Quantification of lactoferrin in human milk using monolithic cation exchange HPLC

Oberčkal, Jernej; Liaqat, Humna; Matijašić, Bojana Bogovič; Rozman, Vita; Treven, Primož

doi:10.1016/j.jchromb.2022.123548

Cited by 6 publications

(3 citation statements)

References 43 publications

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“…Since A. Marshall et al ( 12 ) reported that the composition of the upper chamber had little effect on maintaining membrane barrier function when only the medium in the lower chamber was changed daily, we speculated that MCF10A cells might withstand the challenge with HM, i.e., survive and retain barrier function. Indeed, we showed that the MAGIC model was able to withstand the challenge with heat-treated HM samples; however, there were noticeable differences among the effects of different milk samples, indicating that different bioactive components might be present in HM samples ( 26 ). As expected, when testing the raw pooled milk sample ( Fig.…”

Section: Discussionmentioning

confidence: 96%

In vitro model of human mammary gland microbial colonization (MAGIC) demonstrates distinctive cytokine response to imbalanced human milk microbiota

Treven,

Paveljšek,

Kostanjšek

et al. 2024

Microbiol Spectr

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Despite the established concept of the human mammary gland (MG) as a habitat with its own microbiota, the exact mechanism of MG colonization is still elusive and a well-characterized in vitro model would reinforce studies of the MG microbiota development. We aimed to establish and characterize an in vitro cell model for studying MAmmary Gland mIcrobial Colonization (MAGIC) model. We used the immortalized cell line MCF10A, which expresses the strong polarized phenotype similar to MG ductal epithelium when cultured on a permeable support (Transwell). We analyzed the surface properties of the MAGIC model by gene expression analysis of E-cadherin, tight junction proteins, and mucins and by scanning electron microscopy. To demonstrate the applicability of the model, we tested the adhesion capability of the whole human milk (HM) microbial community and the cellular response of the model when challenged directly with raw HM samples. MCF10A on permeable supports differentiated and formed a tight barrier, by upregulation of CLDN8, MUC1, MUC4, and MUC20 genes. The surface of the model was covered with mucins and morphologically diverse with at least two cell types and two types of microvilli. Cells in the MAGIC model withstood the challenge with heat-treated HM samples and responded differently to the imbalanced HM microbiota by distinctive cytokine response. The microbial profile of the bacteria adhered on the MAGIC model reflected the microbiological profile of the input HM samples. The well-studied MAGIC model could be useful for studies of bacterial attachment to the MG and for in vitro studies of biofilm formation and microbiota development. IMPORTANCE The MAGIC model may be particularly useful for studies of bacterial attachment to the surface of the mammary ducts and for in vitro studies of biofilm formation and the development of the human mammary gland (MG) microbiota. The model is also useful for immunological studies of the interaction between bacteria and MG cells. We obtained pioneering information on which of the bacteria present in the raw human milk (HM) were able to attach to the epithelium treated directly with raw HM, as well as on the effects of bacteria on the MG epithelial cells. The MAGIC cell model also offers new opportunities for research in other areas of MG physiology, such as the effects of bioactive milk components on microbial colonization of the MG, mastitis prevention, and studies of probiotic development. Since resident MG bacteria may be an important factor in breast cancer development, the MAGIC in vitro tool also offers new opportunities for cancer research.

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Section: Discussionmentioning

confidence: 96%

In vitro model of human mammary gland microbial colonization (MAGIC) demonstrates distinctive cytokine response to imbalanced human milk microbiota

Treven,

Paveljšek,

Kostanjšek

et al. 2024

Microbiol Spectr

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“…The accurate, efficient, and robust method has been reported for the detection and quantification of LF in bovine and human milk during lactation, using the technology of monolithic cation exchange HPLC [100]. Herein, due to the positively charged surface of LF at N-terminus domain (peptide lactoferricin), LF strongly bound to the chromatographic column and eluted as last peak during analysis.…”

Section: Milk Proteinsmentioning

confidence: 99%

Advances in separation and identification of biologically important milk proteins and peptides

Št́astná

2023

Electrophoresis

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Milk is a rich source of biologically important proteins and peptides. In addition, milk contains a variety of extracellular vesicles (EVs), including exosomes, that carry their own proteome cargo. EVs are essential for cell–cell communication and modulation of biological processes. They act as nature carriers of bioactive proteins/peptides in targeted delivery during various physiological and pathological conditions. Identification of the proteins and protein‐derived peptides in milk and EVs and recognition of their biological activities and functions had a tremendous impact on food industry, medicine research, and clinical applications. Advanced separation methods, mass spectrometry (MS)‐based proteomic approaches and innovative biostatistical procedures allowed for characterization of milk protein isoforms, genetic/splice variants, posttranslational modifications and their key roles, and contributed to novel discoveries. This review article discusses recently published developments in separation and identification of bioactive proteins/peptides from milk and milk EVs, including MS‐based proteomic approaches.

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“…Currently, Lf detection in dairy products can be classified into three main categories, including chromatography, biosensors, and immunoassays [7]. Among chromatography, high-performance liquid chromatography (HPLC) enables a reliable distinction between different forms of Lf and has been intensively and extensively investigated and implemented [8], as well as the cutting-edge approaches such as monolithic cation exchange HPLC [9], HiTrap TM Heparin HP column into HPLC [10], micro-batch resin extraction HPLC [11]. Recently, Yang et al integrated amino acid-based isotope dilution LC-mass spectrometry into a mass balance approach to achieve SI-traceable purity assessment of bovine Lf with a low level of uncertainty (<2%) [12].…”

Section: Introductionmentioning

confidence: 99%

Self-Responsive Fluorescence Aptasensor for Lactoferrin Determination in Dairy Products

Liu,

Gao,

Qin

et al. 2024

Molecules

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In this study, a self-responsive fluorescence aptasensor was established for the determination of lactoferrin (Lf) in dairy products. Herein, the aptamer itself functions as both a recognition element that specifically binds to Lf and a fluorescent signal reporter in conjunction with fluorescent moiety. In the presence of Lf, the aptamer preferentially binds to Lf due to its specific and high-affinity recognition by folding into a self-assembled and three-dimensional spatial structure. Meanwhile, its reduced spatial distance in the aptamer–Lf complex induces a FRET phenomenon based on the quenching of 6-FAM by amino acids in the Lf protein, resulting in a turn-off of the fluorescence of the system. As a result, the Lf concentration can be determined straightforwardly corresponding to the change in the self-responsive fluorescence signal. Under the optimized conditions, good linearities (R2 > 0.99) were achieved in an Lf concentration range of 2~10 μg/mL for both standard solutions and the spiked matrix, as well as with the desirable detection limits of 0.68 μg/mL and 0.46 μg/mL, respectively. Moreover, the fluorescence aptasensor exhibited reliable recoveries (89.5–104.3%) in terms of detecting Lf in three commercial samples, which is comparable to the accuracy of the HPCE method. The fluorescence aptasensor offers a user-friendly, cost-efficient, and promising sensor platform for point-of-need detection.

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Quantification of lactoferrin in human milk using monolithic cation exchange HPLC

Cited by 6 publications

References 43 publications

In vitro model of human mammary gland microbial colonization (MAGIC) demonstrates distinctive cytokine response to imbalanced human milk microbiota

In vitro model of human mammary gland microbial colonization (MAGIC) demonstrates distinctive cytokine response to imbalanced human milk microbiota

Advances in separation and identification of biologically important milk proteins and peptides

Self-Responsive Fluorescence Aptasensor for Lactoferrin Determination in Dairy Products

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