CE‐LIF‐MSⁿ profiling of oligosaccharides in human milk and feces of breast‐fed babies

Abstract

Mixtures of the complex human milk oligosaccharides (HMOs) are difficult to analyze and gastrointestinal bioconversion products of HMOs may complicate analysis even more. Their analysis, therefore, requires the combination of a sensitive and high-resolution separation technique with a mass identification tool. This study introduces for the first time the hyphenation of CE with an electrospray mass spectrometer, capable to perform multiple MS analysis (ESI-MS(n)) for the separation and characterization of HMOs in breast milk and feces of breast-fed babies. LIF was used for on- and off-line detections. From the overall 47 peaks detected in off-line CE-LIF electropherograms, 21 peaks could be unambiguously and 11 peaks could be tentatively assigned. The detailed structural characterization of a novel lacto-N-neo-tetraose isomer and a novel lacto-N-fucopentaose isomer was established in baby feces and pointed to gastrointestinal hydrolysis of higher-Mw HMOs. CE-LIF-ESI-MS(n) presents, therefore, a useful tool which contributes to an advanced understanding on the fate of individual HMOs during their gastrointestinal passage.

Evolution of milk oligosaccharides and lactose: a hypothesis

“…In addition, Gal(b1 -4)GlcNAc(b1 -6)Gal(b1 -4)Glc (type II) and Gal(b1 -4)[Fuc(a1 -3)]GlcNAc(b1 -6)Gal(b1 -4)Glc (type II) were found in the faeces, although these are not present in human milk (Albrecht et al, 2010). These results suggest that LNT is preferentially digested and that LNB, released from Gal(b1 -3)GlcNAc(b1 -3)[Gal(b1 -4)GlcNAc(b1 -6)]Gal(b1 -4)Glc (LNH) or Gal(b1 -3)GlcNAc(b1 -3){Gal(b1 -4)[Fuc(a1 -3)]GlcNAc(b1 -6)}Gal(b1 -4)Glc (monofucosyl LNH) by the action of lacto-N-biosidase, is utilized by bifidobacteria during the passage of HMO through the gastrointestinal tract when the infant consumes breast milk, so that the above type II saccharides remain in the faeces.…”

Structural characterization of neutral and acidic oligosaccharides in the milks of strepsirrhine primates: greater galago, aye-aye, Coquerel’s sifaka and mongoose lemur

“…We hypothesize that LNB released from type I in preference of type II HMOs by Bifidobacterium bifidum lacto-N-biosidase are utilized by multple strains of colonic bifidobacteria in breast-fed infants, leading to their predominance. Albrecht et al [42] compared oligosaccharides in human milk and in feces of breast-fed babies by capillary electrophoresis with a laser induced fluorescence detector coupled to a mass spectrometer (CE-LIF-MS n ). They found that the relative level of LNT (type I) was markedly lower in the fecal extract than in breast milk, while the relative level of LNnT (type II) was higher in the feces.…”

Analysis and role of oligosaccharides in milk

“…CE with laser-induced fluorescence coupled to mass spectrometry (CE-LIF-MS) is effective for derivatized HMO. Labeling with 8-aminopyrene-1,3,6-trisulfonic acid (APTS) introduces a fluorophore for the LIF detection, while simultaneously adding negative charge needed for the separation (49)(50)(51). However although good separation can be achieved using offline CE-LIF using very fast runs (around 9 minutes), both resolution and separation time have to be compromised when coupling CE to mass spectrometry (52).…”