Bovine Lactoferrin to Prevent and Cure Iron Deficiency and Iron Deficiency Anemia in Complicated Pregnancies

In industrialized and developing countries, iron deficiency (ID) and iron deficiency anemia (IDA) are highly prevalent in pregnant women. ID and IDA, in pregnant women as a consequence of an increased iron requirement, due to enhanced blood volume and development of fetal-placenta unit, represent a high risk for maternal and infant health: preterm delivery, fetal growth retardation, low birth weight, and inferior neonatal health. However, the degree of fetal ID is not always as severe as that in mother, being iron transfer from the mother to the fetus regulated by the placenta. In particular, the placental syncytiotrophoblast acquires ferric iron bound to maternal transferrin at the apical membrane through transferrin receptors (TfR-1), which noticeably increase in pregnant women suffering of ID and IDA. Recently, it has been suggested that most iron transfer to the fetus, occurring after the 30th week of gestation, also involves placental expression of hepcidin and ferroportin, two proteins known to modulate systemic iron homeostasis in adults. As matter of fact, iron homeostasis is tightly regulated through iron absorption, storage and transport. The absorption of nearly all dietary iron (1-2 mg daily), ensuring iron supplies in the bone marrow, at second and third trimester of pregnancy increases until to about 4 and 8 mg/day, respectively. The iron absorption 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) by a ferrireductase (duodenal cytochrome B; (ii) apical uptake by enterocytes followed by trans-cellular trafficking via divalent metal transporter 1; (iii) storage into ferritin; and (iv) basolateral efflux by the iron transporter ferroportin. Ferroportin, the only known cellular iron exporter from tissues into blood, has been found in all cell types involved in iron export, including the enterocytes, hepatocytes, placental cells and macrophages which require ferroportin to daily recycle 20 mg of iron from lysed erythrocytes for erythropoiesis.

Another pivotal component of systemic iron homeostasis is hepcidin, a circulating peptide hormone synthesized by hepatocytes in iron loading conditions and secreted in plasma and urine. Hepcidin regulates the entry of iron into plasma through ferroportin. Hepcidin, by binding to ferroportin, causes ferroportin phosphorylation, internalization and degradation in lysosomes, thus hindering iron export and enhancing cytosolic iron storage in ferritin. Iron homeostasis disorders appear to arise from hepcidin and/or ferroportin dysregulation. Similarly to the regulation of maternal systemic iron homeostasis, fetal hepcidin controls the transfer of maternal iron across the placenta to the fetus and the enhanced placental-fetal iron transport is related to an increased expression of ferroportin on placental basal fetal-facing membrane, consistent with unidirectional mother-fetus iron transport. Even if the interaction of hepcidin with ferroportin can explain the regulation of iron homeostasis at systemic level, the influence of iron metabolism on critical stage of fetal development, is still unknown.

Regulation of hepcidin expression seems to occur at transcriptional level and its production is increased by iron loading and inflammation and decreased by anemia and hypoxia.

Even if the molecular mechanisms of hepcidin regulation by iron, oxygen and anemia are still unclear, it is known that Interleukin 6 (IL-6) induces transcription of the hepcidin gene in hepatocytes. In inflammatory and infection disorders, cytokine induced hepcidin excess, through ferroportin binding, contributes to development of anemia of inflammation, characterized by ID and IDA despite adequate iron stores. When iron export is hindered, iron is stored in host cells. However, inflammation may contribute to ID and IDA by hepcidin-independent mechanism(s) as the down-regulation of ferroportin. Independently on hepcidin synthesis, high levels of serum IL-6 seem to down-regulate ferroportin mRNA expression, thus sequestering iron inside cells and blocking iron flow into plasma. The inability to export iron leads to hypoferremia, decreased pool of serum transferrin-Fe(III) and iron-limited erythropoiesis.

The recent discovery of hepcidin-ferroportin complex has greatly contributed to clarify the enigmatic mechanism of systemic iron homeostasis. Notwithstanding, iron homeostasis disorders, as ID and IDA, are still treated with oral administration of large quantity of iron as fer¬rous sulfate due to its poor bio-availability. Ferrous sulfate oral administration often fails to exert significant effects on ID and IDA, and frequently causes many adverse effects, including gastrointestinal discomfort, nausea, vomiting, diarrhea, and constipation.

Recently, a significant decrease of total serum iron and serum ferritin concentrations related with an increase of serum IL-6 concentration has been observed in pregnant women and in hemodialysis patients, orally treated with ferrous sulfate. Therefore, it can be hypothesized that supplemented iron was not exported from cells to circulation, but it was accumulated inside the cells, thereby increasing inflammatory status, similarly to that reported in animals treated with ferrous sulfate.

The weight of evidences, which let to doubt on the efficacy and safety of ferrous sulfate oral administration, has stimulated the research of more effective approaches to prevent ID and toxicity associated with iron overload. The treatment of ID and IDA during pregnancy should be entirely reconsidered taking into account not only the enhanced blood volume and development of fetal-placenta unit, but also other factors, as IL-6, which, inducing hepcidin up-regulation and ferroportin down-regulation, can play a pivotal role in iron homeostasis disorders.

For this purpose, lactoferrin (Lf), a cationic iron-binding glycoprotein, able to chelate at high affinity (KD w10/20 M) two ferric ions per molecule (30), is emerging as an important regulator of systemic iron homeostasis, able to cure ID and IDA. Lf is synthesized by exocrine glands and neutrophils in infection and inflammation sites. In humans, free iron does not exceed 10-18 M to avoid precipitation, microbial growth and formation of reactive oxygen species. Lf in tissues and secretions and transferrin in blood assure that iron was bound and scarcely available as free-ion.

Even if several functions, dependent and independent on its iron binding ability, have been attributed to Lf, our recent clinical trials have demonstrated that this natural compound may represent an interesting approach in the therapy for ID and IDA in pregnant women. As matter of fact, a milk derivative bovine lactoferrin (bLf) restores the physiological transport of iron from tissues to circulation, thus curing ID and IDA. Moreover, differently from ferrous sulfate, bLf oral administration exerts an anti-inflammatory effect by decreasing serum IL-6 concentration in uncomplicated pregnancies.

The hypercoagulable state represents one of the physiological changes occurring during pregnancy. The hereditary thrombophilia (HT), a genetic predisposition to inappropriately form clots, significantly increases adverse outcomes including recurrent miscarriages, intrauterine fetal death and growth retardation, preeclampsia and placental abruption. It is well known that inflammation promotes coagulation and IL-6 high levels in plasma of HT affecting pregnant women with severe preeclampsia have been found. Moreover, elevated intra-amniotic IL-6 and IL-8 levels have been correlated to preterm birth.

Therefore, iron supplementation via oral administration of ferrous sulfate increasing serum IL-6 levels in uncomplicated pregnancies, could be deleterious in HT affecting pregnant women and could contribute to enhance iron overload in tissues, inflammation and cell damages.

The bLf capability to prevent and cure ID and IDA together with its ability of decreasing serum IL-6 concentration could be effective even in the treatment of ID and IDA in pregnant women affected by HT.

In this clinical trial, pregnant women affected by hereditary thrombophilia (HT), suffering of ID and IDA, receive an oral administration of bovine Lf (bLf) 100 mg/twice a day or ferrous sulfate 520 mg/day. The number of red blood cells, the values of hemoglobin, total serum iron, serum ferritin, haematocrit, serum Interleukin-6 (IL-6) and prohepcidin are assayed before therapy, every 30 days until delivery.