Quantitation of Sialic Acids in Infant Formulas by Liquid Chromatography–Mass Spectrometry: An Assessment of Different Protein Sources and Discovery of New Analogues

Abstract

Glycosidically bound, but not free, dietary sialic acids are used for the biosynthesis of new glycoconjugates in humans, making the quantitation of these two forms in infant food sources important, as in neonates the demand for sialic acid may exceed the de novo biosynthetic supply. Here, a rapid high-performance liquid chromatography-mass spectrometry method was developed to identify and quantitate glycosidically bound and free sialic acids in infant formulas. The sialic acid contents of eight commercially available infant formulas with varying protein source or manufacturer were investigated. The formula protein sources (whey vs casein) did not have a large impact on the ratios of free to bound sialic acids, nor did protein hydrolysis or sample form (solid vs liquid). Hydrolyzed bovine whey protein-based formulas were found to contain the highest amount of the most abundant human sialic acid, 5- N-acetylneuraminic acid (Neu5Ac). O-Acetylated Neu5Ac was quantified in all formulas tested and, for the first time, 2-keto-3-deoxy-d-glycero-d-galacto-nononic acid (Kdn) was detected in several infant formulas.

Influence of sulfonated and diet-derived human milk oligosaccharides on the infant microbiome and immune markers

“…Across both groups, the ratio of ingested Neu5Gc to Neu5Ac remained essentially constant. The actual levels of Neu5Ac and Neu5Gc glycosidically bound to HMOs were quantitated (42) in all samples ( Fig. 1a).…”

“…HMOs were subsequently removed from the proteins and lipids by liquid-liquid extraction and dried before salts and the majority of lactose were removed by solid-phase extraction using graphitized carbon cartridges (ENVI-Carb, Sigma). HMO-containing fractions were freezedried, redissolved in 18 M⍀cm Ϫ1 water, and divided into five equal portions for quantitation of total reducing sugars (62), quantitation of Neu5Ac and Neu5Ac (42), neutral monosaccharide analysis (38), HMO profiling by CE-LIF, and targeted HMO analysis by HPLC-MS (38). All monosaccharide standards were purchased from Carbosynth, and HMO standards were purchased from Carbosynth or Dextra Laboratories, Inc. Where possible, HMO identification was made based on comigration (CE-LIF) or coelution and m/z (HPLC-MS) to these commercially available standards.…”

Assays for the identification and quantification of sialic acids: Challenges, opportunities and future perspectives

“…As such, the discovery, validation and implementation of more stable fluorescent dyes would be a particularly important step towards facilitating analysis and quantification of sialic acid. Recent research has identified 4,5-dimethylbenzene-1,2-diamine (DMBA) as a labelling reagent [97][98][99] that is similar to those previously discussed but does not suffer from the same instability post-derivatisation, whilst offering similar labelling times to DMB and still offering excellent UV and fluorescent activity for detection and measurement.…”

Glycan Utilisation and Function in the Microbiome of Weaning Infants

“…RMOs exist in lower concentrations compared to HMOs [17,18,19], with fewer unique structures having been identified [20,21], as shown in Figure 1. RMOs also have higher ratios of non-fucosylated neutral and sialylated structures, compared to HMOs [19,21,22,23,24]. Of these sialylated oligosaccharides, bovines express more Neu5Gc, which may be associated with inflammation [25], whereas humans express Neu5Ac [26].…”