Simultaneous quantification of sialyloligosaccharides from human milk by capillary electrophoresis

Abstract

The acidic oligosaccharides of human milk are predominantly sialyloligosaccharides. Pathogens that bind sialic acid-containing glycans on their host mucosal surfaces may be inhibited by human milk sialyloligosaccharides, but testing this hypothesis requires their reliable quantification in milk. Sialyloligosaccharides have been quantified by anion exchange HPLC, reverse or normal phase HPLC, and capillary electrophoresis (CE) of fluorescent derivatives; in milk, these oligosaccharides have been analyzed by high pH anion exchange chromatography with pulsed amperometric detection, and, in our laboratory, by CE with detection at 205 nm. The novel method described herein uses a running buffer of aqueous 200 mM NaH 2 PO 4 at pH 7.05 containing 100 mM SDS made 45% (v/v) with methanol to baseline resolve five oligosaccharides, and separate all 12. This allows automated simultaneous quantification of the 12 major sialyloligosaccharides of human milk in a single 35-minute run. This method revealed differences in sialyloligosaccharide concentrations between less and more mature milk from the same donors. Individual donors also varied in expression of sialyloligosaccharides in their milk. Thus, the facile quantification of sialyloligosaccharides by this method is suitable for measuring variation in expression of specific sialyloligosaccharides in milk and their relationship to decreased risk of specific diseases in infants. Keywordshuman milk oligosaccharides; sialyloligosaccharides; capillary electrophoresis INTRODUCTORY STATEMENTHuman milk contains a large number of compounds that provide nutritional support and contribute toward defense of the newborn. Human milk oligosaccharides (HMOS), a principal component of human milk, are found in concentrations of 6 to 12 g/L, and these oligosaccharides vary in size, structure, and abundance [1]. The oligosaccharides of milks from other species vary widely, and the composition of oligosaccharides from human milk is unique [2].HMOS play a potentially protective role to nursing infants. Breastfed infants have a different microbiota than infants fed artificially, and human milk glycans are thought to act as prebiotics that promote the growth of Bifidobacterium bifidus [3]. Breast-fed infants have fewer or less severe gastrointestinal and respiratory infections during the first year of life than formula-fed

Determination of Sialyl and Neutral Oligosaccharide Levels in Transition and Mature Milks of Samoan Women, Using Anthranilic Derivatization Followed by Reverse Phase High Performance Liquid Chromatography

“…Furthermore, we found that during lactation, the concentrations of sialyl oligosaccharides tended to decrease, with the exception of LSTb. Our results for the levels of sialyl HMOs were slightly different from those of Bao et al 26) but similar to those of Coppa et al 29) For instance, the concentration values for 3 0 -SL, LSTc, and LSTb in transition milk obtained in this study were a little higher and those for DSLNT a little lower than those reported by Bao et al 26) With respect to mature milk, the values we obtained are fairly well in agreement with those reported by Coppa et al 29) except that our DSLNT values were lower. This DSLNT level was higher than that described by Asakuma et al 24) for the colostrum of Japanese women.…”

“…The oligosaccharides previously studied by other researchers were similar to those in the present study; their methods involved determination of peak areas in chromatograms using capillary electrophoresis, HPLC, or HPEAC-PAD of these oligosaccharides or their derivatives. 15,24,[26][27][28] Peaks 1 to 7 corresponded to 6 0 -SL, DSLNT, galacturonic acid (internal standard), LSTb, 3 0 -SL, LSTc, and LSTa respectively. The sialyl oligosaccharides isomers, 3…”

Analysis and role of oligosaccharides in milk

“…Several sialylated isomers have been separated with this technique however extensive isomer separation of heterogeneous mixtures still creates problems. Capillary Electrophoresis (CE) separation techniques have also been applied in the analysis of HMO (47,48). CE with laser-induced fluorescence coupled to mass spectrometry (CE-LIF-MS) is effective for derivatized HMO.…”

Individual and Combined Effects of Nucleotides and Human Milk Oligosaccharides on Proliferation, Apoptosis and Necrosis in a Human Fetal Intestinal Cell Line

“…Individual sialylated and fucosylated HMO were purchased from V-Labs (Covington, LA) and combined into a FL mix (85% [10,[13][14][15][16]. For the SL mix, we chose to mimic the total SA content of human milk [11,[17][18][19][20][21]. The 6'SL and 3'SL were added at the reported ratio to each other and the remaining total SA was provided as free SA, assuming that some lipid or protein-bound SA could be released by sialidases in the intestine [22] or by microbial fermentation [23].…”