed erythrocytes [15]. FPN has a molecular weight of 67 kDa and 12 putative transmembrane domains and seems to function as a dimmer [16]. The synthesis of FPN is modulated by transcriptional and post-transcriptional mechanisms. IRE/IRP regulate FPN mRNA. FPN transports Fe(II), which is converted to Fe(III) by multicopper oxidases, including ceruloplasmin and hephaestin, using oxygen as an electron acceptor [17,18]. In the absence of a ferroxidase, iron does not get exported through FPN. As a matter of fact, subjects carrying mutations in ceruloplasmin gene show iron-overload in parenchymal tissues and, in the absence of hephaestin, iron remains within the mucosa [19]. The iron transport across the enterocytes and its export into the blood by FPN are shown in Fig. 1. Another pivotal component of systemic iron metabolism is hepcidin, a circulating peptide hormone synthesised by hepatocytes and secreted in plasma and urine. It has been reported that hepcidin mRNA is also present in the heart, pancreas and

haematopoietic cells, although the biological relevance of this expression is not understood. The active form of hepcidin, derived from an 84-amino-acid precursor, is a 25-amino-acid peptide containing eight cystein residues. The bioactive human hepcidin is a 25-amino-acid peptide first identified in human urine [20] and plasma [21]. The urine also contains minor 20- and 22-amino-acid forms truncated at the Nterminus [22]. Hepcidin regulates the entry of iron into plasma through FPN [23,24]. Hepcidin, by binding to FPN, causes FPN phosphorylation, internalisation and degradation in lysosomes [25,26], thus preventing iron export and enhancement of cytosolic iron stored in ferritin. The influence of hepcidin concentration on iron export from cells into the blood is shown in Fig. 2. Therefore, increased expression of hepcidin leads to IDA [27,28]. The expression of hepcidin can be regulated in several ways [29]. Hypoxic conditions decrease hepcidin expression, resulting in increased iron export into the plasma [27]. Moreover, hepcidin production is also suppressed when iron stores are low, and consequently, FPN is displayed on basolateral membranes of enterocytes allowing iron transport to plasma. On the contrary, when iron stores are adequate or high or when oral or parenteral iron is loaded, the liver produces hepcidin, which circulates to the small intestine thus hindering iron export to circulation [27]. Similarly, in macrophages recycling senescent red blood cells, hepcidin-induced degradation of FPN results in iron trapping by macrophages. Moreover, hepcidin synthesis can be markedly induced by inflammatory status. In particular, interleukin-6 (IL-6) and probably other cytokines, induce transcription of the hepcidin gene in hepatocytes [30]. Transcriptional activation occurs in response to the binding of signal transducer and activator of transcription-3 (STAT3) to the hepcidin gene promoter. Increased hepcidin gene expression and consequent decrease in FPN levels result in decreased plasma iron concentration. Hypoferremia in this instance is characterised by iron retention in the intestinal mucosa and in macrophages. The inability to export iron leads to a decreased pool of serum transferrinFe(III) and iron-limited erythropoiesis. Indeed, iron and inflammation have both been reported to suppress FPN mRNA expression independently of hepcidin [31e33]. Therefore, the dysregulation of hepcidin, FPN, ceruplasmin or hephaestin results in a spectrum of iron disorders [13]. In inflammatory disorders and infections, cytokine-induced hepcidin up-modulation contributes to the development of anaemia of inflammation, characterised by hypoferremia and anaemia despite adequate iron stores [34]. 3. Hypoferremia and iron deficiency anaemia in pregnancy

Fig. 1. Iron transport across the duodenal enterocyte and its export into the blood by ferroportin. Ferric ion is reduced to ferrous ion by a ferroreductase (DCYTB) located in the apical side of enterocytes. Ferrous ion is transported in duodenal enterocytes by divalent metal transporter (DMT1) and then sequestered by ferritin. The ferrous ion is exported from enterocytes into the blood by ferroportin. Ferrous ion is oxidised to ferric ion by a membrane-associated ferroxidase, hephaestin.

Pregnancy is characterised by an increased iron requirement, due to enhanced blood volume and development of foetaleplacenta unit [35]. Hypoferremia and anaemia are still prevalent among pregnant women in both developing and industrialised countries, and represent an important risk factor for maternal and infant health. However, the degree of foetal ID is not always as severe as that in mother [36].

R. Paesano et al. / Biochimie 91 (2009) 44e51

47

Fig. 2. Hepcidin regulates iron export from cells into the blood. When hepcidin concentration is low, iron is exported from cells into the blood by ferroportin. When hepcidin concentration is high, hepcidin binds to ferroportin and induces its internalisation and degradation thus inhibiting iron export from cells to the blood.

Iron transfer from the mother to the foetus is supported by a substantial increase in maternal iron absorption during pregnancy and is regulated by the placenta [37,38]. Most iron transfer to the foetus occurs after the 30th week of gestation and likely involves placental expression of those proteins known to mediate systemic iron homeostasis. The placental syncytiotrophoblast acquires ferric iron bound to transferrin at the apical membrane through TfR-1 [11]. The synthesis of TfR-1 increases in pregnant women suffering of ID and IDA [38]. FPN is found on the placental basal foetal-facing membrane, consistent with unidirectional motherefoetus iron transport [39]. Ferritin is strongly expressed in the stroma and contributes to iron accumulation in foetal tissues [39]. In contrast, maternal serum ferritin usually markedly decreases between 12th and 25th week of gestation, probably as a result of iron utilisation for expansion of the maternal red blood cell mass. Concomitantly with the enhanced placentalefoetal iron transport, controlled by foetal hepcidin, an increased

expression of placental FPN is also observed. Therefore, similarly to the regulation of systemic iron homeostasis, high levels of hepcidin could induce internalisation and degradation of placental FPN, thus decreasing iron transport to foetus. Interestingly, it has been reported that hepcidin synthesis increases during inflammation, mostly due to the stimulatory effect of IL-6 on hepcidin promoter. The enhanced hepcidin production leads to a decrease in plasma iron level, thus contributing to hypoferremia and anaemia induction. In this respect, the level of pro-inflammatory cytokines, including IL-6, synthesised by peripheral blood cells in uncomplicated pregnancy and in unexplained recurrent spontaneous abortion has been compared [40]. In this study, significantly increased levels of pro-inflammatory cytokines were found in women undergoing unexplained recurrent spontaneous abortion as compared to normal pregnant women at similar stages of pregnancy. Furthermore, it has been demonstrated that significantly higher levels of IL-6 (range 0e77 pg/ml) are present in the

R. Paesano et al. / Biochimie 91 (2009) 44e51

48

plasma of patients with preeclampsia as compared to healthy pregnant women (0e19 pg/ml) [41]. Overall, these data strongly suggest that an increased expression of pro-inflammatory cytokines, including IL-6, occurring during pregnancy could play a role in the establishment of systemic iron homeostasis disorders, such as ID and IDA. In light of these observations, the treatment of ID and IDA during pregnancy and postpartum should be entirely reconsidered taking into account not only the enhanced blood volume and development of foetaleplacenta unit, but also the pivotal role that factors regulating iron transport from tissues to blood, i.e. FPN and hepcidin, might play in these events. 4. Oral administration of lactoferrin in pregnant women suffering of ID and IDA The most used treatment for ID and IDA currently consists in oral administration of iron as ferrous sulphate. However, ferrous sulphate administration often fails to exert any significant effects on these pregnancy-associated pathologies, and frequently causes several adverse effects (gastrointestinal discomfort, nausea, vomiting, diarrhoea, constipation). This is likely due to the poor bio-availability of inorganic iron requiring the administration of large quantity of ferrous sulphate [42,43]. Lf, a prominent protein in milk, many other secretory fluids and white blood cells, is a monomeric 80-kDa glycoprotein, synthesised by exocrine glands and by neutrophils at infection and inflammation sites. The molecule is folded into homologous N- and C-terminal lobes, each comprising two domains that enclose a conserved iron-binding site [44]. Lf by virtue of its iron-binding ability, ensures that free iron concentration in human secretions does not exceed 1018 M, thus preventing ROS formation and inhibiting microbial growth. Although Lf iron-binding at high affinity (KD w10/20 M) is, without any doubt, a key property of this protein, and accounts for some of its many biological roles in host defence [2], it is now clear that Lf exhibits other functions besides iron sequestration, such as a strong capacity to modulate the inflammatory response. Lf, by virtue of its capacity to reduce pro-inflammatory cytokine expression in vivo, including IL-6 [45], might represent a novel and promising natural compound to be used in the prevention and cure of pregnancy-associated ID and IDA. In this

respect, we have recently reported that oral administration of bovine Lf could represent an effective therapy in preventing and curing ID and IDA in uncomplicated pregnancy [46]. In this study, representing the first clinical trial on the therapeutic effect of bovine Lf on systemic iron homeostasis, we have clearly demonstrated the efficacy of this natural compound in rescuing iron homeostasis in pregnant women suffering of ID and IDA. Independently of pregnancy trimester, oral administration of 100 mg of Lf (about 30% iron-saturated) twice a day before the meal, increased the total serum iron and haemoglobin concentrations at a greater extent than that observed after oral administration of ferrous sulphate. In contrast to the administration of ferrous sulphate, Lf oral administration did not result in any side effect [46]. The potent effect exhibited by Lf as compared with ferrous sulphate was neither due to the amount of iron upplied by Lf (8.8 mg/day), lower than that provided by ferrous sulphate (156 mg/day) [46], nor to enhanced iron absorption, equally well absorbed from Lf and ferrous sulphate [47]. As shown in Table 1, in a further clinical trial involving 143 pregnant women suffering of ID and IDA, we found that oral administration of Lf also increases the number of red blood cells and serum ferritin concentrations at a higher extent with respect to orally administered ferrous sulphate. In order to gain further information on the therapeutic potentiality of orally administered Lf in the control of systemic iron homeostasis, we performed a study on five pregnant women suffering of ID and IDA. The enrolled subjects were treated for 30 days with Lf followed by further 30 days of ferrous sulphate treatment. The number of red blood cells, as well as haemoglobin, total iron, ferritin and IL-6 concentrations in serum were assayed. As shown in Table 2, all the blood parameters tested were found to be increased after Lf therapy, while these values decreased already after 30 days of ferrous sulphate treatment. Concerning IL-6, its concentration significantly decreased after 30 days of Lf treatment, while newly increased after 30 days of ferrous sulphate treatment. In keeping with these results, an inflammatory response to oral ferrous sulphate administration has been also observed in healthy volunteers receiving 120 mg of iron per day [42]. High levels of IL-6 well correlated with low values of total serum iron and serum ferritin. Conversely, low levels of IL-6 detected after Lf treatment well related with increased values of total serum iron and serum ferritin.

Table 1 Haematological values of 143 pregnant women suffering of ID and IDA untreated or treated with lactoferrin or ferrous sulphate orally administered for 30 days Pregnant women (33)

P

Refusing therapy

Red blood cells (mmc) Haemoglobin (g/dl) Total serum iron (mg/dl) Serum ferritin (ng/ml)

Time 0

After 30 days

3.869.000 11.6 52 11

3.688.000 10.9 32 6

Pregnant women (60)

P

Treated with lactoferrin

0.0940 0.0042 0.0017 0.0066

Time 0

After 30 days

3.786.000 11.5 51 12

4.045.000 12.6 96 27

All the values are expressed as mean values. Statistical analysis was performed using ANOVA test.

Pregnant women (50)

P

Treated with ferrous sulphate

<0.0001 <0.0001 <0.0001 <0.0001

Time 0

After 30 days

3.6760.00 10.9 44 5

3.626.000 11.9 51 3

0.56 <0.0001 0.51 0.0008

R. Paesano et al. / Biochimie 91 (2009) 44e51

49

Table 2 Haematological values of five pregnant women suffering of ID and IDA treated with lactoferrin for 30 days, followed by 30 days of ferrous sulphate treatment PW

1 2 3 4 5

Red blood cells 103/mmc

Haemoglobin (g/dl)

T0

T30

T60

T0

T30

T60

T0

T30

T60

T0

T30

T60

T0

T30

T60

3.000 3.450 3.250 2.900 3.340

3.500 4.200 3.900 3.500 4.400

3.600 4.100 3.800 3.600 3.300

9.5 10.8 10.0 10.8 11.0

10.5 11.4 10.5 11.5 11.9

10.4 11.2 10.6 11.2 11.5

68 45 56 54 35

110 70 95 90 70

70 30 30 34 30

12 10 6 12 13

30 18 9 26 18

15 10 3 10 10

19 25 33 21 28

10 12 15 10 17

18 29 35 27 32

Total serum iron (mg/dl)

Serum ferritin (ng/ml)

IL-6 (pg/ml)

PW ¼ pregnant women. T0 corresponds to the beginning of the treatment, T30 corresponds to 30 days of lactoferrin treatment and T60 corresponds to the following 30 days of ferrous sulphate treatment.

5. Conclusions The result of the clinical trials carried out in pregnant woman suffering of ID and IDA unravelled the strong therapeutic potential of bovine Lf, indicating that this natural compound represents an efficient alternative to ferrous sulphate therapy. As a matter of fact, iron supplementation with ferrous sulphate only restores haemoglobin concentration [6], while does not significantly increase the number of red blood cells, total serum iron and serum ferritin (Table 1). The failure of ferrous sulphate therapy in increasing total serum iron concentration indicates that this therapeutic regiment fails in restoring iron transport from tissues to blood, differently from that observed after Lf therapy. These intriguing results support the idea that Lf can modulate systemic iron homeostasis independently of the concentration of Lf-bound iron, which is considerably lower than that supplied by ferrous sulphate [46]. It is now well established that systemic iron homeostasis is mainly regulated by iron absorption and inflammation. Concerning iron absorption, Lf and ferrous sulphate

exhibit a similar capacity in iron absorption [47]. However, a relevant difference resides in the fact that while Lf significantly reduces IL-6 concentration in serum of pregnant woman suffering of ID and IDA, ferrous sulphate rather exhibits an opposite effect (Table 2). The recent advances on the role of inflammation in iron homeostasis, as well as of FPN in iron transport from tissues to plasma, may provide an explanation for the failure of the classical therapy with ferrous sulphate in restoring physiological concentrations of total serum iron and serum ferritin. Under inflammatory conditions, FNP expression at the cell surface might be negatively regulated by its interaction with hepcidin [31e33] as well as by other factors, thus impairing iron release into plasma in patients treated with ferrous sulphate. Since hepcidin synthesis is under the control of various factors, including IL-6, and IL-6 is increased in pregnancy, the hepcin-IL-6 axis could represent another key molecular link between inflammation and hypoferremia/anaemia. Although a correlation between hepcidin serum levels and haematological parameters characterising ID and IDA has not been found yet [48,49], the capacity of

Fig. 3. A putative interplay of lactoferrin with key proteins of systemic iron homeostasis. Lactoferrin, orally administered, in pregnant women suffering of iron deficiency anaemia (hypoferremia) decreases. IL-6 thus restoring iron export from cells into the blood by ferroportin.

50

R. Paesano et al. / Biochimie 91 (2009) 44e51

Lf to rescue systemic iron homeostasis through factors as hepcidin, other than IL-6 and FPN, cannot be excluded. A putative interplay of lactoferrin with key proteins of systemic iron homeostasis is illustrated in Fig. 3. In conclusion, these clinical trials demonstrate that oral administration of Lf exerts a potent effect to counteract ID and IDA in pregnant women. We speculate that this effect could be due to the capacity of Lf to decrease pro-inflammatory cytokines leading to the rescue of haematological parameters and reduction of adverse pregnancy outcomes in women suffering of ID and IDA.

References [1] J.J. Bullen, H.J. Rogers, P.B. Spalding, C.G. Ward, Iron and infection: the heart of the matter, FEMS Immunol. Med. Microbiol. 43 (2005) 325e330. [2] P. Valenti, G. Antonini, Lactoferrin: an important host defence against microbial and viral attack, Cell Mol. Life Sci. 62 (2005) 2576e2587. [3] T. Ganz, E. Nemeth, Regulation of iron acquisition and iron distribution in mammals, Biochim. Biophys. Acta. 1763 (2006) 690e699. [4] T.H. Bothwell, Iron requirements in pregnancy and strategies to meet them, Am, J. Clin. Nutr. 72 (2000) 257e264. [5] P. Aisen, Transferrin receptor 1, Int. J. Biochem. Cell. Biol. 36 (2004) 2137e3. [6] B. Mackenzie, M.D. Garrick, Iron imports: II. Iron uptake at the apical membrane in the intestine, Am. J. Physiol. Gastrointest. Liver Physiol. 289 (2005) G981eG986. [7] E.C. Theil, Ferritin: at the crossroads of iron and oxygen metabolism, J. Nutr. 133 (2003) 1549e1553. [8] M.W. Hentze, M.U. Muckenthaler, N.C. Andrews, Balancing acts: molecular control of mammalian iron metabolism, Cell 117 (2004) 285e297. [9] J.L. Casey, M.W. Hentze, D.M. Koeller, S.W. Caughman, T.A. Rouault, R.D. Klausner, J.B. Harford, Iron-responsive elements: regulatory RNA sequences that control mRNA levels and translation, Science 240 (1988) 924e928. [10] E.A. Leibold, H.N. Munro, Cytoplasmic protein binds in vitro to a highly conserved sequence in the 50 untranslated region of ferritin heavy- and light-subunit mRNAs, Proc. Natl. Acad. Sci. U.S.A. 85 (1988) 2171e2175. [11] E.W. Mullner, L.C. Kuhn, A stem-loop in the 30 untranslated region mediates iron-dependent regulation of transferrin receptor mRNA stability in the cytoplasm, Cell 53 (1988) 815e825. [12] N.C. Andrews, Molecular control of iron metabolism, Best. Pract. Res. Clin. Haematol. 18 (2005) 159e169. [13] I. De Domenico, M.D. Ward, J. Kaplan, Regulation of iron acquisition and storage: consequences for iron-linked disorders, Nat. Rev. Mol. Cell Biol. 9 (1) (2008) 72e81. [14] A. Donovan, C.A. Lima, J.L. Pinkus, G.S. Pinkus, L.I. Zon, S. Robine, N.C. Andrews, The iron exporter ferroportin Slc40a1 is essential for iron homeostasis, Cell Metab 1 (2005) 191e200. [15] X.B. Liu, F. Yang, D.J. Haile, Functional consequences of ferroportin 1 mutations, Blood Cells Mol. Dis. 35 (2005) 33e46. [16] I. De Domenico, D.M. Ward, G. Musci, J. Kaplan, Evidence for the multimeric structure of ferroportin, Blood 109 (2007) 2205e2209. [17] S. Osaki, D.A. Johnson, E. Frieden, The possible significance of the ferrous oxidase activity of ceruloplasmin in normal human serum, J. Biol. Chem. 241 (1966) 2746e2751. [18] H.P. Roeser, G.R. Lee, S. Nacht, G.E. Cartwright, The role of ceruloplasmin in iron metabolism, J. Clin. Invest. 49 (1970) 2408e2417. [19] Z.L. Harris, L.W. Klomp, J.D. Gitlin, Aceruloplasminemia: an inherited neurodegenerative disease with impairment of iron homeostasis, Am. J. Clin. Nutr. 67 (1998) 972Se977S.

[20] C.H. Park, E.V. Valore, A.J. Waring, T. Ganz, Hepcidin, a urinary antimicrobial peptide synthesized in the liver, J. Biol. Chem. 276 (2001) 7806e7810. [21] A. Krause, S. Neitz, H.J. Magert, A. Schulz, W.G. Forssmann, P. Schulz-Knappe, K. Adermann, LEAP-1, a novel highly disulfidebonded human peptide, exhibits antimicrobial activity, FEBS Lett. 480 (2000) 147e150. [22] H.N. Hunter, D.B. Fulton, T. Ganz, H.J. Vogel, The solution structure of human hepcidin, a peptide hormone with antimicrobial activity that is involved in iron uptake and hereditary hemochromatosis, J. Biol. Chem. 277 (2002) 37597e37603. [23] O. Loreal, C. Haziza-Pigeon, M.B. Troadec, L. Detivaud, B. Turlin, B. Courselaud, G. Ilyin, P. Brissot, Hepcidin in iron metabolism, Curr. Protein Pept. Sci. 6 (2005) 279e291. [24] T. Ganz, Hepcidinda regulator of intestinal iron absorption and iron recycling by macrophages, Best Pract. Res. Clin. Haematol. 18 (2005) 171e182. [25] I. De Domenico, D.M. Ward, C. Langelier, M.B. Vaughn, E. Nemeth, W.I. Sundquist, T. Ganz, G. Musci, J. Kaplan, The molecular mechanism of hepcidin-mediated ferroportin down-regulation, Mol. Biol. Cell 18 (2007) 2569e2578. [26] E. Nemeth, M.S. Tuttle, J. Powelson, M.B. Vaughn, A. Donovan, D.M. Ward, T. Ganz, J. Kaplan, Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization, Science 306 (2004) 2090e2093. [27] G. Nicolas, M. Bennoun, A. Porteu, S. Mativet, C. Beaumont, B. Grandchamp, M. Sirito, M. Sawadogo, A. Kahn, S. Vaulont, Severe iron deficiency anemia in transgenic mice expressing liver hepcidin, Proc. Natl Acad. Sci. U.S.A. 99 (2002) 4596e4601. [28] D.A. Weinstein, C.N. Roy, M.D. Fleming, M.F. Loda, J.I. Wolfsdorf, N.C. Andrews, Inappropriate expression of hepcidin is associated with iron refractory anemia: implications for the anemia of chronic disease, Blood 100 (2002) 3776e3781. [29] G. Nicolas, C. Chauvet, L. Viatte, J.L. Danan, X. Bigard, I. Devaux, C. Beaumont, A. Kahn, S. Vaulont, The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation, J. Clin. Invest. 110 (2002) 1037e1044. [30] E. Nemeth, S. Rivera, V. Gabayan, C. Keller, S. Taudorf, B.K. Pedersen, T. Ganz, IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hepcidin, J. Clin. Invest. 113 (2004) 1271e1276. [31] I. De Domenico, D.M. Ward, M.C. di Patti, S.Y. Jeong, S. David, G. Musci, J. Kaplan, Ferroxidase activity is required for the stability of cell surface ferroportin in cells expressing GPI-ceruloplasmin, EMBO J. 26 (2007) 2823e2831. [32] D.M. Frazer, S.J. Wilkins, E.M. Becker, T.L. Murphy, C.D. Vulpe, A.T. McKie, G.J. Anderson, A rapid decrease in the expression of DMT1 and Dcytb but not Ireg1 or hephaestin explains the mucosal block phenomenon of iron absorption, Gut. 52 (2003) 340e346. [33] E. Nemeth, G.C. Preza, C.L. Jung, J. Kaplan, A.J. Waring, T. Ganz, The N-terminus of hepcidin is essential for its interaction with ferroportin: structure-function study, Blood 107 (2006) 328e333. [34] E. Nemeth, T. Ganz, Regulation of iron metabolism by hepcidin, Annu. Rev. Nutr. 26 (2006) 323e342. [35] T.O. School, Iron status during pregnancy: setting the stage for mother and infant, Am. J. Clin. Nutr. 81 (2005) 1218e1222. [36] E.D. Harris, New insights into placental iron transport, Nutr. Rev. 50 (1992) 329e331. [37] Y. Cheng, O. Zak, P. Aisen, S.C. Harrison, T. Walz, Structure of the human transferrin receptor-transferrin complex, Cell 116 (2004) 565e576. [38] J. Bradley, E.A. Leibold, Z.L. Harris, J.D. Wobken, S. Clarke, K.B. Zumbrennen, R.S. Eisenstein, M.K. Georgieff, Influence of gestational age and fetal iron status on IRP activity and iron transporter protein expression in third-trimester human placenta, Am. J. Physiol. Regul. Integr. Comp. Physiol. 287 (2004) R894eR901. [39] J. Bastin, H. Drakesmith, M. Rees, I. Sargent, A. Townsend, Localisation of proteins of iron metabolism in the human placenta and liver, Br. J. Haematol. 134 (2006) 532e543.

R. Paesano et al. / Biochimie 91 (2009) 44e51 [40] R. Raghupathy, M. Makhseed, F. Azizieh, A. Omu, M. Gupta, R. Farhat, Cytokine production by maternal lymphocytes during normal human pregnancy and in unexplained recurrent spontaneous abortion, Hum. Reprod 15 (2000) 713e718. [41] M.D. Peter Takacs, L. Kermic, B.S. Green, N. Athip, W. Scott, Kauma Richmond V., Increased vascular endothelial cell production of interleukin-6 in severe preeclampsia, Am. J. Obstet. Gynecol. 188 (2003) 741e744. [42] K. Schu¨mann, T. Ettle, B. Szegner, B. Elsenhans, N.W. Solomons, On risks and benefits of iron supplementation recommendations for iron intake revisited, J. Trace Elem. Med. Biol. 21 (2007) 147e168. [43] M. Makrides, C.A. Crowther, R.A. Gibson, R.S. Gibson, C.M. Skeaff, Efficacy and tolerability of low-dose iron supplements during pregnancy: a randomized controlled trial, Am. J. Clin. Nutr. 78 (2003) 145e153. [44] E.N. Baker, H.M. Baker, Molecular structure, binding properties and dynamics of lactoferrin, Cell. Mol. Life Sci. 62 (2005) 2531e2539.

51

[45] F. Berlutti, S. Schippa, C. Morea, S. Sarli, B. Perfetto, G. Donnarumma, P. Valenti, Lactoferrin downregulates pro-inflammatory cytokines upexpressed in intestinal epithelial cells infected with invasive or noninasive Escherichia coli strains, Biochem. Cell Biol. 4 (2006) 351e357. [46] R. Paesano, F. Torcia, F. Berlutti, E. Pacifici, V. Ebano, M. Moscarini, P. Valenti, Oral administration of lactoferrin increases hemoglobin and total serum iron in pregnant women, Biochem. Cell Biol. 84 (2006) 377e380. [47] B. Lonnerdal, A. Bryant, Absorption of iron from recombinant human lactoferrin in young US women, Am. J. Clin. Nutr. 83 (2006) 305e309. [48] K.B. Hadley, L.K. Johnson, J.R. Hunt, Iron absorption by healthy women is not associated with either serum or urinary prohepcidin, Am. J. Clin. Nutr. 84 (1) (2006) 150e155. [49] M.L. Jacober, R.L. Mamoni, C.S. Lima, B.L. Dos Anjos, H.Z. Grotto, Anaemia in patients with cancer: role of inflammatory activity on iron metabolism and severity of anaemia, Med. Oncol. 24 (3) (2007) 323e329.