lactoferrin and ferric-chloride

The iron-lactoferrin complex is a common food ingredient because of its iron-solubilizing capability in the presence of hydrogen carbonate ions. However, it is unclear whether the formation of a stable iron-binding complex is limited to lactoferrin. In this study, we investigated the effects of bovine serum albumin (BSA) on iron solubility and iron-catalyzed lipid oxidation in the presence of hydrogen carbonate ions. BSA could solubilize >100-fold molar equivalents of iron at neutral pH, exceeding the specific metal-binding property of BSA. This iron-solubilizing capability of BSA was impaired by thermally denaturing BSA at ≥ 70 °C for 10 min at pH 8.5. The resulting iron-BSA complex inhibited iron-catalyzed oxidation of soybean oil in a water-in-oil emulsion measured using the Rancimat test. Our study is the first to show that BSA, like lactoferrin, forms a soluble iron-binding complex in the presence of hydrogen carbonate ions.

Topics: Antioxidants; Bicarbonates; Chlorides; Emulsions; Ferric Compounds; Hydrogen-Ion Concentration; Iron; Lactoferrin; Lipid Metabolism; Oxidation-Reduction; Serum Albumin, Bovine; Solubility

Lactoferrin (Lf), present in colostrum and milk is a member of the transferrin family of iron-binding glyco-proteins, with stronger binding capacity to ferric iron than hemoglobin, myoglobin or transferrin. Unlike hemoglobin and myoglobin, iron-bound Lf is reasonably stable to gastric and duodenal digestive conditions. Unlike ferrous iron, ferric iron is not directly reactive with oxygen supporting the capacity of Lf capture of heme iron to suppress reactive oxygen species (ROS) production. We therefore hypothesized that bovine Lf could capture and thereby terminate the cycle of ROS production by heme iron. The transfer of heme iron from either intact or digested forms of hemoglobin and myoglobin and from intact ferritin was demonstrated by in vitro methods, monitoring Fe-saturation status of Lf by changes in absorptivity at 465 nm. The results are discussed in the context of new proposed opportunities for orally administered Lf to regulate oxidative damage associated with heme iron. In addition to potentially suppressing oxidative heme-iron-mediated tissue damage in the lumen, Lf is expected to also reverse the overload of ferritin-bound iron, that accompanies chronic inflammation and aging. These new proposed uses of Lf are additional to known host defense functions that include anti-microbial, anti-viral properties, immune and cancer cell growth regulation. The findings and interpretations presented require clinical substantiation and may support important additional protective and therapeutic uses for Lf in the future.

Topics: Animals; Biological Transport; Cattle; Chlorides; Duodenum; Electrophoresis, Polyacrylamide Gel; Ferric Compounds; Ferritins; Heme; Hemoglobins; In Vitro Techniques; Iron; Iron, Dietary; Lactoferrin; Models, Biological; Myoglobin; Oxidation-Reduction; Oxidative Stress; Reactive Oxygen Species

Secretory diarrhea caused by cholera toxin (CT) is initiated by binding of CT's B subunit (CTB) to GM1-ganglioside on the surface of intestinal cells. Lactoferrin, a breast milk glycoprotein, has shown protective effect against several enteropathogens. The aims of this study were to determine the effect of bovine-lactoferrin (bLF) on CT-induced intestinal fluid accumulation in mice, and the interaction between bLF and CT/CTB with the GM1-ganglioside receptor. Fluid accumulation induced by CT was evaluated in the mouse ileal loop model using 56 BALB/c mice, with and without bLF added before, after or at the same time of CT administration. The effect of bLF in the interaction of CT and CTB with GM1-ganglioside was evaluated by a GM1-enzyme-linked immunosorbent assay. bLF decreased CT-induced fluid accumulation in the ileal loop of mice. The greatest effect was when bLF was added before CT (median, 0.066 vs. 0.166 g/cm, with and without bLF respectively, p<0.01). We conclude that bLF decreases binding of CT and CTB to GM1-ganglioside, suggesting that bLF suppresses CT-induced fluid accumulation by blocking the binding of CTB to GM1-ganglioside. bLF may be effective as adjunctive therapy for treatment of cholera diarrhea.

Topics: Animals; Cattle; Chlorides; Cholera Toxin; Dose-Response Relationship, Drug; Enterotoxins; Escherichia coli; Female; Ferric Compounds; G(M1) Ganglioside; Gangliosides; Intestinal Mucosa; Intestines; Lactoferrin; Mice; Protein Binding; Receptors, Cell Surface

Lactoferrin, a multifunctional protein with antimicrobial activity, is a component of the innate immune system. It may possibly prevent clinical isolates of Pseudomonas aeruginosa from developing biofilm, but this hypothesis is yet to be widely accepted. We evaluated the in vitro effects of lactoferrin on biofilm formation by various clinical isolates of P. aeruginosa using a modified method of the microtiter plate biofilm assay. Lactoferrin significantly inhibited biofilm formation in these isolates. The effect was the most marked at 2 mg/ml, which suggested that an optimal concentration of lactoferrin might exist. Lactoferrin inhibited biofilm formation in eight of nine clinical isolates after 1 day of incubation; however, the inhibitory effects were maintained until 7 days of incubation in only two of those eight strains. Suppression of biofilm formation may be caused by a mechanism that is independent of the bactericidal effects of lactoferrin because the number of viable bacteria was not influenced by lactoferrin under the experimental conditions. Supplementation of lactoferrin to preformed biofilm demonstrated a reduction in biofilm, which suggests that lactoferrin may have a destructive effect on biofilm. Pretreatment with ferric chloride partially restored biofilm formation, suggesting an iron-chelating action may be involved in the inhibitory mechanism of lactoferrin. These results suggest that lactoferrin provides inhibitory effects on biofilm formation in many clinical isolates of P. aeruginosa and that it may also have destructive effects on preformed biofilm, but further research using multiple clinical strains should be undertaken to clarify if those effects are universal.

Topics: Anti-Bacterial Agents; Biofilms; Cell Proliferation; Chlorides; Ferric Compounds; Humans; Lactoferrin; Microbial Sensitivity Tests; Pseudomonas aeruginosa; Pseudomonas Infections

Lactoferrin (Lf), an iron-binding salivary glycoprotein, plays an important role in human innate defense against local mucosal infection. We hypothesized that Lf interferes with initial oral bacterial attachment to surfaces by iron sequestration, so inhibiting subsequent biofilm formation. The objective was to investigate the effect of Lf on the early stages of single-species and multi-species oral biofilm development.. Streptococcus gordonii, Streptococcus mutans, Fusobacterium nucleatum and Porphyromonas gingivalis were used in this study. Glass disks of a two-track flow cell coated with flowing artificial saliva (0.3 ml/min) with and without Lf (100 microg/ml) were used for studying bacterial attachment (3 h, 37 degrees C). Attachment was also examined by incubating single or multiple species of test bacteria (10(7) colony-forming units/ml) with Lf-coated (20-100 microg/ml) and uncoated glass slides. The effects of beta-lactoglobulin, 2,2'-dipyridyl (25-100 microg/ml), an iron chelator, and FeCl3 on attachment were also examined.. Lf inhibited the initial attachment of S. gordonii (50.3%, P < 0.05) but not that of F. nucleatum and P. gingivalis. However, the attachment of a dual-species biofilm containing S. gordonii (i.e. S. gordonii/F. nucleatum or S. gordonii/P. gingivalis) was significantly reduced (48.7% or 62.1%, respectively, P < 0.05) in the presence of Lf. beta-Lactoglobulin did not affect the attachment of S. gordonii. In the presence of 100 microm 2,2'-dipyridyl, attachment of S. gordonii was reduced by 53.87%. No reduction in attachment was noted in S. gordonii pretreated with Lf (100 microg/ml) and FeCl3 (20-200 microm).. Lf suppresses initial attachment of S. gordonii and S. gordonii coaggregates by iron sequestration. This may lead to subsequent inhibition of oral biofilm development.

Topics: 2,2'-Dipyridyl; Bacterial Adhesion; Biofilms; Chlorides; Colony Count, Microbial; Ferric Compounds; Fusobacterium nucleatum; Humans; Image Processing, Computer-Assisted; Iron Chelating Agents; Lactoferrin; Lactoglobulins; Microbial Sensitivity Tests; Mouth; Porphyromonas gingivalis; Saliva, Artificial; Salivary Proteins and Peptides; Streptococcus; Streptococcus gordonii; Streptococcus mutans

Actinobacillus actinomycetemcomitans, a pathogen associated with oral and extra-oral infections, requires iron to grow under limiting conditions. Although incapable of producing siderophores, this pathogen could acquire iron by direct interaction with compounds such as haemin, haemoglobin, lactoferrin and transferrin. In this work the ability of different A. actinomycetemcomitans strains to bind and use different iron sources was tested. None of the strains tested used haemoglobin, lactoferrin or transferrin as sole sources of iron. However, all of them used FeCl(3) and haemin as iron sources under chelated conditions. Dot-blot binding assays showed that all strains bind lactoferrin, haemoglobin and haemin, but not transferrin. Insertion inactivation of hmsF, which encodes a predicted cell-envelope protein related to haemin-storage proteins produced by other pathogens, reduced haemin and Congo red binding drastically without affecting haemin utilization as an iron source under chelated conditions. Biofilm assays showed that all strains tested attached to and formed biofilms on plastic under iron-rich and iron-chelated conditions. However, scanning electron microscopy showed that smooth strains formed simpler biofilms than rough isolates. Furthermore, the incubation of rough cells in the presence of FeCl(3) or haemin resulted in the formation of more aggregates and microcolonies compared with the fewer cell aggregates formed when cells were grown in the presence of the synthetic iron chelator dipyridyl. These cell responses to changes in extracellular iron concentrations may reflect those that this pathogen expresses under the conditions it encounters in the human oral cavity.

Topics: 2,2'-Dipyridyl; Aggregatibacter actinomycetemcomitans; Bacterial Outer Membrane Proteins; Biofilms; Chelating Agents; Chlorides; Electrophoresis, Polyacrylamide Gel; Ferric Compounds; Hemin; Hemoglobins; Humans; Iron; Iron Compounds; Lactoferrin; Microscopy, Electron, Scanning; Mutation; Protein Binding; Species Specificity; Stomatognathic Diseases

Human serum has been reported to inhibit the growth of several fungal pathogens. Serum inhibitory activity has predominantly been associated with transferrin-mediated iron binding. We found that serum from several animal species (human, dog, mouse and fetal bovine) inhibited the growth of the dimorphic fungal pathogen, Blastomyces dermatitidisin vitro. In initial studies, we found no correlation between the total iron-binding capacity of various sera and their inhibitory activity against B. dermatitidis yeast. In addition, we were unable to abrogate the inhibitory activity of human serum against B. dermatitidis yeast by the addition of ferric chloride (10-200 microM). We found that apo-transferrin had little or no inhibitory activity against B. dermatitidis yeast. Furthermore, serum from hypotransferrinemic mice (hpx/hpx) had inhibitory activity against B. dermatitidis yeast that was equivalent to that of normal mouse serum. These findings are consistent with our initial findings and suggest that serum inhibitory activity against B. dermatitidis yeast is transferrin independent. Although high concentrations (3.5-5 mg/ml) of lactoferrin did have inhibitory activity against B. dermatitidis yeast, these concentrations were orders of magnitude greater than those found in serum under normal physiological conditions, suggesting that lactoferrin was not likely to contribute to serum inhibitory activity against B. dermatitidis yeast. Our findings suggest that host iron-binding proteins may be relatively ineffective in innate host defense against infection with B. dermatitidis yeast.

Topics: Animals; Blastomyces; Blood Physiological Phenomena; Cattle; Chlorides; Coloring Agents; Dogs; Ferric Compounds; Humans; Lactoferrin; Mice; Mice, Inbred ICR; Oxazines; Transferrin; Xanthenes

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.

Topics: Animals; Cattle; Chlorides; Drug Administration Schedule; Drug Interactions; Escherichia coli Infections; Female; Ferric Compounds; Iron; Lactoferrin; Lactoglobulins; Male; Mice; Mice, Inbred Strains

Results from experiments in this laboratory using 59Fe suggest that bovine lactoferrin (Lf) has no effect on iron absorption in rats. A study was therefore carried out in newborn infants to measure the effects of Lf on iron retention. Bovine Lf was labeled with the stable isotope 58Fe and fed to 7-day-old infants in a standard milk formula. Iron retention was estimated by measuring the unabsorbed 58Fe excreted in the feces during the following 3 days using neutron activation analysis. The results were compared with those obtained from a group of infants fed a similar level of iron as ferric chloride, labeled with 58Fe, together with 30 mg ascorbic acid. There was a very wide variation in percent iron retention amongst the infants but no overall difference between the Lf and ferric chloride groups. This confirms the previous findings in rats that Lf does not influence the availability of nonheme iron.

Topics: Absorption; Biological Availability; Chlorides; Female; Ferric Compounds; Humans; Infant, Newborn; Iron; Iron Isotopes; Lactoferrin; Lactoglobulins; Male

Previously it was shown that tissue injury occurring in acute immune-complex-induced vasculitis, which is complement and neutrophil-dependent, is significantly attenuated by the presence of catalase, suggesting the pathogenic role of H2O2 generated from activated neutrophils. We now show that significant protection is also afforded by pretreatment of animals with apolactoferrin , a naturally occurring chelator of iron. Iron-saturated lactoferrin is devoid of protective effects. Deferoxamine mesylate, a synthetic iron chelator, also has protective effects. Infusion of ionic iron, especially Fe(III), potentiates the tissue injury. Significant protection from tissue injury is also produced by treatment of rats with dimethyl sulfoxide, a potent hydroxyl radical scavenger. Morphologically, animals treated with these protective interventions show the influx of neutrophils into sites of immune complex deposition, but there is markedly attenuated edema, little or no hemorrhage, and little evidence of endothelial cell injury, in contrast to the findings in nonprotected animals. These data support the suggestion that immune-complex-induced injury may be linked to generation of H2O2 from activated neutrophils and the subsequent conversion of H2O2 to the hydroxyl radical.

Topics: Animals; Apoproteins; Arthus Reaction; Chlorides; Deferoxamine; Dimethyl Sulfoxide; Drug Evaluation, Preclinical; Ferric Compounds; Ferrous Compounds; Free Radicals; Hydroxylation; Immune Complex Diseases; Lactoferrin; Male; Quaternary Ammonium Compounds; Rats; Rats, Inbred Strains; Specific Pathogen-Free Organisms; Vasculitis

Lactoferrin was examined for its effect on the growth of a human colon adenocarcinoma cell line (HT 29) in culture and its action was compared to that produced by transferrin and two different iron solutions (ferrous sulfate and ferric chloride). When transferrin was replaced by either iron solutions the cell grew in proportion to the quantity added and the maximal effect obtained was identical to that produced by transferrin alone. When transferrin was replaced by lactoferrin the cells were unable to proliferate for a long time. However, in the presence of low-concentration iron solutions, lactoferrin stimulated the cell growth, and the effect was more pronounced with the ferric chloride solution.

Topics: Adenocarcinoma; Cell Division; Cell Line; Chlorides; Colonic Neoplasms; Ferric Compounds; Ferrous Compounds; Humans; Kinetics; Lactoferrin; Lactoglobulins; Transferrin

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 nonsupplemented milk diet (approximately 1 mg Fe/L) for 4 weeks had a microcytic, hypochromic anemia and low tissue iron concentrations. Iron supplementation of the diet with lactoferrin-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.

Topics: Anemia, Hypochromic; Animals; Biological Availability; Cattle; Chlorides; Female; Ferric Compounds; Food, Fortified; Hematocrit; Iron; Lactoferrin; Lactoglobulins; Mice; Milk; Weaning

Lactoferrin isolated fom normal human tears was shown to inhibit the complement-mediated lysis of antibody-coated red cells. The anti-complementary effect of lactoferrin on serum complement could be reversed by adding Fe3+ but not by Mg++ or Ca++. Lactoferrin did not inhibit the formation of EAC14 but markedly blocked the assembly of the EAC142 enzyme. Once the C3 convertase was formed lactoferrin did not affect the function of the enzyme and only had a minor effect on the intrinsic decay of C2 from the convertase. Inhibition of the C3 convertase formation was not seen by preincubating EAC14 intermediates with lactoferrin, but only occurred when lactoferrin and C2 were incubated together with EAC14 cells. Our findings suggest that lactoferrin may play an anti-inflammatory role by modulating activation of the complement system.

Topics: Calcium Chloride; Chlorides; Complement Activating Enzymes; Complement C3-C5 Convertases; Complement C4; Complement Inactivator Proteins; Ferric Compounds; Humans; Immunoelectrophoresis; Lactoferrin; Lactoglobulins; Magnesium; Magnesium Chloride; Tears