Glycoconjugate stability in human milk: glycosidase activities and sugar release

Many human milk glycoconjugates (glycolipids, glycoproteins, mucins, glycos aminoglycans) and oligosaccharides are biologically active, and their activ ity depends on the precise structure of the glycan. The sugars on the termi nus of the glycan are vulnerable to cleavage by glycosidases. Because glyco conjugates incubate together with endogenous glycosidases in the breast bet ween feedings, and in expressed milk during storage, the presence and activ ity of glycosidases in human milk was investigated. alpha -L-Fucosidase, al pha -D-galactosidase, beta -D-galactosidase, beta -D-glucosidase, N-acetyl- beta -hexosaminidase, beta -D-glucuronidase, and neuraminidase were measure d in milk samples from 4 donors by use of synthetic fluorogenic glycosides; fucosidase and hexosaminidase displayed the highest activity. The cataboli c activity of the major glycosidases was confirmed by measuring the corresp onding free sugars in milk. Free fucose, N-acetylneuraminic acid, and N-ace tylhexosamines were measured and their identities were confirmed by high-pe rformance liquid chromatography, gas chromatography, and gas chromatography -mass spectrometry. Incubation of samples for 16 hr at 37 degreesC and 20 d egreesC, but not at 4 degreesC, resulted in time-dependent increases in the amount of free fucose, N-acetylneuraminic acid, and N-acetylhexosamines, c onsistent with their enzymatic release by the endogenous glycosidases. Stor ed frozen milk had the same levels of these sugars as did samples analyzed immediately after collection, indicating that glycosidases are inactive in the frozen milk. Samples analyzed immediately after collection contained on ly small amounts of free sugars, suggesting that glycoconjugate degradation during the typical residence time of milk in the breast is modest. (C) 200 1 Elsevier Science Inc. All rights reserved.