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Abstract

During the last decade an enormous progress regarding knowledge about the composition and properties of human milk (HM) has been made. The three main fractions of HM are proteins, fats and carbohydrates. They combine a large variety of properties and functions, like lowering the risk of infections or balance/teach the developing immune system of newborns. The project presented describes the establishment and improvement of a glycoanalysis technique, based on multiplexed capillary gel electrophoresis with laser induced fluorescence detection (CGE-LIF), which allows for high-throughput (HTP) characterization of human milk oligosaccharides (HMOS). The HMOS fraction primarily consists of lactose and a large variety of neutral and acidic oligosaccharides. The characterization of HMOS is essential to understand the relationship between structure and biological effect. Therefore, a rapid and sensitive HTP-method for the analysis of the HMOS fraction composition was established. The method comprises protein-precipitation, clean-up, derivatization as well as the analysis by multiplexing CGE-LIF, using a “ABI PRISM 3100 Genetic AnalyzerTM ”. In combination with an in-house HMOS database, the generated HMOS “fingerprints” (normalized electropherograms) can be used for in-depth analysis, i.e.: structural elucidation of each single compound and its relative quantification. The method developed was tested on various HM samples (time series of different donors). Several factors were identified to be responsible for differences in HMOS-composition, including origin of the mother, lactation time point and genetic determinants (secretor-gene (Se) and lewis-gene (Le)). The combination of these two antigenic determinants defines four human milk types [1]. It turned out, that multiplexed CGE-LIF is a valuable alternative to the existing time consuming analysis techniques. The method and system developed [2], allows a straightforward, fast, unequivocal and sensitive analysis of HMOS fraction composition including an easy determination of secretor and lewis status of milk donors. [1]Thurl, S. et al., Variation of human milk oligosaccharides in relation to milk groups and lactational periods. British Journal of Nutrition (2010) 104, 1261-1271. [2]Schwarzer, J.; Rapp, E.; Reichl, U.; N-Glycan Analysis by Capillary Gel Electrophoresis - Profiling Influenza A Virus Hemagglutinin N-Glycosylation during Vaccine Production. Electrophoresis (2008) 29, 4203-4214.