Weronika Hewelt-Belka, an assistant professor at Gdańsk University of Technology in Poland, answers questions about her research analyzing GM3 gangliosides in human milk, elucidating their molecular distribution and dynamic changes throughout lactation.
Q: At what point of the lactation period does information about ganglioside content and, specifically, GM3 composition in human milk (HM) generally become more difficult to quantify? How does this knowledge gap affect our full understanding of the developmental properties of HM?
A: Ganglioside analysis is challenging at every stage of lactation. Milk itself is a very complex sample containing a wide range of biomolecules, interfering with gangliosides, such as glycerophospholipids. Moreover, gangliosides in human milk are at very low concentration level. It is known that the total amount of GD3 gangliosides decrease during the lactation period (with the highest content in colostrum), while the GM3 ganglioside content increases. This altogether hinders the quantitative analysis of gangliosides. Before the liquid chromatography–mass spectrometry (LC–MS) analysis, the gangliosides have to be isolated from human milk and enriched, usually by employing time-consuming and multistep procedures consisting of liquid-liquid extraction (LLE) and subsequent solid-phase extraction (SPE) clean-up. Another limiting factor is the availability of standards, which usually are a mixture of ganglioside species of a specific class, and originating from bovine milk. This limits the absolute quantification of particular ganglioside species instead of quantifying the total content of gangliosides. To fully understand the developmental properties of milk and biological role of its compounds, it is important to comprehensively describe the composition of lipid species, including gangliosides. This refers to gangliosides' compositional differences between individual mothers as well as during the lactation period. Along with the new knowledge about the chemical structure of gangliosides, we should investigate how this compositional diversity impacts infant health. Are these changes significant for the infant's development? Answering these questions will help us increase awareness about the health benefits of breastfeeding as well as to optimize the infant formula to better mimic human milk.
Q: How did you arrive at the multifaceted analysis approach of reversed-phase liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (RPLC–QTOF-MS), what are the advantages of this method, and did you have to endure significant trial-and-error to get desired results? Why have previous studies failed to produce more information?
A: We decided to use the RPLC–QTOF-MS approach since we were interested in investigating how the ganglioside profile changes during lactation, with the focus on ganglioside species differing in ceramide moiety structure. Employing the RPLC mode allowed us to separate gangliosides accordingly to the hydrophobicity of the ceramide part. High-resolution mass spectrometry (HRMS) enables us to identify gangliosides based on accurate mass measurement and fragmentation patterns, even without standards available.
Luckily, during method development and validation we didn’t have to start from scratch, since we found previous studies regarding the analysis of human milk gangliosides with the use of LC–MS. We had to optimize the method to fit the sensitivity of our QTOF mass spectrometer. Another problem was the variance of concentration of gangliosides in human milk samples originating from different mothers. To find a suitable amount of sample needed for the analysis and to optimize the sample preparation protocol, we had to test several samples from different donors and lactation stages.
I think that the scarce information about the dynamics of ganglioside profiles of human milks results from the lack of interest of the scientific community. But this is changing now, as has our knowledge about the importance of these compounds, along with the awareness of the possible biological effect of lipids varying in FA compositions. Moreover, the lack of commercially available standards hinders the analysis of human milk gangliosides. Although bovine gangliosides can be useful to the method optimization and validation, it should be noted that its profile differs from human milk. Another issue is the availability of human milk samples collected at the advanced lactation stage, especially after one year postpartum. Although the World Health Organization (WHO) recommends continuing to breastfeed for up to two years of age or beyond, many women resign from breastfeeding before this time, from different reasons. We had the possibility to collect these samples within a project aiming to evaluate the human milk lipidome dynamics, and human milk donors were under the care of certified lactation consultants throughout the whole lactation period. This helped sustain lactation and collect samples even in its very late stages.
Q: You say this is the first study that to your knowledge examines GM3 content in “advanced” lactation, in some cases past 24 months. What may be the hypothetical developmental advantages this stage of lactation can bring to a child still consuming HM?
A: This has to be further investigated. We observe that the profile of GM gangliosides changes during the lactation, but maybe what is more important is that it differs between individual mothers. It is believed that human milk composition is tailored for the special needs of the child. It is assumed that human milk gangliosides play a role in neurological development, intestinal protection, and inhibition of enterotoxins; thus, their presence in human milk even at advanced lactation suggest that they are still necessary for proper child development and health. The benefits for children from intake of gangliosides from human milk, and their biological functions, have to be investigated more thoroughly.
I think it is important to raise awareness about the presence of compounds in human milk, and its role to promote the health benefits we already know. Additionally, an in-depth understanding of ganglioside composition and dynamics, among other components, can significantly contribute to the development of milk formulas that more accurately mimic human milk, aligning with the nutritional needs of children.
Q: Do you have any specific recommendations for future research?
A: In my opinion, the biological significance of the structural diversification in human milk gangliosides should be explained. We observe changes in the composition of gangliosides, but we don’t know how these impact the functionality of the milk, and how they affect the infant's health. Without this knowledge, we are not able to optimize formula milk composition to mimic human milk as close as possible, and to switch from standardized protocols to individualized nutrition of infants.
(1) Hewelt-Belka, W.; Młynarczyk, M.; Garwolińska, D.; Kot-Wasik, A. Characterization of GM3 Gangliosides in Human Milk throughout Lactation: Insights from the Analysis with the Use of Reversed-Phase Liquid Chromatography Coupled to Quadrupole Time-Of-Flight Mass Spectrometry. J. Agric. Food Chem. 2023, 71 (46), 17899–17908. DOI: 10.1021/acs.jafc.3c04489