Capillary electrophoresis of acidic oligosaccharides from human milk

Abstract

Interest in defining the array of oligosaccharides of human milk has been increasing. Pathogens that bind glycans on their host mucosal surfaces may be inhibited by human milk oligosaccharides. It has been postulated that acidic oligosaccharides in human milk may inhibit binding by pathogens that bind acidic glycans in the gut, but testing this hypothesis requires their reliable quantification in milk. Sialyloligosaccharides of human milk have been quantified by HPLC and CE. A recent CE technique uses the MEKC mode with direct detection at 205 nm to resolve and quantify, in the native form, the 12 most dominant sialyloligosaccharides of human milk in a single 35-min run. The method gives a linear response from 39 to 2500 microg/mL with a coefficient of variation between 2 to 9% and accuracy from 93 to 109%. This was used to detect variation in expression of specific sialyloligosaccharides in milk. Individual sialyloligosaccharide concentrations in milk differ among individual donors and between less and more mature milk. Thus, CE can be used to measure variation in sialyloligosaccharide expression in milk, and thereby test the relationship of this variation-to-variation in risk of specific diseases in breastfed infants.

Quantification of neutral human milk oligosaccharides by graphitic carbon high-performance liquid chromatography with tandem mass spectrometry

“…However, the oligosaccharide fraction of human milk is a large complex mixture of molecules that are very polar, many are highly branched, contain multiple structural isomers, and no intrinsic chromophore, presenting a challenge to both their resolution and quantitative detection. HPLC is most commonly used in the analysis of oligosaccharides [13-24], with some capillary electrophoresis (CE) as well [25-28]. Derivatives of neutral oligosaccharides can be separated on both normal phase [15] and reversed-phase columns, followed by quantification by their UV absorbance [13; 16; 18; 20].…”

Effects of Recombinant Human Prolactin on Breast Milk Composition

“…24 Therefore, r-hPRL administration is associated with compositional changes that mirror those in women undergoing normal lactogenesis in the first 2 to 10 days after birth. 7,18,[30][31][32][33] Furthermore, r-hPRL administration increased oligosaccharide levels, whereas total daily IgA levels did not change, which potentially improved the antimicrobial properties of breast milk. These findings suggest that r-hPRL improves breast milk quantity, maturity, and immunity for mothers with lactation insufficiency.…”

“…17,30,31 Acidic oligosaccharide levels were measured by using capillary electrophoresis. 32 Citrate, sodium, calcium, and lactose assays were performed after defatting and deproteination, as described previously. 6 Lactose and citrate levels were measured by using a commercial enzymatic assay (RBiopharm, Darmstadt, Germany) validated for human milk.…”

“…12 The glycan component of oligosaccharides resembles the cell surface glycans of the intestine and competitively binds enteric pathogens. 12,32 Oligosaccharides are associated with lower levels of diarrhea in breastfed infants. 14 Oligosaccharide concentrations are highest immediately after birth and decrease exponentially in the first 2 months of breastfeeding.…”

Advances in Fractionation and Analysis of Milk Carbohydrates

“…Baross et al [43] determined the lactose in milk and milk products by CE with indirect UV detection. More recently, HMOs have been detected by single CE with laser-induced fluorescence (LIF) [42,44] or additionally coupled online with MS [45][46][47]. Labeling of HMO with 8-aminopyrene-1,3,6-trisulfonic acid introduces a fluorophore for the LIF detection, simultaneously adding the negative charge needed for the separation.…”