Human Milk Glycosaminoglycans in Feces of Breastfed Newborns: Preliminary Structural Elucidation and Possible Biological Role

Dear Editor:

Much evidence has been obtained that several human milk glycans (glycoproteins, glycolipids, and especially oligosaccharides), behaving as cell surface receptor homologs, are able to inhibit binding of pathogens and thus protect the newborn against several enteric infections. ¹ Moreover, recent studies have focused attention on human milk complex sulfated polysaccharides, the glycosaminoglycans (GAGs), as well as their structure and their possible biological roles.^2,3 However, no data are yet available regarding the metabolic fate of GAGs and their possible presence in the feces of breastfed newborns or regarding their composition and structure, which would be useful to shed light on their metabolism.

Following written parental consent, morning fecal samples were obtained from 12 term breastfed newborns (six females, six males) between days 5 and 33 postpartum (mean±SD, 13.92±7.89 days). Samples were immediately stored at −20°C and then lyophilized. GAGs were extracted and purified, and disaccharide mapping was determined according to previous methodology ² to obtain GAG structural characterization.

We determined the presence of chondroitin sulfate (CS) (approximately 67%) and dermatan sulfate (approximately 4%) for a total content of approximately 32 mg/100 g of feces, and the total amount of heparan sulfate (HS)/heparin (Hep) (approximately 29%) was approximately 13 mg/100 g of feces. Disaccharide evaluation of CS showed the presence of a large percentage of disaccharide sulfated in position 4 with a lower content of disaccharide sulfated in position 6 and of the nonsulfated disaccharide for a charge density value of approximately 0.81 (Table 1).

The nonsulfated disaccharide 4,5-unsaturated uronic acid-N-acetylglucosamine, typical of HS, was found to constitute the main species (approximately 28%) present in feces along with the three main monosulfated (approximately 23%), the three main disulfated (approximately 35%), and the trisulfated (approximately 14%) disaccharide typical of Hep (Table 1). Because of this peculiar disaccharide composition, the HS extracted from human newborn feces was found to have a charge density value (approximately 1.40) similar to the polymer characterized in human milk showing a high content of disulfated and trisulfated disaccharides and a high charge density. ²

Our results demonstrate the presence in the feces of breastfed newborns of a highly sulfated HS molecule rather similar in structure and disaccharide composition to the polyanion characterized in human milk. ² However, the CS structure was found to be different, in particular because of a higher content of 4-sulfated disaccharide and a lower contribution of the nonsulfated species for a generally high charge density, with respect to CS from human milk (0.81 vs. 0.36 of human milk). This primary structure of feces CS may be the result of the partial absorption of its chains, in particular those lacking in sulfate groups as demonstrated in previous studies. ⁴ In fact, the partial (and preferential) absorption of CS chains/oligomers may be the result of its degradation by enzymes produced by intestinal bacteria, such as chondroitinases produced by Bacteroides strains from the human colon able to degrade CS of various structures exhibiting broad specificity. ⁵

The presence of Bacteroides species from the human intestine suggests that the catabolism of human milk GAGs might partially take place in newborns. However, we still found the presence in newborns' feces of CS and, in particular, HS/Hep structurally similar to those evaluated in human milk, demonstrating that a part of these polymers could be excreted as unmodified macromolecules. This is new information enabling us to understand more fully the role and the presence of these complex macromolecules in human milk and their relationship with the health of newborns. In fact, Newburg et al. ⁶ have demonstrated that GAGs in human milk are able to inhibit the binding of the human immunodeficiency virus envelope glycoprotein gp120 to the cellular CD4 receptor. From these preliminary data, we can suppose that human milk GAGs, in particular HS/Hep, pass partially undigested through the entire digestive system of newborns, acting as possible soluble receptors able to inhibit the binding of different pathogens to the intestinal mucosa, thus protecting the infant from infections.

However, although feces HS/Hep seems likely to be derived from ingested milk, the origin or potential modification of CS remains unclear. In fact, intestinal cells produce large amounts of GAGs that may be shed in the feces. Further studies are necessary to clarify this point, such as the structural evaluation of feces GAGs between breastfed and formula-fed infants or between infants and adults.