CD BioGlyco provides an integrated One-stop Solution for HMO Research. We customize systematic solutions according to clients' scientific research needs.
NEC is a devastating disease of premature infants that also occurs as a major cause of death and morbidity. Neurodevelopmental outcomes are worse after NEC than after meningitis or sepsis. Caring for a baby with NEC has a financial, practical, and emotional toll on the family. Despite substantial investment in NEC research, rates have not declined. The pathogenesis of NEC is complex and not fully understood, but is believed to occur when complex interactions between the immune system, gut microbiome, and diet lead to an inflammatory process within the gut.
Human milk oligosaccharides (HMO) are produced by the mammary gland and are present in varying amounts in different kinds of breast milk. Studies in human preterm infants have shown that a specific HMO called DSLNT is lower in the breast milk of infants who go on to develop NEC.
DSLNT, a specific HMO, has been reported to reduce NEC in animal models. Clinical investigations found that DSLNT deficiency in the mother's breast milk was the cause of nearly all cases of advanced NEC in pediatric outpatient clinics. DSLNT appears to be required to maintain gut health in infants, but the underlying mechanism is unknown.
Fig.1 Structure of DSLNT. (Bode, 2018)
Chromatography and mass spectrometry are the most commonly used methods for the analysis of HMO, such as liquid chromatography/mass spectrometry (LC/MS). CD BioGlyco provides a combined method to analyze DSLNT.
The lipid layer is removed by centrifugation. Ethanol is added and the protein is precipitated from the aqueous phase after centrifugation. Remove ethanol from the HMO-containing supernatant by rotary evaporation. Lactose and salts are removed by gel filtration chromatography using a semi-automated fast protein liquid chromatography (FPLC) system.
HMOs are separated by charge using anion exchange chromatography. Separation is monitored by fluorescence high-performance liquid chromatography (HPLC-FL). Differently charged HMO fractions are further separated by size using gel filtration chromatography and monitored by HPLC-FL. Fractions containing the same but no other HMOs are pooled and lyophilized.
HMOs are fluorescently labeled with 2-aminobenzamide (2AB) and separated by HPLC.
2AB-labeled HMO peaks are collected, dried, and mixed with matrix, and spot on MALDI plates for analysis.
Dissolve HMO in DMSO and perform para-O-methylation. Partially methylated alditol acetates are extracted with dichloromethane and analyzed by GC-MS. Identification is by a combination of established retention time and mass fragmentation patterns.
Fig.2 The workflow diagram for DSLNT analysis by two-dimensional chromatography. (CD BioGlyco)
Chromatography and MS are the most commonly used methods for HMO Profiling. CD BioGlyco provides an efficient and accurate LC/MS method to analyze DSLNT. If you are interested in our services, please contact us for more details without any hesitation.
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