Transferrins constitute the most important iron regulation system in vertebrates and some invertebrates. Soluble transferrins, such as bovine lactoferrin and hen egg-white ovotransferrin, are glycoproteins with a very similar structure with lobes that complex with iron. In this in vitro study, a comparison of bovine lactoferrin and ovotransferrin was undertaken to confirm the comparability of biological effects. An in vitro gastric barrier model using gastric epithelial cells GTL-16 and an in vitro intestinal barrier model using CaCo-2 cells was employed to evaluate iron absorption and barrier integrity. An analysis of the molecular pathways involving DMT-1 (divalent metal transporter 1), ferritin and ferroportin was also carried out. These in vitro data demonstrate the activity of both 15% saturated and 100% saturated ovotransferrin on the iron regulation system. Compared with the commercial bovine lactoferrin, both 15% saturated and 100% saturated ovotransferrin were found to act in a more physiological manner. Based on these data, it is possible to hypothesise that ovotransferrin may be an excellent candidate for iron supplementation in humans; in particular, 15% saturated ovotransferrin is the overall best performing product.
It is commonly accepted that human lactoferrin is an important component of innate immunity, and can interact with microorganisms by the iron sequestration mechanism and microbial molecules at the intestinal lumen. Human lactoferrin is a glycoprotein found in milk and is also present in most exocrine secretions such as tears, saliva, intestinal mucus, and genital secretions, which have been demonstrated to have several properties including antibacterial, immuno‐modulating, and anti‐inflammatory properties because the main function has a protective effect on intestinal barrier integrity.
The bovine lactoferrin (bLF) effects depend on its relative resistance to proteolytic digestion; indeed, in vitro tests have shown that it is not totally degraded in the luminal environment of the stomach and at the level of the small intestine, allowing it to bind to specific receptors in the brush border membranes. These receptors specifically mediate the uptake of bLF into enterocytes and crypt cells. In vitro, bLF is transported from the intestinal lumen to the bloodstream, and acts not only at the luminal intestinal level but also systemically. Ovotransferrin, a protein abundant in hen egg white, shares many of the same activities as human/bovine lactoferrin. Ovotransferrin combines iron transport and the defence functions of mammalian serum transferrin and lactoferrin, respectively. It also shares approximately 50% of the sequence homology with each protein.
However, the structural analogy between ovotransferrin and lactoferrin is much closer than the sequence homology and similar clusters of positively charged residues responsible for different activities such as antiviral/bacterial, immunomodulatory, antioxidant and anti‐inflammatory properties. In addition, this protein has been demonstrated to be able to supply iron to cells. Ovotransferrin is readily digested by pepsin in the stomach and the bioactive forms of ovotransferrin are readily transported into human intestinal cells. Despite a large body of evidence that supports the beneficial impact of various lactoferrin preparations on human health, there is still very little information regarding the mode of action of lactoferrin, and whether saturation of the protein with Fe affects its function and is also similarly observed with ovotransferrin. Given this premise, in this study we analysed the release, absorption, and biological activity of ovotransferrin from hen egg white with a different iron saturation rate and bovine lactoferrin using a well‐characterised in vitro model of the digestive system , mimicking the human gastro‐intestinal system both in the presence or absence of Fe3+. The findings obtained in this study demonstrate that holo‐OvT was able to maintain a higher significant beneficial effect on cell viability and integrity compared to the other holo‐OvT and bLact tested, in both gastric and intestinal compartments, confirming the important hypothesis of the iron saturation rate to obtain beneficial effects.
The knowledge that the administration of ovotransferrin influences iron absorption and metabolism is a noteworthy finding. Therefore, the passage and the real absorption rate of all forms of holo‐OvT mimicking oral intake, compared to bLact, confirmed a slow release. This is important data for defining the dose form and the posology in humans to prevent the rebound effect. According to the data obtained, holo‐OvT promotes iron absorption while also preventing the body from being damaged by excessive iron; this condition was observed during pre‐treatment with Fe3+. Furthermore, the concentration to obtain the beneficial effects, while simultaneously preventing the negative effects of accumulation, was shown to be 30 μg/mL of holo‐OvT. Because iron metabolism is influenced by various conditions, the importance of the different test products on iron uptake (DMT1 analysis) and on the transfer across the cell monolayer (ferritin light chain for transportation and ferroportin to extrude) was investigated according to a standard protocol. In addition, the tests on the molecular mechanisms demonstrate that holo‐OvT can physiologically regulate iron metabolism. Indeed, it was observed that holo‐OvT requires iron by transferrin, which is released as ferrous ions translocating via DMT1 into the cytoplasm where it is sequestered by ferritin. The release of iron from this protein to the cytoplasm occurs after the reduction of ferric to ferrous ions. Then, ferrous ions are exported into plasma by ferroportin.
The epithelium of the intestinal mucosa is one of the most important barriers in terms of extension that separates the intestine and the internal organs. For this reason, it is a dual target of any toxic insult from drugs or substances in the diet. In fact, it is known that the mucosal alterations cause not only damage to the tissue itself but can also represent an uncontrolled passage of potentially toxic substances from the intestinal lumen to blood. In particular, as reported in the literature, bLact can have a direct effect on the intestinal epithelium and play a beneficial role on the intestinal epithelial barrier through the NK‐κB signalling pathway or by preserving the integrity of the intestinal barrier. bLF is transported from the intestinal lumen to the bloodstream and functions not only in the intestinal lumen but also systemically. The formation of tight junctions (TJs) in epithelial cells plays a pivotal role in the intestinal barrier. TJs mediated by proteins such as claudins, occludin, and zonula occludens (ZO) are necessary for epithelial barrier maintenance. For this reason, disruption of the intestinal epithelial barrier can increase intestinal permeability.
Therefore, the effect of bLact on the three TJs was confirmed in the present study, and the effect of OvT to significantly increase the expression of the TJ proteins claudin‐1, occludin and ZO‐1 were demonstrated, effectively strengthening the integrity and the barrier function of the two cellular models used. Furthermore, the stress potentially caused by all forms of Ovt was maintained at a physiological level, thus ensuring safety without adverse or harmful effects on the intestinal epithelium, which is very important for maintaining the integrity of TJ. Therefore, the current data indicate that OvT significantly increases the expression of TJ proteins and protects the function of the intestinal epithelial barrier, and even improves the integrity of the intestinal epithelial barrier, as demonstrated by the increase in TEER values, both in the stomach and in the intestine. The same data were observed in presence of iron, and these observations indicate that OvT formulation is suitable for gastric and intestinal epithelial cells, exerting a beneficial role in terms of iron absorption and metabolism and gastro‐intestinal barrier integrity.
In conclusion, this in vitro study demonstrates for the first time the ability of hen egg-white ovotransferrin, in two different forms, the apo‐ (iron‐free) and holo‐form (containing iron at different saturation percentages), to actively enhance absorption of iron at the gastric and intestinal level without accumulation and gastric‐intestinal irritability, indicating the huge potential of ovotransferrin in new dietary supplementation strategies. The demonstrated action is similar to that of bLact but with a greater effect. Major human iron markers such as DMT‐1, ferritin and ferroportin were used to investigate the underlying iron uptake mechanism. Overall, both apo‐ovotransferrin and holo‐ovotransferrin 15% saturated showed better performance compared to the tested bovine‐lactoferrin. In detail, the 15% saturated holo‐ovotransferrin showed the best activity at the lowest concentration among those used, i.e., 30 μg/mL. Furthermore, ovotransferrin decreased the negative effects of the presence of iron, maintaining the integrity of the gastrointestinal barrier. Based on these in vitro data, we can conclude that the natural protein ovotransferrin from hen egg white is an excellent candidate for iron supplementation, as an iron absorption enhancer, and as an alternative to bovine‐lactoferrin.
In vivo studies should be performed to confirm the in vitro data presented in this study.
Rebecca Galla1, Paride Grisenti2, Mahitab Farghali1, Laura Saccuman 1, Patrizia Ferraboschi3 and Francesca Uberti1,*
1. Laboratory Physiology, Department of Translational Medicine, University of Eastern Piedmont, Via Solaroli 17, 28100 Novara, Italy
2. Bioseutica B.V., Landbouwweg 83, 3899 BD Zeewolde, The Netherlands
3 Department Medical Biotechnology and Translational Medicine, Università degli Studi di Milano, Via Saldini 50, 20133 Milan, Italy
*Author to whom correspondence should be addressed. Academic Editor: H. P. Vasantha Rupasinghe
Received: 22 September 2021 / Revised: 19 October 2021 / Accepted: 21 October 2021 / Published: 26 October 2021
Bioseutica BV; Ovotransferrins for use in the treatment of iron deficiency anaemia. EP 21158368.7, 22 February 2021
BIOSEUTICA B.V, The Netherlands (www.bioseutica.com), partially sponsored the study. In addition, this study was (partially) funded by the Italian Ministry of Education, University and Research (MIUR) program “Departments of Excellence 2018– 2022”, AGING Project–Department of Translational Medicine, Università del Piemonte Orientale.