Background. Human milk contains many biologically active glycoconjugates and oligosaccharides. Human milk also contains many enzymes, but those capable of modifying oligosaccharides and glycoconjugates have not been well studied.

Objectives. This study is to determine if human milk contains glycosidic hydrolases and to measure the extent to which they modify human milk oligosaccharides.

Methods. Four lactating mothers donated milk directly into test tubes, whereupon the milk was flash frozen and stored at -80°C until analysis. The activities of seven glycosidases were measured using 4-methylumbelliferyl fluorogenic substrates. Milk samples were incubated at 4°, 20°, and 37°C for various times, and the release of free sugars was assessed by chromatographic and mass spectrometric techniques.

Results. Human milk contains appreciable fucosidase and hexosaminidase, but their activities show high variability among donors. Fucosidase varied from about 250 nmol/ml in one donor to 2000 nmol/ml in another. Hexosaminidase activity was less variable (800-2000 nmol/ml milk). Free fucose, N-acetylhexosamines and NANA were present in milk, and incubation at 20° and 37°C increases the concentrations of these sugars, consistent with release by enzymatic hydrolysis.

Discussion. High fucosidase activity is associated with high free fucose in milk. A reciprocal relationship between free fucose and NANA was apparent. Oligosaccharides of human milk are modified between the time of their synthesis in the breast and delivery of the milk to the infant; modification increases with elapsed time. Free sugar concentrations relative to oligosaccharides are low; however, protective oligosaccharides are also present in quite low amounts, suggesting possibile biological relevance to the protective potential of breast milk. Supported by HD 13021.