LACTOFERRIN PRESENTATION

by Dr Jean-Paul Perraudin, PhD

CONTENENTS

INTRODUCTION

 

  1. INTRODUCTION

BIOEF, working with the Belgian company Biopole, tries to develop the Romanian market and to sell innovative, high quality products and concepts, based on its scientific experience gained in the last decade in the field of human and animal secretory molecules and resulted from many scientific studies performed for identifying the properties and possible practical uses of these molecules.

 The liquid secretions, that have mainly the same role and approximately the same molecules in their composition contain, beside other bases, active substances like lactoferrin, immunoglobulins, lacto-peroxidase, liposomes and growth factors. These beneficial effects in the development of the body have been demonstrated in many clinical studies made in renowned European and American research centers and universities.

This is why our first concern is to introduce the lactoferrin and the colostrum in our products for newborns, because of their high concentration of active principals (immunoglobulins, growth factors and of course lacto-peroxidase). Both the lactoferrin and the colostrum are products of animal origin, collected and extracted from attentively chosen raw materials, to avoid their microbiological, physical and chemical contamination.

In the following we will show some up to date information from the specialty literature concerning studies made on lactoferrin and colostrum.

For your knowledge, lactoferrin is already used in the children’s powder milk formulas, in yogurt, liquid yogurt, oral healthcare products and food supplements in other countries. The main actions for which we recommend the use of lactoferrin in diets are: 

  • Antimicrobial and antiviral action
  • The regulation of iron absorption and facilitation of its transport and absorption in the body
  • Antioxidant action
  • Prebiotic action -  the promotion of normal saprophytic intestinal flora

 

1.1 THE PRESENCE OF LACTOFERRIN IN THE BODY

The lactoferrin is a protein that ties iron molecules, and it is present in the majority of mucous secretions (milk, saliva, tears, plasma, genital secretions, bile, gastric juice, amniotic liquid, urine), but also in neutrophil cells. The content of lactoferrin in milk varies according to the specie, the animal’s age, the number of births, frequently being inversely proportional with the level of transferrin. For the human colostrum, the initial value of lactoferrin immediately after birth is 6 mg/ml, decreasing in the first week of lactation to a level of approximately 0,8 mg/ml in the mature milk. The same phenomenon was observed for the cow’s colostrum as well, it having an initial content of 5 mg/ml and rapidly decreasing to 0,35-0,20 mg/ml in the mature milk.

1.2 THE STRUCTURE OF LACTOFERRIN

 The lactoferrin is a glycoprotein with a molecular mass of about 77 000Dka. The complete aminoacidic sequence of lactoferrin is of approximately 680 residual amino acids varying for a species to another through different structures of the glycans chain.

Each lactoferrin molecule can reversibly tie 2 atoms of iron (FeIII). The 2 tying places of the iron are similar, but not identical. The tying of iron depends on the concurrent tie of the synergic anions, like the bicarbonate that has an essential part in the tying of the metal in its place and also in its untying. Lactoferrin can take 2 forms: untied to iron and saturated in iron.

The iron is an essential element in nutrition. Iron deficiency or anemia is a common problem in children and adults, especially in developing countries, more so for the bodies in the stage of rapid growth in the first weeks of life.

Anemia doesn’t mean just the decrease of hemoglobin and other hemoglobin indices, it can also imply immediate immune responses of lymphocytes and granulocytes and it can affect the functions of some organs, as iron is essential for many enzymes.

If iron is a very important metal for life, as easily it can become poison. In ionic form, too much iron can be a danger to the body. In fact, it is an essential element for the growth and colonization of pathogen bacteria and tumor cells. It also influences the development of some forms of cancer and plays an essential role in the chronicity of arthritis. There is an important relationship between the metal and immunity, meaning that the cells of the immune system protect the tissues against the toxicity of the iron, thus playing a primordial role in controlling the concentration of this metal. The utility of the presence of such cells capable of liberating proteins as lactoferrin, transferrin and ferritin that can neutralize iron is highlighted by their effect of eliminating iron’s toxicity by binding it.

R.K. Chandua, B. Au. G. Woodfort, P. Ham: “Iron level, immune response and infection susceptibility” Dr Charles A. Janeway Child Health Center and Memorial University of Newfoundland

Abstract: Nutritional factors can modulate immune answers. Iron’s concentration among other nutrients influences the host’s defense mechanisms. In experimental induction of iron deficiency in animals, their morbidity and mortality in the face of bacterial attack rose several times. Cellular mediated immunity and the destruction of bacteria at intracellular level by the polymorphonuclear leucocytes are weakened in the individuals with iron deficiency. This weakening is most probably mediated by the effect of missing iron for the proliferation of cells, DNA synthesis and activating iron dependent enzymes and those that contain iron that are involved in the destruction and elimination of microbes.

Contrary, iron’s availability is a critical determinant for bacteria multiplication. It is not surprising that clinical and epidemiological data about the frequency of bacterial, fungal and other infections in groups of healthy individuals with iron deficiency and with supersaturated iron levels differ very much. Infection vulnerability, based on the individual iron levels  Vulnerability to infection, based on the individual iron level, its influence on microbial growth on the one hand, and host immunocompromite on the other. The key in maintaining interactions between physiological limits is a diet with optimal iron intake.

  1. M. J. Barry, A. W. Reeve et all.:” Increased incidence of Gram-negative neonatal sepsis with intramuscular iron administration”, Department of Pediatrics, Hawke's Hospital Bourd, Memorial Hospital, New Zealand – published in Pediatrics (1977) 60:908-912

Abstract: During a five-year period, the incidence of neonatal sepsis was 20 times higher in Polynesian newborns compared with European newborns (11 per 1,000 vs. 0.6 per 1,000 total births). This high incidence in Polynesians was confined to a period when the infants were being given intramuscular iron. When the iron administration was stopped the incidence of disease in Polynesians decreased from 17 per 1,000 to 2.7 per 1,000 total births. An analysis of the Polynesian iron-treated and non-iron-treated groups showed a statistically significant difference in the incidence of sepsis, the type of causative organism, and mortality.

 

The data suggest that the iron dextran injections have impaired the immunity of the treated infants, making them more susceptible to Escherichia coli sepsis.

Oppenheimer. S. J.: “Iron and infections: clinical evidence”, Acta Paediatrics Suppl 361: 53. 1989

Abstract: Iron deficiency is prevalent throughout childhood mainly in the developed and developing countries. In many countries, there have been introduced programs of presumptive therapy with a role in supplementing and fortifying the diet. The unresolved issue regarding the interaction of iron and infections in a clinical environment needs the reevaluation of these practices. The situations of iron overdose are associated with elevated sensitivity to some infections. The exact mechanism can vary with the baseline pathology. Iron treatment has been associated with acute exacerbation of the infection, mainly with malaria. In most cases parenteral iron has been used. The administration of parenteral iron in newborns has been associated with severe E-coli infections. In countries with malaria epidemics, parenteral iron administration has been associated with the rise of malaria rate and of child morbidity caused by respiratory illnesses. Contrary, in countries where there is no malaria, studies regarding the oral intake of iron have shown a decrease in morbidity. The methodological issues in the above-mentioned reports show the continuous need of perspective controlled studies with the exact recording of morbidity if there have been made documented recommendations.

  1. THE BIOLOGICAL FUNCTIONS OF LACTOFERRIN

2.1 DOES LACTOFERRIN RESIST ABSORPTION IN THE SMALL INTESTINE?

The studies about the functions of the lactoferrin ingested by newborns are difficult to be conducted without knowing if the protein survives the intestinal transit. Brock and his contributors have studied in detail the in vitro effects of trypsin and chymotrypsin on cow’s and human lactoferrin. Cow’s lactoferrin saturated in iron was more resistant to digestion than the apoprotein. The human apoforma of lactoferrin remained almost intact, judging by its capacity to bind iron and its bacteriostatic action on E-coli. Spick confirmed cow’s and human’s lactoferrin’s stability in the gastric and duodenal fluids obtained from children aged between 1 and 3 months. The proteins have not been hydrolyzed in the gastric fluid and only partially in the duodenal fluid.

When the milk formula has been supplemented with cow’s and human lactoferrin, the iron saturated proteins were approximately 50% more resistant to digestion than those partially saturated and much more cow’s lactoferrin was excreted than human. This happened because of a higher resistance of cow’s lactoferrin. The lactoferrin purified from fecal was found to be immunological active, but partially hydrolyzed, corresponding to some fragments of molecular weight. These fragments were capable to continue to bind iron, showing that their antimicrobial action remained intact.

  1. S. Goldman, C. Garza, R. J. SCHANLERat all.: “Molecular forms of lactoferrin in breast fed children’s stool and urine”, Department of pediatrics. Human biological Chemistry and Genetics. and Microbiology. The University of Texas published in Pediatric Res (1990) 27:252-255

Abstract: Lactoferrin’s molecular forms have been examined from the stool and urine collected at 2,5 or 5 weeks from newborns with very small birth weight and that were fed with cow’s milk formula or human milk fortified formula. In the Western blotting test, lactoferrin was not found in the excretions of children fed with cow’s milk. Contrary, intact or fragmented lactoferrin was detected in the stools or concentrated urine of each child fed with human milk. Only intact lactoferrin was found in the human milk fortified formula, where many types of lactoferrin fragments were present in the stools and urine. The molecular approximate weight of the most significant fragments was 44, 38, 34 and 32 kD. Anyway, fragments with low molecular weight were found in the stool that were not found in the urine of these children.

Lactoferrin fragments in those secretions were similar to those produced in vitro trough limited apolactoferrin digestion with trypsin. Furthermore, fragments formed through the in vivo proteolyze were immune-reactive to the lactoferrin ELISA test. This is why stool lactoferrin fragments in breast-fed children with very low birth weight seem to be the result of in vivo protolyze, and resembling lactoferrin fragments coming from the gastrointestinal tract. It remains unclear if the whole lactoferrin molecules derived from the lactoferrin ingested from human milk or partially from the lactoferrin produced by the baby in response to human milk.

 

2.2 ANTIMICROBIAL ACTION

It is a known fact that lactoferrin has a role in the growth of microorganisms. Very many mentioned studies demonstrated the in vitro bacteriostatic effect and in some cases the bactericide effect of lactoferrin on a large variety of microorganisms, including gram-positive and gram-negative, aerobe, anaerobe bacteria, fungi and viruses, as are the human cytomegalovirus (HCMV), the human herpes simplex virus (HSV-1) and the human immunodeficiency virus (HIV). The bacteriostatic effect of lactoferrin was associated with its ability to bind iron. Almost all bacteria need iron and as a result of its binding properties, lactoferrin can impede the use of iron by bacteria. Other mechanisms are also involved in lactoferrin’s action, namely: the blockage of carbohydrates metabolism to microbes or the destabilization of the bacteria cell wall, probably by binding the calcium and magnesium and also through an interaction between lactoferrin and microorganisms. Lactoferrin acts together with other antibacterial proteins such as the lysozyme in milk. In this case, even the saturated form of iron is active and its inhibition comes from the agglutination of the bacteria done by the lactoferrin, whose cellular walls have been modified by the lysosomes.

In vivo, lactoferrin from milk can exercise its inhibitory effect on the microbial growth in the newborn’s intestine. It is frequently suggested that the antimicrobial action of lactoferrin plays an important role in the selection of the intestinal flora in a newborn and prevents the colonization with enteropathogenic organisms. Anyway, if the tests of establishing lactoferrin’s in vivo antimicrobial role are often contrasting, this happens mainly because of the protocols used by scientists. In fact, it is not at all easy to analyze what happens in vivo in the intestine and to make a correlation with the analysis of the fecal. But one can observe a difference between an inhibition state of the bacteria in the intestine and the same bacteria coming from the fecal samples cultivated in a rich environment (that anyway contains iron). All the analysis systems used by different scientists can lead to different results.

This remark does not refer to comparing the intestinal flora of a breast-fed newborn with that of a milk formula fed newborn. Nonetheless, all scientists have agreed upon that human milk is the ideal reference that contains not only lactoferrin, but also other agents that are completely absent in the commercial formulas. This remark implies the feeding of a newborn with a formula whose sole difference from a group to another is the presence of lactoferrin and/or inorganic iron.

With the help of the European Community there has been conducted a study on children with chronic diarrhea. The results of the test have shown the efficacy of lactoferrin combined with the lysosome added to the milk formula, without concentrating on the intestinal flora, but just on physiological criteria induced by the pathological status of children.

J.J. Bullens: “The significance of iron in infection”, National Institute for Medical Research, London, 1981 published in Biochimica et Biophysica Acta. 718 (1982)42-48

Abstract: Proteins that bind iron, as lactoferrin and transferrin restrict the quantity of ionic iron available in the bodily fluids at 10 -18 M. this quantity is not sufficient for the normal growth of the bacteria and the pathogenic ones obtain iron either by producing iron chelator agents, or by using the hem’s components. The proteins that bind iron, combined with antibodies often have a bacteriostatic in vitro effect and are essential to the protection against many infections. It appears that lactoferrin is essential for the polymorphonuclear leucocytes’ bactericide action against. The fever reduces the quantity of iron in the serum and favors the resistance to infection. The release of the hem’s components can amplify the clinical infections.

  1. T. Ellisson III, Th. J. Giehl et all. :”The destruction of the external membrane of gram-negative Enterobacteriaceae by lactoferrin and transferrin” Medical Service, Veterans Administration medical Center, and Division of infectious diseases, Department of Medicine, University of Colorado School of Medicine, Denver, Colorado.

Abstract: Many studies have shown that lactoferrin and transferrin have an antimicrobial action against the gram-negative bacteria, but the action mechanism was not completely defined. It is assumed that the protein that binds iron can affect the external membrane of the gram-negative bacteria in a similar way of that of EDTA chelator. The lactoferrin’s and transferrin’s property of releasing radioactive marked lipopolysaccharides (LPS) from the UPD – epimerase deficient galactose of mutant Escherichia coli and wild Salmonella typhimurium was tested. Initial studies made on barbital acetate have shown that EDTA and lactoferrin produce significant releases of LSP in all 3 species.  The studies have shown that the release of LPS was blocked by iron saturated lactoferrin produced at a pH between 6.0 - 7.5, which lead to a decrease of the bacterial concentration at 104 and 107 CFU/ml because of the growth of the lactoferrin concentration. Studies based on the use of the Hank reactive with salt, without calcium and magnesium have shown that also transferrin can release LPS. In addition, lactoferrin and transferrin enhance the antibacterial effect of the sub-inhibitory concentration of rifampin, o substance that does not penetrate the external membrane of the bacteria. This experiment demonstrates that these proteins that bind iron destroy the external membrane of the gram-negative bacteria and affect the permeability of the external bacterial membrane.

  1. Zagulski. P. Lipiriski. A. Zagulska. S. Broniek and Z. Jarzabek: “Lactoferrin can protect the mice against the lethal dose of Escherichia coli in in vivo experimental infections”, published in Br. J; Exp. Path. (1989) 70. 697-704

Abstract: Experiments were undertaken to demonstrate and partially explain the protective effect of bovine lactoferrin (LB) when administered intravenously to mice 24 h before a challenge with a lethal dose of Escherichia coli. About 70% of mice pretreated with LB survived challenge. The survival rates in control mice treated with E. coli alone and pretreated with bovine serum albumin (BSA), were 4 and 8%, respectively. Human lactoferrin (LH) had almost the same protective effect as LB. Sufficient amounts of ferric ions were given to mice, in single and multiple doses, for full serum transferrin saturation 30 min before or after E. coli administration. The multiple dose of ferric ions did not change considerably the survival rate of mice pretreated with LB. In contrast, a single dose of ferric ions gradually decreased the survival rate of the mice after the first week of experiment. From day 14 this decrease was statistically significant in all groups of mice treated with a single dose of ferric ions when compared with mice pretreated only with LB, and the difference ranged from 25 to 35% on day 30. The possible mechanism(s) of protective effect of LB and role of iron ions are discussed.

Smita S. Naidu1, Janos Erdei, Eva Czirek et all.: “The specific binding of lactoferrin in Escherichia Coli isolated in the human intestine”, Departments of Medical Microbiology and Infectious Diseases, Malme General Hospital, University of Lund, Suedia – published in Nutrition Research (1983) 3, pp.373-384

Abstract: The degree of human (HLf) and bovine (BLf) lactoferrin’s binding was examined in 169 species of Escherichia Coli isolated in the human intestine. Statistically, the binding of the HLf and BLf onto the cellular walls of the bacteria situated between 3.7 - 73.4%, respectively 4.8 - 61.6%, the binding being stronger for BLf than for HLf (p <0.001). The enterotoxin species have shown a HLf binding significantly higher than the enteropathogenic ones (p <0.01), entero-invasive (p <0.001) or enterohemorrhagic (p <0.01). The enteropathogenic species belonging to the serotypes 044 and 0127 have shown a significantly higher binding in HLf compared to serotypes 026, 055, 0111, 0119 and 0126.

After that Lf’s binding mechanism was studied in an enteropathogenic specie, E34663 (serotype 0127), that demonstrated a strong binding for both lactoferrin types, HLf and BLf. The binding was stable at a pH between 4.0 and 7.5 and was not affected by the presence of sodium chlorate 2M or urine 2M solutions, achieving complete saturation in 110 minutes. The interaction between lactoferrin and the wall can be covered depending on the dosage by either one of the HLf or BLf. The apoformas and the iron saturated forms of lactoferrin manifested similar bindings. The inhibition of the tie with different types of proteins and carbohydrates suggested the specific nature of the interaction. The Scatchard – Plot test showed 5400 spots of specific binding of HLf per cell, with a constant affinity (Ka) of 1.4 x 10"7 M. The data showed the existence of a specific binding mechanism of lactoferrin to Escherichia Coli isolated in the human intestine.

Gun-Britt Fransson, Cari L. Keen, and Bo Lennerdal: “Supplementation of Milk with Iron Bound to Lactoferrin Using Weanling Mice: I. Effects on Hematology and Tissue Iron”, Department of Nutrition, University of California, SUA – published in Journal of Pediatrics Gastroenterology and Nutrition (1983) 2:693-700

Abstract: Lactoferrin is an iron-binding protein present in high concentrations in human milk. The efficacy of supplementing iron bound to lactoferrin to iron-deficient and iron-sufficient young mice was evaluated in comparison with supplementation of iron as iron chloride. Mice fed a non-supplemented milk diet (1 mg Fe/L) for 4 weeks had a microcytic, hypochromic anemia and low tissue iron concentrations. Iron supplementation of the diet with lactorferrin-iron. or iron chloride at a level of 5 mg Fe/L prevented the anemia and resulted in tissue iron levels similar to levels found for mice fed a stock commercial diet. There was no significant difference in any of the parameters analyzed between the groups of mice receiving the two iron supplements following a diet deficient in iron. Apolactoferrin when supplemented to the diet had no negative effect on the iron status of the mice. These results show that lactoferrin may be a useful vehicle for supplementation of iron.

 

2.3. BACTERIA INHIBITION BY LACTOFERRIN AND OTHER IRON CHELATING AGENTS

In their iron non-saturated form, human, bovine and goat lactoferrin have manifested a bacteriostatic action on Bacillus stearothermophilus and Bacillus subtilis, both in the presence and absence of insignificant quantities of metals. Bovine lactoferrin inhibited the spores’ germination and the growth of the vegetative forms of B. stearothermophilus. The bacteriostatic action of lactoferrin was suppressed by the iron (II) ion and amplified by the zinc (II), cobalt (II), Mangan (II), nickel (II) and copper (II) ions. B. stearothermophilus was also inhibited by 8-hidroxichinolina and I,10-fenantrolin. Other agents that bind iron, including some transferrin did not have an inhibiting action. B. stearothermophilus and T. subtilis were also inhibited by a complex with big molecular weight isolated from cow’s milk that did not breast feed. The complex contained lactoferrin and casein and its action was suppressed by adding iron and it had a slightly higher stability than purified lactoferrin and it didn’t deposit because of the lactoferrin’s antibodies action.

  1. T. Allison, F. M. LAFORGE, Th.J. Ghiehl et all.: ”The destruction of the gram-negative’s external membrane by Ca2+ and Mg2+ modulated lactoferrin and transferrin”, Department of Medicine, University of Colorado

The lactoferrin and transferrin have an antimicrobial action against some gram-negative bacteria, but their action mechanism has not been defined. This study aims at evaluating lactoferrin’s and transferrin’s property to destroy the gram-negative bacteria’s external membrane and to show how this is done. The release of LPS by the proteins can be blocked by adding calcium and magnesium ions. Adding Ca2+ blocked lactoferrin’s ability to rise Escherichia coli’s sensitivity to rifampicin. As opposed to lactoferrin, the transferrin raised the gram-negative bacteria’s sensitivity to deoxycholate, with an inversion of the sensitivity to the exposure to Ca2+ or Mg2+. When studied at the electronic microscope, polymyxin B produced deformations to the bacteria’s membrane, but there were no morphologic alterations in the cells exposed al EDTA, lactoferrin or transferrin.

Soukka T, Lumikari M, Tenovuo J: “Combined inhibitory effect of lactoferrin and lactoperoxidase system on the viability of Streptococcus mutans, serotype C” – published in Scand J Dent (1990) 98: 125 - 129

Abstract: We have studied the effects of iron-free lactoferrin (apo LF) and lactoperoxidase system (lacto-peroxidase, LP/SCN-/H2O2), separately and together, on the viability of Streptococcus mutans (serotype c) in vitro. The bacteria were incubated in buffered KCl (pH 5.5) with and without the above components which were used at concentrations normally present in human saliva. Both apo LF and LP-system had a bactericidal effect against S. mutans at low pH. Together they showed an additive, but not a synergistic, antibacterial effect against S. mutans. Apo LF enhanced the LP enzyme activity but decreased the yield of the antimicrobial component, hypothiocyanite (HOSCN/OSCN-), when incorporated into the reaction mixtures. This decrease, which was most pronounced at low pH, was due to an LP-independent reaction between apo LF and HOSCN/OSCN-. Our study indicates that the LP-system and apo LF can be combined to combat oral S. mutans.

  1. Satyanarayan Naidu, J. Miedzobrodzki, M. Andersson et all.: “Bovine lactoferrin binding to six species of coagulase-negative staphylococci isolated from bovine intra-mammary infections”, Journal of Clinical microbiology, (1990) 95, 2312 - 2319

Abstract: Bovine lactoferrin (BLf), an acute-phase iron-binding secretary protein present in secretions of the bovine udder, was demonstrated to bind to the following staphylococcal species associated with bovine intra-mammary infections: S. epidermidis, S. warneri, S. hominis, S. xylosus, S. hyicus, and S. chromogens. The degree of '251-labeled BLf uptake significantly varied among the blood agar-grown cells of all six species of coagulase-negative staphylococci tested. Isolates identified as S. xylosus demonstrated the highest (mean, 35.1 X 106 ± 13.3 x 106 nmol) and S. hyicus the lowest (mean, 10.7 X 106 ± 5.9 x 106 nmol) binding to 125I- BLf. BLf binding was optimum at an acidic pH, with time-dependent binding saturation ranging from 70 min for S. warneri to 240 min for S. hominis. The BLf-binding mechanism was specific, with affinity constants (Ka values) ranging between 0.96 x 106 and 11.90 x 106 liters/mol. The numbers of BLf-binding sites per cell, as determined by using Scatchard analysis, were as follows: S. epidermidis, 3,600; S. warneri, 1,900; S. hominis, 4,100; S. xylosus, 4,400; S. hyicus, 6,100; and S. chromogens, 4,700. '251-BLf binding to all species was inhibited by unlabeled BLf and unlabeled human lactoferrin, whereas none of the various plasma, connective tissue, or mucosal secretory proteins or carbohydrates tested caused significant interference. BLf-binding receptors of the six coagulase-negative staphylococcal species demonstrated marked differences in patterns of susceptibility to proteolytic or glycolytic enzyme digestion and to heat or periodate treatment. These data suggest that the BLf-binding components in S. epidermidis and S. warneri are proteins containing glycidyl residues. In the remaining four species, the proteinaceous nature of the BLf-binding component was evident, but the involvement of glycidyl residues was not clear. Results of this study establish the presence of specific binding components for BLf on coagulase-negative staphylococci isolated from bovine intra-mammary infections.

  1. Hasegawa, W. Motsuchi, S. Tanaka, et all.: “Inhibition with lactoferrin of in vitro infection with human herpesvirus (HSV – 1)– Journal of Medical Science and Biology, (1994) 47, 73 – 85

Abstract: Human lactoferrin (hLF) as well as bovine lactoferrin (bLF) inhibited infection of tissue culture cells with human cytomegalovirus (HCMV) and human herpes simplex virus-1 (HSV-1). The addition of lactoferrin (LF) inhibited both in vitro infection and replication of HCMV and HSV-1 in human embryo lung host cells. The maximum inhibition by more than six exponentials of TCID50 for HCMV and four exponentials for HSV-1 was obtained at a concentration in a range from 0.5 to 1 mg of LF per ml of medium. The antiviral activity of LF was associated with its protein moiety, but not with its iron molecule or sialic acid. None of other transferrin gene family members bound to ferrous ions or sialic acid possessed significant antiviral activity. Additionally, we found that LF prevented virus adsorption and/or penetration into host cells, indicating an effect on the early events of virus infection. Preincubation of host cells with LF for 5 to 10 min was sufficient to prevent HCMV infection, even when LF was removed after addition of virus. These results suggest that LF possesses a potent antiviral activity and may be useful in preventing HCMV and HSV-1 infection in humans.

  1. Kawakami, M. Hiratsuka, S. Dasaxo: “Effects of Iron-saturated Lactoferrin on Iron Absorption”, Snow Brand Milk Products Co.. Ltd.. Technical Research Institute, Japan

Abstract: Iron absorption from iron-saturated lactoferrin was compared to that from ferrous sulfate in iron-deficient anemic rats. One group of rats was given 50 µg of iron orally once a day and changes in red blood cell density, hematocrit, and hemoglobin values were measured at 14-day intervals for 70 days. A statistically significant increase in these values was demonstrated for the rats fed iron-saturated lactoferrin (50 µg Fe/35mg lactoferrin/day), while the ferrous sulfate group showed no improvement in these values. The results suggest that iron from iron-saturated lactoferrin is absorbed across the intestinal mucosa by a mechanism other than the one by which soluble iron salts are absorbed.

 

2.4. LACTOFERRIN’S EFFECT ON THE ITESTINAL FLORA

The results of many experiments have given solid arguments in favor of supplementing milk formulas with bovine lactoferrin.

On the other side, it is important to highlight that one of the most used medical practices for preventing premature anemia in the new-born is supplementing the powder milk with iron, an essential nutrient for the development of the body and the reduction of the pathogen intestinal flora. Dr Mevissen-Verhage established that the new-born fed with a milk formula enriched with inorganic iron favored the development of the pathogen intestinal bacteria and in many cases the babies had intestinal inflammations. The results obtained by Dr Mevissen drove him to depose at the European Community Directive no 420/86 asking to stop any inorganic iron supplementation of the powder milk formulas destined for the new-born.

E.A.E. Mevissen-Verhage, J.H. Marcelis, W.C.M. Harmsen-Van Amerongen, N.M. de Vos, J.Verhoef: “ The effect of iron on the new-born intestinal flora in the first 3 months of life”, Journal Officiel des Communautes Europennes, 28th of May 1986

Abstract: In order to study the effect of the milk supplemented with iron on the intestinal flora of the new-born, feces from 10 breast-fed babies, 6 babies that received bovine milk preparation supplemented with iron (5mg/l) and 7 that received unfortified cow’ milk preparation. (iron concentration<0,5 mg/l) have been studied in the first 12 weeks of life. Those on breast milk had low fecal pH, high counts of bifid-bacteria and low counts of Enterobacteriaceae, Bacteroides and clostridia. Infants receiving fortified cow-milk preparation had a high fecal pH and high counts of Enterobacteriaceae and putrefactive bacteria such as Bacteroides and Clostridia. Counts of bifid-bacteria were also high. In those on unfortified cow-milk preparation a slow rise was observed in counts of Enterobacteriaceae followed by an increase in counts and isolation frequency of bifid-bacteria: the latter was still rising on day 7. It is concluded that a low iron content in standard preparations of cow's milk enhances resistance of the neonatal gut to colonization.

2.5. THE TROFIC EFFECT OF LACTOFERRIN ON THE INTESTINE

It is long known that the colostrum and the milk of many species favor the development of the intestines. Widdowson and many other scientists like Berseth, Heird established that the milk and the colostrum that piglets, mice, rabbits and dogs are fed with increase the weight and length of the intestines and the stomach compared to the animals fed with an artificial diet. This effect of natural feeding was well documented, suggesting that the milk and the colostrum can be important for the adaptation of the sick species to the ectopic life. The milk component responsible for the stimulation has been identified by Nichols as being lactoferrin. In fact, Nichols showed that the presence of lactoferrin could stimulate the taking over of the thymidine marked in the cells’ DNA, suggesting lactoferrin’s part as a promotor of growth in the process of the gastro-intestinal tract maturation.

The formulas based on bovine milk, soya and hydrolyzed casein inhibited the DNA, but adding lactoferrin to the formula decreased this effect. This test was made with a baby’s milk formula    that contains lactoferrin and that is now marketed.

On the other side, a test of this role of bovine lactoferrin was conducted in vivo on children that were hospitalized for gastro-intestinal issues. Using the biopsy, pediatricians showed that the babies that received the lactoferrin experienced a beneficial renewal of the enterocytes in the recovery phase of an acute diarrhea.

Moreover, lactoferrin has bifidogenic properties. Lactoferrin has an effect on the growth of the bifidobacterial populations, as shown further.

  1. Saito, H. Mtyakawa, N. Ishibashi, et all.: “The effect of free or bind lactoferrin forms on the growth of Bifidobacterium, Escherichia. coli and Staphylococcus aureus “, Nutritional Science Laboratory, Japan,

Abstract: The effects of the iron free lactoferrin (apo-LF) and of some forms of this protein’s bind metals (Fe-LF, Co-LF, Zn-LF) on the growth of Bifidobacterium, Escherichia coli and Staphylococcus aureus have been investigated. Apo-LF, Co-LF and Zn-LF have inhibited the growth of E. coli and S. aureus. The inhibiting effect of Co-LF and Zu-LF was much stronger that apo-LF’s, Fe-LF being inefficient or less efficient. On the other side, apo-LF, Co-LF and Fe-LF stimulated the growth of the species of Bifidobacterium, as indicated by the growth in OD culture of the number of viable cells. This growth stimulation effect disappeared when the lactoferrin was hydrolyzed by pancreatin. 

Lactoferrin (LF) has an antibacterial action, stimulates the iron absorption, influences the immune system and acts as a growth factor for some animal tissues. The antibacterial action of lactoferrin was explained by its property to bind and steal iron that is essential for the growth of bacteria. Recent studies have shown that the LF molecule acts directly on the bacterial cells. An antibacterial fragment was isolated from the peptic digestion of LF and its structure was determined. The antibacterial mechanism of LF remains unclear.  While native LF can bind metals, as it has 2 spots per molecule that can bind a metal ion, it is of real interest to study the influence of the metal forms of LF on the growth of bacteria.

The bifid-bacteria is considered one of the most beneficial bacteria for the human and animal intestine, while E. coli and S. aureus are potentially harmful bacteria. The effects of different forms of LF on these bacteria have been compared.

Lactoferrin, a milk antibacterial protein, is known as an inhibitor of the growth of a large variety of microorganisms. Its antibacterial mechanism was attributed to its iron binding properties, iron being essential to the microbial growth. In our experiments, apo-LF, Co-LF and Zn-LF considerably inhibited the growth of de E. coli and S. aureus. Also, a bactericide effect of apo-LF and Co-LF was revealed for E. coli. While Fe-LF didn’t inhibit the growth and didn’t have an anti-bactericide effect, it seems that the antibacterial effect of LF is based on its property to bind iron. Anyway, it is very difficult to explain the difference between the effects of apo-LF, Co-LF and Zn-LF. Also, the bactericide effect of LF appears to be different regarding the mechanism of iron elimination. In 1992, Bellamy and his contributors isolated a fragment with bactericide properties of a peptic digestion of LF. They determined the structure of this peptide and named it “lactoferricin”. They supposed that LF’s bactericide effect is mediated by this fraction of the molecule. The differences between the antimicrobial properties on binding different metal ions can result from the changes in the 3D structure of the LF molecule. However, how does LF inhibit the growth and how does it manifest its bactericide action remains unclear. In order to clarify the antibacterial mechanism, it appears that it’s necessary to further investigate the connection between the antibacterial action and the 3D structure of apo-LF and its metal bind forms. Also, the behavior of the metals bind in the growth environment should be studied.

It has been seen that the growth of the bifid-bacteria is stimulated by adding apo-LF, Co-LF, or Fe-LF. From the tested species, B. breve and B. bifidum were the most sensitive. When a pancreatic digest of LF was added to the environment, there was no seen effect of growth stimulation.  It is hard to explain LF’s growth mechanism on the bifid-bacteria. Enzymatic digested LF didn’t show this property, it constituents (amino acids, the bind carbohydrate complex) appearing to be inefficient. Bellamy and his contributors have tested lactoferrin’s bactericide effect and showed its strong inhibitor effect on the growth of many types of bacteria, including E. coli, clostridium, bacilli and others, while not showing any significant action on the bifid-bacteria. Taking into consideration the prevalence of LF in the environment, for example as a component of breast milk and saliva and its opposed effects on bifid-bacteria and E. coli or S. aureus, one can say that LF may be important in connection with the gastro-intestinal health of humans.

2.6. LACTOFERRIN’S ROLE IN TRANSPORTING IRON

Until 6 months, the iron deficit was seldom seen in breast fed newborns, as they are born with a good iron reserve. The piglets don’t get anemia as fast because the pigs’ milk contains more iron than the humans’.

Not being sustained by the low iron content, the high bio-availability of iron in the human milk becomes a good source of iron compared to the bovine or goat milk with comparable iron levels. It is generally known that the human milk is a better source of iron than the bovine milk or bovine milk preparations even if the total amount of iron is similar. Schulz has repeatedly infused marked iron to cows and fed babies with the milk produced by them. The percentage of iron absorption was calculated from the recovery of marked iron in the feces. The balance of these studies showed that between 3 and 17% of iron was used by the babies and iron retention was better correlated with the hemoglobin formation. Babies with iron deficiency have absorbed 3-4 times more iron than those without deficiency and 3 times more than the adults. The conclusion that was reached was that anemia in babies fed with cow’s milk was caused by a low iron content and an insufficient iron use. The bio-availability of iron coming from cow’s milk was not only low, but the milk showed it was directly interfering with the iron intake. Human milk doesn’t only interfere with the iron absorption, but it also reduces it from 18 to 8,5% in babies with normal iron deposits and from 26 to 8,5% in those without deposits. In contrast with inorganic iron, hemoglobin as an alternative iron source was not been inhibited by bovine milk. Heinrich discovered that some babies absorb around 18% on a 5mg inorganic iron dose administered from human milk and only 4,5% from bovine milk. Saarinen confirmed this data by dosing the traces of marked iron either during breast-feeding (I), after breast-feeding (II) or during the feeding with pasteurized bovine milk (III). Iron absorption was measured in the whole body. Iron absorption for group (I) was 48%, for (II) 38% and for (III) just 20%.

One of the first attempts of associating iron absorption with LF was made by Fransson and his colleagues. They showed that iron absorption was as efficient from iron saturated LF as the one from the iron sulfate in naturally anemic piglets and experimental diet anemic mice. The discovery of a specific LF receptor sustains the argument that this protein plays an essential role in iron absorption. On the other side, Vet showed that apo-LF has an inhibiting effect in iron absorption and concluded that LF can play a role in regulating the intestinal iron absorption inhibition for the prevention of excess metal absorption. The conclusion is that LF controls more than stimulates the iron absorption in newborns. If other experiments about LF’s role in iron absorption are subject to controversy, it is important to place the illnesses in this experiment in context. In fact, it is very difficult to compare the in vivo results of these experiments by using different types of patients. The most controversial experiments are those referring to the LF’s iron transporting role and shows that iron binding LF’s bio-availability was no better than that of inorganic iron.

 

  1. RESEMBLNCE BETWEEN BOVINE AND HUMAN LACTOFERRIN

3.1. STRUCTURE

There are now known the complete primary structures of human and bovine lactoferrin. These two molecules are made of an only one polypeptide chain of 689 and 692 amino acids. The human lactoferrin sequence has 69% resemblance with the bovine. All lactoferrins are glycoproteins. The difference between human and bovine lactoferrin resides in number of glycosides types:  the human LF has 2 glycosides sites and the bovine has 4. The human LF glycan structure was discovered to be heterogeneous because the biantennary glycans N-acetyl-lactose-amine contain 1 or 2 sialic acids and 1 or 2 fuscous residues. The bovine LF glycan structure is heterogenous. In this molecule, the oligomannosidic glycans contain between 3 and 9 mannose residues.

3.2 ACTION

Tomita and contributors have demonstrated that the active part of LF responsible for its antimicrobial action is located in the first 52 amino acids. From another point of view, Dr Spick showed that the sequence that ties LF to the LF receptor of different cells as are the enterocytes was located in the region from 4 to 52. Because the comparative analysis pf the primary structure of the polypeptide chain on the residues from 4 to 53 from bovine LF presents a high resemblance percentage with human LF, it was assumed that the bovine LF from bovine source is recognized by the human LF receptors. Dr Spick demonstrated that the inhibition in humans and cows was effectively similar and didn’t take place in other types of lactoferrins, such as mice. It was also shown that when you compare the conformation of the peptides from 4 to 52 one can observe an important resemblance, which indicates that the human and bovine form LF peptides are identical. These results indicate that the bovine LF is capable of recognizing the human LF receptors and concur the human LF molecules. This suggests that in vivo, the human and bovine LF should play the same biological roles.

 

  1. CONCLUSIONS

The main reason for adding iron in a product is to stop or prevent the anemia related issues in children. We now have the possibility to replace this iron by a natural molecule, a protein that binds iron. LACTOFERRIN is now produced at an industrial scale. Its use will allow the powder babies’ milk formulas to be more human and the milk structure to resemble the human milk.

Anyway, there are differences between the studies of different groups of scientists in regard to the important issue of adding iron to the final produces.

  • The growth of the pathogen intestinal flora
  • The production of free radicals as the hydroxyl
  • The occurrence of the intestinal inflammations
  • The iron transport, even if it is not necessary, can create hematocrits.

On the other hand, it was proven that bovine lactoferrin that has a big resemblance to human lactoferrin, can be used for iron transportation. But bovine LF can also remove the iron addition issue and:

  • Favorizes the intestinal flora environment
  • Eliminates the production of free radicals by binding iron
  • Regulates iron transportation, also preventing the metal’s excess concentration in the body
  • Eliminates the intestinal inflammation problems

Dr Jean-Paul Perraudin

 

  1. BIBLIOGRAPHY