Proteolysis of vaginally administered bovine lactoferrin: clearance, inter-subject variability, and implications for clinical dosing.

This report describes proteolytic fragmentation and clearance of bovine lactoferrin (bLF) upon intravaginal administration in premenopausal women. Tablet formulations (MTbLF) containing 300 mg of bLF progressed through three phases: Pre-Dissolution, Dissolution, and Washout, over a 30-h time course. Tablets dissolved slowly, replenishing intact 80 kDa bLF in vaginal fluid (VF) as proteolysis occurred. bLF was initially cleaved approximately in half between its N- and C-lobes, then degraded into sub-fragments and small peptides. The extent of proteolysis was less than 10-20% across multiple subjects. Concentrations of both intact 80 kDa bLF and smaller fragments decreased in VF with a similar time course suggesting washout not proteolysis was the main clearance mechanism. Concentrations of intact and/or nicked 80 kDa bLF peaked between 4 and 8 h after administration and remained above 5 mg/mL for approximately 24 h. Experiments with protease inhibitors in ex vivo VF digests suggested an aspartyl protease was at least partially responsible for bLF cleavage. However, digestion with commercial pepsin or in vivo in the human stomach, demonstrated distinctly different patterns of fragments compared to vaginal proteolysis. Furthermore, the 3.1 kDa antimicrobial peptide lactoferricin B was not detected in VF. This suggests pepsin-like aspartyl proteases are not responsible for vaginal proteolysis of bLF.

Characterization of proteolytic degradation products of vaginally administered bovine lactoferrin.

When bovine lactoferrin (bLF) contacts human vaginal fluid (VF) it is subjected to proteolytic degradation. This report describes fragmentation patterns of bLF dosed vaginally in clinical trials or incubated ex vivo with VF. A consensus pattern of fragments was observed in samples from different women. The 80 kDa bLF molecule is initially cleaved between its homologous 40 kDa domains, the N-lobe and C-lobe, and then degraded into sub-fragments and mixtures of small peptides. We characterized this fragmentation process by polyacrylamide gel electrophoresis, western blotting, chromatographic separation, and mass spectral sequence analysis. Common to most VF fragmentation patterns were large amounts of an N-lobe 37 kDa fragment and a C-lobe 43 kDa fragment resulting from a single cleavage following tyrosine 324. Both fragments possessed full sets of iron-ligand amino acids and retained iron-binding ability. In some VF samples, alternative forms of large fragments were found, which like the 37+43 kDa pair, totaled 80 kDa. These included 58+22 kDa, 18+62 kDa, and 16+64 kDa forms. In general, the smaller component was from the N-lobe and the larger from the C-lobe. The 18+62 kDa pair was absent in some VF samples but highly abundant in others. This variability suggests multiple endopeptidases are involved, with the 18 kDa fragment's presence dependent upon the balance of enzymes. Further action of VF endopeptidases produced smaller peptide fragments, and we found evidence that exopeptidases trimmed their N- and C-termini. The 3.1 kDa antimicrobial peptide lactoferricin B was not detected. These studies were facilitated by a novel technique we developed: tricolor western blots, which enabled simultaneous visualization of N- and C-terminal epitopes.

Lactoferrin-Loaded Alginate Microparticles to Target Clostridioides difficile Infection.

Some forms of bovine lactoferrin (bLf) are effective in delaying Clostridioides difficile growth and preventing toxin production. However, therapeutic use of bLf may be limited by protein stability issues. The objective of this study was to prepare and evaluate colon-targeted, pH-triggered alginate microparticles loaded with bioactive bLf and to evaluate their anti-C difficile defense properties in vitro. Different forms of metal-bound bLf were encapsulated in alginate microparticles using an emulsification or internal gelation method. The microparticles were coated with chitosan to control protein release. In vitro drug release studies were conducted in pH-simulated gastrointestinal conditions to investigate the release kinetics of encapsulated protein. No significant release of metal-bound bLf was observed at acidic pH; however, on reaching simulated colonic pH, most of the encapsulated lactoferrin was released. The application of bLf (5 mg/mL) delivered from alginate microparticles to human intestinal epithelial cells significantly reduced the cytotoxic effects of toxins A and B as well as bacterial supernatant on Caco-2 and Vero cells, respectively. These results are the first to suggest that alginate-bLf microparticles show protective effects against C difficile toxin-mediated epithelial damage and impairment of barrier function in human intestinal epithelial cells. The future potential of lactoferrin-loaded alginate microparticles against C difficile deserves further study.

Minimizing the impact of Joule heating as a prerequisite for the reliable analysis of metal-protein complexes by capillary electrophoresis.

Herein we report on a drastic release of metal ions from the Fe-bound transferrin, and Fe- or Mn-bound lactoferrin, observed upon the increase in the separation voltage during CE-based analysis. To verify whether this process is caused directly by electric field, we developed an Isothermal Voltage Increase approach (IVI), which is the extension of methods reported by Krylov et al. IVI ensures isothermal conditions while increasing separation voltage by a hydrodynamic pushing of the injected sample to the actively cooled capillary section, combined with a rationale choice of cooling temperature, dependent on the value of current. Interestingly, the application of IVI revealed that the previously observed effect was caused solely by the insufficient dissipation of Joule heating - the saturation of each protein remained unchanged despite a significant rise in the electric field. This outcome demonstrates how crucial is to ensure an effective temperature control for preventing systematic errors in the analysis of biomolecular complexes. IVI seems also to be a simple and useful tool for discovering new potential processes that may be stimulated directly by electric field.

The impact of lactoferrin with different levels of metal saturation on the intestinal epithelial barrier function and mucosal inflammation.

Translocation of bacteria, primarily Gram-negative pathogenic flora, from the intestinal lumen into the circulatory system leads to sepsis. In newborns, and especially very low birth weight infants, sepsis is a major cause of morbidity and mortality. The results of recently conducted clinical trials suggest that lactoferrin, an iron-binding protein that is abundant in mammalian colostrum and milk, may be an effective agent in preventing sepsis in newborns. However, despite numerous basic studies on lactoferrin, very little is known about how metal saturation of this protein affects a host's health. Therefore, the main objective of this study was to elucidate how iron-depleted, iron-saturated, and manganese-saturated forms of lactoferrin regulate intestinal barrier function via interactions with epithelial cells and macrophages. For these studies, a human intestinal epithelial cell line, Caco-2, was used. In this model, none of the tested lactoferrin forms induced higher levels of apoptosis or necrosis. There was also no change in the production of tight junction proteins regardless of lactoferrin metal saturation status. None of the tested forms induced a pro-inflammatory response in Caco-2 cells or in macrophages either. However, the various lactoferrin forms did effectively inhibit the pro-inflammatory response in macrophages that were activated with lipopolysaccharide with the most potent effect observed for apolactoferrin. Lactoferrin that was not bound to its cognate receptor was able to bind and neutralize lipopolysaccharide. Lactoferrin was also able to neutralize microbial-derived antigens, thereby potentially reducing their pro-inflammatory effect. Therefore, we hypothesize that lactoferrin supplementation is a relevant strategy for preventing sepsis.

Selective separation of ferric and non-ferric forms of human transferrin by capillary micellar electrokinetic chromatography.

The previously published method allowing the separation of non-ferric (iron-free) and ferric (iron-saturated) forms of human serum transferrin via capillary electrophoresis has been further developed. Using a surface response methodology and a three-factorial Doehlert design we have established a new optimized running buffer composition: 50mM Tris-HCl, pH 8.5, 22.5% (v/v) methanol, 17.5mM SDS. As a result, two previously unobserved monoferric forms of protein have been separated and identified, moreover, the loss of ferric ions from transferrin during electrophoretic separation has been considerably reduced by methanol, and the method selectivity has been yet increased resulting in a total separation of proteins exerting only subtle or none difference in mass-to-charge ratio. The new method has allowed us to monitor the gradual iron saturation of transferrin by mixing the iron-free form of protein with the buffers with different concentrations of ferric ions. It revealed continuously changing contribution of monoferric forms, characterized by different affinities of two existing iron binding sites on N- and C-lobes of protein, respectively. Afterwards, the similar experiment has been conducted on-line, i.e. inside the capillary, comparing the effectiveness of two possible modes of the reactant zones mixing: diffusion mediated and electrophoretically mediated ones. Finally, the total time of separation has been decreased down to 4min, taking the advantage from a short-end injection strategy and maintaining excellent selectivity.

Separation of iron-free and iron-saturated forms of transferrin and lactoferrin via capillary electrophoresis performed in fused-silica and neutral capillaries.

A capillary electrophoresis-based method for the cost-effective and high efficient separation of iron-free and iron-saturated forms of two members of transferrin family: transferrin and lactoferrin has been developed. The proposed qualitative method relying on the SDS application allowed us to separate iron-free and iron-saturated forms of these proteins, as well as human serum albumin, used as an internal standard. Owing to the distinct migration times under established conditions, the combination of transferrin and lactoferrin assays within a single analytical procedure was feasible. The performance of the method using a fused-silica capillary has been compared with the results obtained using the same method but performed with the use of a neutral capillary of the same dimensions. Neutral capillary has been used as an alternative, since the comparable resolution has been achieved with a concomitant reduction of the electroosmotic flow. Despite of this fact, the migration of analytes occurred with similar velocity but in opposite order, due to the reverse polarity application. A quantitative method employing fused-silica capillary for iron saturation study has been also developed, to evaluate the iron saturation in commercial preparations of lactoferrin.

A high-throughput method for the quantification of iron saturation in lactoferrin preparations.

Lactoferrin is considered as a part of the innate immune system that plays a crucial role in preventing bacterial growth, mostly via an iron sequestration mechanism. Recent data show that bovine lactoferrin prevents late-onset sepsis in preterm very low birth weight neonates by serving as an iron chelator for some bacterial strains; thus, it is very important to control the iron saturation level during diet supplementation. An accurate estimation of lactoferrin iron saturation is essential not only because of its clinical applications but also for a wide range of biochemical experiments. A comprehensive method for the quantification of iron saturation in lactoferrin preparations was developed to obtain a calibration curve enabling the determination of iron saturation levels relying exclusively on the defined ratio of absorbances at 280 and 466 nm (A(280/466)). To achieve this goal, selected techniques such as spectrophotometry, ELISA, and ICP-MS were combined. The ability to obtain samples of lactoferrin with determination of its iron content in a simple and fast way has been proven to be very useful. Furthermore, a similar approach could easily be implemented to facilitate the determination of iron saturation level for other metalloproteins in which metal binding results in the appearance of a distinct band in the visible part of the spectrum.