Protective effect of human lactoferrin in the gastrointestinal tract

Objective: To describe mechanisms of action of human lactoferrin to protect gastrointestinal morbidities. Data sources: Nonsystematic literature review using the following databases: SciELO, Lilacs and Medline from 1990 to 2011. The key-words used were lactoferrin, human milk/breastfeeding, gastrointestinal, and immunity, in Portuguese and English. Data synthesis: Lactoferrin is the second predominant protein in the human milk, with higher concentrations in the colostrum (5.0 to 6.7mg/mL) if compared to mature milk (0.2 to 2.6mg/mL.) In contrast, cow’s milk has lower levels, with 0.83mg/mL in the colostrum and 0.09mg/mL in the mature milk. Lactoferrin has several physiological functions to protect the gastrointestinal tract. The antimicrobial activity is related to the ability to sequester iron from biological fluids and/or to destruct the membrane of microorganisms. Lactoferrin also has the ability to stimulate cell proliferation. The anti-inflammatory action exercised by lactoferrin is associated with its ability to penetrate the core of the leukocyte and to block the Kappa B nuclear factor Endereço para correspondência: Valterlinda Alves de O. Queiroz Rua Araújo Pinho, 32 – Canela CEP 40110-150 – Salvador/BA E-mail: valterlinda.oliveira@gmail.com Conflito de interesse: nada a declarar Recebido em: 12/1/2012 Aprovado em: 29/5/2012


Introduction
At birth, the immune system of the child is still immature, the stomach has less ability to eliminate pathogens, and the intestine lacks the gut microbiota.Based on these considerations, it is reasonable to assume that the child is dependent on exogenous protection.In that sense, the human milk represents the ideal food as it has immunological, nutritive and digestive components that favor the maturity process of the intestinal mucosa and counterbalance the little competence for adaptive response of the digestive system, as well as the immaturity of other body systems, strongly contributing with the body defenses in the first two years of life (1) .
In the neonatal period, the unfavorable effects of the immunological immaturity are compensated by physiologic mechanisms, such as the transplacentary passage of immunoglobulin G (IgG) from to mother to the fetus during pregnancy and the intake of immunological components of the human milk.Breastfeeding keeps the maternal-fetal link of protection after birth, favoring the transfer of maternal factors that modulate the immune system.This contributes for the child´s immune competence during a period that is crucial for the development of its own immunity (2) .
The protection provided by the human milk is due the presence of a variety of functional proteins, including the immunoglobulin A (IgA), lactoferrin, growth factors and cytokines, which play an important role in the maturation of the child´s gastrointestinal tract (3) .
Among the milk components, lactoferrin has great importance in the defense line against diseases, in particular the gastrointestinal ones (4)(5) .Lactoferrin is a glycoprotein from the transferrin family that has been attracting growing scientific interest since the late 1950´s, due to its high concentration in human milk (6) and its physiological functions, specially the antimicrobial, anti-inflammatory and immunomodulating functions on the gastrointestinal tract (7) .Thus, the objective of this review is to report, using the analysis of the literature, the mechanisms of action of the human lactoferrin in the protection of the gastrointestinal tract.

Data collection
The search of the references was performed using the electronic databases Scientific Electronic Library On-line (SciELO), Literatura Latino-Americana e do Caribe em Ciências da Saúde (Lilacs) and Medical Literature Analysis and Retrieval System Online (MedLine) of the National Library of Medicine, in the period from 1990 to 201.The references of the selected articles were also consulted to identify classical studies on the subject.
Therefore, because of the relevance of the contributions, six studies that were published outside the predetermined search period were detected and included in this review.The keywords used in Portuguese, and their correspondents in English, were: lactoferrin (lactoferrina), breast milk/ human milk (leite materno/humano), gastrointestinal (gastrintestinal), and immunity (imunidade).The search was performed using the keywords both alone and in combination.

Data synthesis
The structure of lactoferrin Lactoferrin is the second predominant protein of the human milk (4) , whose concentration is higher in the colostrum.Each lactoferrin molecule consists of one unique polypeptide chain with two globular lobes located in each of the terminals, named lobe C (acetyl) and lobe N (amino), which are linked by an α-helix, with each lobe containing one binding site (7)(8) .Each one of them consists of two sub-lobes named N1, N2, C1 and C2 (8) .
Each lobe can bind to a metal in synergy with the bicarbonate ion, which is essential for the binding of the iron ion with the lactoferrin.The metals that usually bind to lactoferrin are Fe +2 or Fe +3 , but others (Cu +2 , Zn +2 , Mn +2 ) can also bind to the lactoferrin lobes (9) .

Concentration of lactoferrin in the human milk
Lactoferrin is widely distributed in the body fluids, especially in the human milk (5) .Its concentration varies among the animal species, with the human and other primates' milk showing the highest concentrations in comparison with cow´s milk (6) .
The concentration of lactoferrin varies widely (10) , from 5.0-6.7mg/mL in the colostrum and 0.1-2.6mg/mL in the human mature milk (11)(12) .In contrast, cow´s milk shows lower concentrations of lactoferrin, with 0.83mg/mL in the colostrum and 0.09mg/mL in the mature milk (13) .

Digestion of the lactoferrin
Studies have shown that not only the active form of lactoferrin has biological activity.Digestion of lactoferrin produces lactoferricin, a peptide that derives from the N-terminal of lactoferrin and has strong activity against Gram-positive and Gram-negative pathogenic bacteria (14) .Therefore, both lactoferrin and the product derived from its digestion show antimicrobial activity.It is probable that lactoferricin has bactericidal effect stronger than lactoferrin due to its size, which facilitates its direct action against the bacterial cell membrane (15) .

Antimicrobial properties
Diarrhea is, usually, an infection caused by bacteria, viruses or parasites, and represents the second cause of death in children (16) .The infections caused by rotavirus are the main cause of severe diarrhea in children under five years old worldwide (17) .Rotavirus is responsible for about 527.000 deaths and 2 million hospital admissions due to dehydration per year (18) .
Among the well-recognized preventive measures against diarrhea is the adoption of the breastfeeding practice, exclusive in the six first months, and complementary for at least two years (16) .In addition to avoiding the contamination from other foods, the efficacy of the breastfeeding as a preventive measure can be attributed to multiple anti-inflammatory, anti-infectious and immunomodulating factors, which are present in the human milk (19,20) and act in the protection of the child (21) .Among these protective factors, lactoferrin is well known by its antimicrobial properties, mainly related to its ability to sequester iron from biological fluids, and/or disrupt the membrane of the microorganisms, acting on the body´s defense against bacteria, viruses and protozoa (22) .The effect of lactoferrin on the intestinal cells may be one of the mechanisms against enteric infections (23) .

Antibacterial effect
The bactericidal action of lactoferrin is attributed to two distinct protection mechanisms.The first one is the bacteriostatic action related to the affinity of the protein for the iron molecules, which inhibit the growth of the bacteria that need this nutrient (24) , including several Gram-positive and Gram-negative bacteria (25) .
The bacteriostatic effect of lactoferrin is also dependent of its degree of saturation (24) : the lesser the iron saturation, the greater its ability to sequester iron (26) .Bearing this is mind, the importance of the lactoferrin that is present in the human milk must be emphasized, as its saturation ranges from 5 to 8% and, therefore, has greater bacteriostatic action (23) in comparison with cow´s milk lactoferrin, whose saturation ranges from 15 to 20% (8) .The lactoferrin with less than 5% iron saturation is known as apolactoferrin (apo), while the lactoferrin with higher saturation is known as hololactoferrin (holo) (8) .In the human milk, lactoferrin predominates in the form of apolactoferrin (90%) (27) .
The second protective mechanism against bacterial overgrowth that can be attributed to lactoferrin is related to its interaction with the bacterial surface (28) .Studies on the molecular structure of lactoferrin have shown that it interacts directly with the anionic lipid A, which is a component of the lipopolysaccharides that, in their turn, constitute part of the Gram-negative cell wall (29) .The enhanced affinity of lactoferrin with the lipopolysaccharides is related to its N-terminal, differently from the C-terminal, which shows low affinity (30) .
Such interaction can damage the bacterial membrane, affecting its permeability and promoting the release of lipopolysaccharides (31,32) .These alterations facilitate the action of lactoperoxidase and other proteins of defense against the bacteria.The interaction of lactoferrin with the lipopolysaccharides also potentiates the action of other natural antibacterial factors, such as lysozyme (28) .
According to in vivo studies conducted by Kruzel et al, the administration of human lactoferrin to mice one hour before the injection of lipopolysaccharides increased significantly the survival, reducing the mortality from 83.3 to 16.7% in the animals who were pre-treated with lactoferrin.The histopathologic analysis of the intestine of the pre-treated animals showed greater resistance to the damages caused by the lipopolysaccharides.In the animals not treated with human lactoferrin, severe atrophy and edema of the intestinal vilosities, in addition to epithelial vacuolization, were observed (30) .
Recent studies have shown that the action of the lipopolysaccharides on the activation of the nuclear factor Kappa B (NF-kB) is insignificant in the presence of human lactoferrin.The NF-kB plays an essential role in the regulation of the immune system and the inflammatory response.In the same experiment, it was also observed that human lactoferrin can induce the NF-kB activation in concentrations much lower than those found in the human milk.According to the authors, it is probable that human lactoferrin acts as a trigger of the tool-like receptor 4 (TLR4) in the intestine of breastfed babies.The TLR4 can detect the lipopolysaccharides, and is important on the activation of children´s innate immune system (33) .
The mechanism of action of lactoferrin against Grampositive bacteria is similar to that described for Gram-negative bacteria, but acting on the lipoteichoic acid, which is a component of the cell wall of the Gram-positive bacteria (34) .According to Leitch and Willcox, lactoferrin and lysozyme exert combined effect against Gram-positive bacteria (34) .

Antiviral effect
Lactoferrin can inhibit the replication of several viruses.The antiviral mechanisms of action of lactoferrin have not been elucidated yet.One of the most accepted theories is that lactoferrin prevents viral invasion of the host cells by either blocking the viral receptors or by directly binding to the virus, thus avoiding the infection (9) .
Lactoferrin is effective in the control of infections caused by rotavirus, a double helix RNA virus from the Reoviridae family which infects mature erythrocytes.These infections are the most frequent cause of gastroenteritis in children worldwide (35) .
The apo form of lactoferrin has been shown to be more potent than the holo form, but the reasons for this have not been elucidated yet.It is speculated that most enzymes need metallic ions to exert their actions, and that apolactoferrin is more efficient to capture metallic ions from the environment in comparison with the saturated forms of lactoferrin (9) , which are found in greater concentrations in cow´s milk.

Antiparasite effect
The action of lactoferrin against parasitic diseases is still unclear.Studies have attributed its effects to the action of lactoferrin on the integrity of the protozoa membrane (36) .In vitro studies have shown that apolactoferrin is the milk protein with greater activity against Entamoeba histolytica, as the binding of the protein to the trophozoite´s membrane causes its rupture, and subsequent damage to the protozoan (37) .

Estimulation of the healthy microbiota
The development of the intestinal microbiota in children who are breastfed is quite different from those on artificial feeding.Exclusively breastfed babies present the intestinal flora pattern consisting of high percentages of lactobacilli, especially Lactobacillus bifidus, while those who are fed with cow´s milk or formulas have their microbiota similar to that of the adults (1) .
The human milk contains substances that have probiotic action and stimulate the growth of beneficial bacteria.These substances, originally named bifidus factor, can promote the growth of bifidobacterium and lactobacilli, protecting the intestine by limiting the multiplication of several different pathogens due to the decrease of the intestinal pH (38) .
Evidences indicate that the oligosaccharides present in the human milk have bifidogenic activity (39) , that act together with the proteins, peptides and nucleotides in the development of lactobacilli and bifidobacterium in the gastrointestinal tract of children (40) .
The antimicrobial activity of lactoferrin exerts beneficial effect on the intestinal microbiota, because its bacteriostatic action does not impair the growth of bacteria that produce lactic acid, as they have low iron requirements (41,42) .
In vitro studies conducted by Liepke et al showed the presence of peptides after the digestion of human milk with pepsin.Two of them originated from lactoferrin and from the secretory IgA.The bifidogenic effect of these peptides were superior to that of N-acetyl-glucosamine, a well know bifidogenic factor (43) .

Promotion of cell proliferation
The epithelial growth factor is a polypeptide found in the human colostrum (200µg/L) and in the mature milk (30-50µg/L); it is observed in much lower concentrations in cow´s milk (44) .
According to Playford et al, the growth factor of the human milk stimulates the proliferation and the differentiation of the child´s intestinal cells (45) .Studies from Corps et al have demonstrated that the presence of the (purified) growth factor per se cannot explain its mitogenic action.The authors concluded that the growth factor present in the human milk, together with other compounds, has greater mitogenic activity than the purified factor (46) , pointing to a synergic action of the growth factor and other milk compounds.
The experiments conducted by Hagiwara et al showed that lactoferrin can promote the proliferation of cells from the gastrointestinal tract.According to the authors, there is a synergic action between lactoferrin and the epithelial growth factor on cell proliferation (47) .

Anti-inflammatory activity
Lactoferrin presents great ability to penetrate the leucocyte nucleus and to block the transcription of the NF-kB, which in its turn induces the release of the pro-inflammatory cytokines interleukin 1 beta (IL1β), tumor necrosis factor alpha (TNFα), interleukin 6 (IL6) and interleukin 8 (IL8) (48) .
Furthermore, lactoferrin is part of the immune system homeostasis that can diminish the molecular oxidative stress, and control the excessive inflammatory response.The oxidative stress develops when the production of the potentially destructive oxygen reactive species exceeds the body´s natural anti-oxidant defenses, resulting in cell damage (49) .
One study performed in vivo using lactoferrin concentrations similar to those of the human colostrum showed that lactoferrin blocked the development of the inflammatory process induced by Shigella flexnery in the intestine of rabbits (50) .
The in vivo experiments of Haversen et al (51) confirmed the anti-inflammatory activity of lactoferrin.Laboratory mice with colitis induced by dextran sulfate and treated with human lactoferrin showed lesser faecal occult blood, as well as lesser damage to the rectal mucosa, less evident colon shortening, reduced plasma levels of IL1β, and less amount of TNFα producing cells (51) .

Genetic engineering and the synthesis of lactoferrin
The human milk is protective to the child´s health, and there is a consensus that it represents the ideal food, that it must be the only source of feeding in the first six months of life and offered in complementation for at least two years.Nevertheless, many children do not benefit from breastfeeding, either totally or partially, for different reasons.Thus, the industry has been developing technologies trying to adjust the composition of the infant formulas to that of the human milk, not only regarding the nutrients composition, but also the bioactive compounds.
The evolution of the genomic sciences and the consequent disclosing of the human genome have favored the development of technologies that use gene sequences which codify the proteins of the human milk.Such technological progress favors the expression of bioactive proteins of plants and animals, and allows the large scale production of these compounds (52) .
Genetic engineering has allowed the expression of recombinant proteins of the human milk in fruits (banana), cereals (rice and barley) e tubercles (potatoes).Among these studies, the rice lactoferrin takes greater importance.The use of this cereal is justified because it has no toxic compounds, in addition to the low allergenic potential, and its intake can be easily incorporated by children all around the world.In vitro studies have shown that the recombinant human lactoferrin of the rice has activity and stability properties that are similar to those of the native proteins (53) .
Transgenic animals represent another alternative for the expression of the bioactive compounds of the human milk.In this sense, studies have shown that the recombinant human lactoferrin obtained from transgenic cows has identical ability to bind to iron when compared with the human lactoferrin, and showed the same efficacy in three different models of in vivo infection (54) .
Considering the possibility of obtaining large scale recombinant biological compounds of the human milk by using plants and genetically modified animals for the enrichment of infants' formulas, further studies to evaluate the efficacy and safety of these compounds both in vitro and in vivo are necessary.

Conclusions
Human lactoferrin is a natural peptide that shows great potential to prevent morbidities, particularly the gastrointestinal diseases, as they present antimicrobial, antiinflammatory, and cell proliferative activities, and favors the development of the bifidus intestinal flora.
The scientific evidences of the protective effects of the human lactoferrin suggest that this molecule can be involved in the protection of the gastrointestinal tract of breastfed infants, which reinforces the recommendations for the practice of breastfeeding, particularly in the first years of life.This life period is more vulnerable to harm due to the fragility of the adaptive response of the digestive system, and the immaturity of other body systems.
Efforts must be done to develop technologies that allow the expression of the recombinant human protein in plants and animals, with the aim of favoring children that, for various reasons, cannot totally or partially consume human milk.