Objectives and study:
The healthy development of neonates is supported by human milk (HM). Among many bioactive compounds, HM comprises more than 1000 individual oligosaccharide structures also including Galactosyllactoses (GLs). GLs may appear in form of several structurally distinct isomers like for example 3’-galactosyllactose (3’-GL) and 6’-Galactosyllactose (6’-GL) which only differ in the gycosidic linkage of the terminal galactose.
3’-GL and other GLs have been reported to modulate major immunologic pathways of immature human intestinal cells  and to attenuate inflammation . Anti-inflammatory effects of e.g. 3’-GL may also be mediated via inhibition of toll like receptor 3 (TLR3) signalling .
To learn more about abundances of Galactosyllactoses in HM and their impact on the healthy development of infants, improved analytical approaches are needed. Currently, new insights are hampered by sensitivity limitations and difficulties in the analytical distinction of low abundant GL-isomers in HM.
To overcome these drawbacks at least in part, we developed an LC-MS approach with enhanced structural selectivity which can distinguish many low abundant GL-isomers. This progress in analytical methodology may help to assign new early life health benefits to known or yet unknown GL-isomers present in HM or infant milk formula.
Fresh human milk volunteer samples from different stages of lactation were spiked with internal standards including e.g. α-Arabinopentaose. Then, anonymized spiked milk specimens were diluted 1:10 with H2O and subjected to ultrafiltration (UF) with 3kDa cut off. UF permeates were analysed by liquid chromatography electrospray ionisation tandem mass spectrometry (LC-ESI-MS2). LC-separation of GLs and other human milk compounds was facilitated by 2.1 x 30mm + 2.1 x 10mm porous graphitized carbon HPLC columns connected in line with a linear ion trap mass spectrometer. Gradient elution of GLs started with 0.3% NH4OH in in 2.5% methanol H2O at 0min and ended with 0.3% NH4OH in 95% methanol at 27 min. Constant flow rate was at 0,4 ml/min and columns were kept at 45 degrees Celsius. Eluting GLs were characterized by negative ion multiple reaction monitoring (MRM) LC-ESI-MS2. Employed MRM transitions had been determined before by MS/MS experiments with commercially available pure GL-standards using collision induced dissociation (CID) fragmentation. Standard addition methodology was applied to calculate GL-abundances in HM.
We successfully developed a label-free negative ion mode LC-ESI-MS2 approach allowing for targeted analysis and distinction of native Galactaosyllactose-isomers directly in HM. By exploiting diagnostic CID-fragments in a multiple reaction monitoring (MRM) LC-ESI-MS2 setup, enhanced label free characterization of GL-isomers could be accomplished. The new targeted MRM LC-ESI-MS2 approach was applied to several human milk samples and required only minimal sample pre-treatment.
With our novel approach, we were able to detect 3’-GL and other GL-isomers directly in human milk samples from various stages of lactation. The abundance of 3’-GL appeared to be relatively stable between colostral and mature milks whereas other GLs behaved differently.
We would like to thank Mr. John M. A. Gonsalves for his excellent technical advice and support in the practical execution of LC-MS experiments.
All authors of this abtracts are employees of Danone Nutricia Research, 3584 CT Utrecht, The Netherlands.
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