Breast milk oligosaccharides: effects of 2'-fucosyllactose and 6'-sialyllactose on the adhesion of Escherichia coli and Salmonella fyris to Caco-2 cells.

Background: It is well known that human milk oligosaccharides play an important role as prebiotics, anti-inflammatory, and anti-infective agents. In the last few years, several studies have been performed using specific oligosaccharides, such as 2'-fucosyllactose and 6'-sialylactose, to evaluate their biological functions. Objectives: The aim of the present study is to evaluate the anti-adhesive effect of the above oligosaccharides on Escherichia coli and Salmonella fyris. Methods: Adhesion experiments were performed in the presence of 2'-fucosyllactose and 6'-sialyllactose as potential inhibitors of Escherichia coli and Salmonella fyris adhesion to Caco-2 cells. The oligosaccharides were used at different concentrations and the adhesion experiments were performed in triplicate and repeated at least three times. Results: A significant reduction of Escherichia coli adhesion was observed in the presence of 2'-fucosyllactose and 6'-sialyllactose at the human milk concentration. On the contrary, no positive effects were observed in both oligosaccharides on Salmonella firis. Conclusions: Our results suggest that the supplementation in infant formulas of 2'-fucosyllactose and 6'-sialyllactose, actually commercially available and absent in cow milk, could play positive effects in artificially fed infants.

12 year follow up of enzyme-replacement therapy in two siblings with attenuated mucopolysaccharidosis I: the important role of early treatment.

BACKGROUND: Mucopolysaccharidosis type I is an autosomal recessive disorder caused by deficiency of α-L-iduronidase and characterized by a progressive course with multisystem involvement. Clinically, Mucopolysaccharidosis type I is classified into two forms: severe (Hurler syndrome), which presents in infancy and is characterized by rapid progressive neurological involvement and attenuated (Hurler/Scheie and Scheie syndromes), which presents with slower progression and absent to mild nervous system involvement. The specific treatment for attenuated Mucopolysaccharidosis type I consists of enzyme-replacement therapy with laronidase (human recombinant α-L-iduronidase, Aldurazyme). We present here the clinical and laboratory results in an 12-year-old patient affected by the attenuated form of Mucopolysaccharidosis type I treated by enzyme-replacement therapy from the age of 5 months, compared with his 17 year old affected sister, who started therapy at 5 years of age. CASE PRESENTATION: Clinical evaluation of these siblings shows that initiation of therapy prior of the onset of clinically detectable disease resulted in considerable improvement in outcome in the young sibling. After 12 years of enzyme-replacement therapy, facial appearance, linear growth rate, and liver and spleen volumes were normal; moreover, the degree of joint disease, vertebral, and cardiac valvular involvement were only minimal compared with those of his sister. CONCLUSION: This study demonstrates that early diagnosis and early initiation of enzyme-replacement therapy substantially modify the natural history of the attenuated form of Mucopolysaccharidosis type I.

Metabolic fate of milk glycosaminoglycans in breastfed and formula fed newborns.

In this study, the content, structure and residual percentages of glycosaminoglycans (GAGs) in the feces of seven breastfed newborns after ingesting a known amount of milk were studied. A comparison was made with five newborns fed with formula milk. Characterization of GAGs from milk and feces samples was performed according to previous methodology. Compared to the ingested GAGs present in milk, residual feces GAGs of breastfed newborns were <0.4 %, contrary to formula milk fed children, where the residues were ~4 %. As a consequence, >99 % of human milk GAGs are utilized as opposed to ~96 % of formula milk. Hyaluronic acid utilization was found to be fairly similar contrary to chondroitin sulfate/dermatan sulfate and heparan sulfate, which were found to be ~10-18 times lower in formula milk fed children. Our new results further demonstrate that the elevated content of human milk GAGs passes undigested through the entire digestive system of newborns, possibly protecting the infant from infections. In the distal gastrointestinal tract, these complex macromolecules are catabolized by a cohort of bacterial enzymes and constituent monosaccharides/oligosaccharides utilized for further metabolic purposes potentially useful for bacteria metabolism or internalized by intestinal cells. Thanks to their elevated structural heterogeneity, milk GAGs are used differently depending on their distinct primary structure. Finally, a different utilization and availability was observed for human milk GAGs compared to formula milk due to their various composition and structural heterogeneity.

Human milk glycosaminoglycans inhibit in vitro the adhesion of Escherichia coli and Salmonella fyris to human intestinal cells.

BACKGROUND: Breast-fed infants have a lower incidence of acute gastroenteritis due to the presence of several anti-infective factors in human milk. The aim of this work is to study the capacity of human milk glycosaminoglycans (GAGs) to inhibit the adhesion of some common pathogenic bacteria. METHODS: GAGs were isolated from a pool of milk samples collected from different mothers during the first month of lactation. Experiments were carried out to study the ability of GAGs to inhibit the adhesion of two intestinal micro-organisms (enteropathogenic Escherichia coli serotype 0119 and Salmonella fyris) to Caco-2 and Int-407 cell lines. RESULTS: The study showed that the GAGs had an anti-adhesive effect on the two pathogenic strains studied with different degrees of inhibition. In particular, in the presence of human milk GAGs, the adhesion of S. fyris to Caco-2 cells and to Int-407 cells of both tested strains was significantly reduced. CONCLUSION: Our results demonstrated that GAGs in human milk can be one of the important defensive factors against acute diarrheal infections in breast-fed infants.

Effect of holder pasteurisation on human milk glycosaminoglycans.

OBJECTIVES: The benefits of human milk for preterm infants are mainly the result of its nutritional characteristics and the presence of biologically active compounds. Among these compounds, glycosaminoglycans (GAGs) play an emerging leading role. When mother's milk is unavailable or in short supply, pasteurised donor milk represents an important nutritional alternative. The aim of this study was to evaluate the effect of Holder pasteurisation on the concentration of different GAGs in preterm human milk. METHODS: Milk samples collected from 9 mothers having delivered preterm were divided into 2 parts. One part of each sample was immediately frozen (-80°C), whereas the other part was pasteurised with the Holder method before being frozen at -80°C. Specific analytical procedures were applied to evaluate the amount, composition, and structure of main human milk GAGs. RESULTS: No significative differences were measured between not-treated and pasteurised samples for total GAGs content, relative percentages of chondroitin sulfate and heparan sulfate, and main parameters related to galactosaminoglycans structure, even if a slight decrease of total GAGs content of ∼18% was observed in treated samples. CONCLUSIONS: Our results indicate that the Holder pasteurisation does not significatively affect the concentration of the main human milk GAGs.

Capillary electrophoresis separation of human milk neutral and acidic oligosaccharides derivatized with 2-aminoacridone.

Human milk is a unique fluid in glycobiology due to the presence of many free structurally complex oligosaccharides emerging as important dietary factors during early life and having many biological and protective functions. Methods that allow accurate profiling of oligosaccharide mixtures in this complex biological fluid with quantification of the four known genetically determined groups are welcomed. A high-voltage CE separation and detection at 254 nm of 17 neutral and acidic human milk oligosaccharide (HMO) standard along with lactose derivatized with 2-aminoacridone, using a BGE containing 20% methanol as an organic modifier and borate, able to form on-capillary anionic borate-polyol complexes, is reported. This CE approach was able to separate both neutral HMOs and acidic HMOs, with the sialic acid residue, also in the presence of lactose in high content. This method was applied to the four secretory groups individually extracted by a rapid and simple preparative step. LODs were found ranging from ∼50 to 700 fmol. We were able to measure HMO content also in the presence of excess fluorophore, or interference from proteins, peptides, salts, and other impurities normally present in this complex biological fluid. Overall, CE equipped with a UV detector is a common analytical approach and this simple CE separation offers high resolution and sensitivity for the differentiation of human milk samples related to genetic groups and days of lactation by considering that important changes in HMO content are a reflection of the lactation day.

Plasmatic dermatan sulfate and chondroitin sulfate determination in mucopolysaccharidoses.

The evaluation of plasmatic galactosaminoglycans, dermatan sulfate (DS) and chondroitin sulfate (CS) can be helpful in the early identification of MPS patients, also considering that primary storage of one type of GAG can lead to secondary accumulation of other lysosomal substrates. We explore the possibility to determine plasmatic DS and CS in numerous healthy pediatric (and sometimes adult) subjects depending on age and in patients affected by various forms of MPS. A highly sensitive HPLC separation and fluorescence detection was applied for plasma/serum DS and CS determination after a specific enzymatic treatment able to release their constituent disaccharides. DS and CS content decrease significantly with age in controls having high values in the first year (~8 µg/mL). A highly significant decrease was observed for 1-5-year-old (∼-33%) and 5-10-year-old (∼-65%) healthy subgroups. No further decrease was determined showing a stabilization after 5 years of age. MPS I Scheie and Hurler patients showed rather similar DS and CS content significantly higher than controls matched for age. Similarly, MPS II, III and IV subjects all presented significantly higher plasmatic DS and CS content compared to healthy subjects matched for age. The same trend was determined for the only patient affected by MPS VI. Plasmatic DS and CS analyzed by the present procedure may be a useful diagnostic and screening marker for various forms of MPS.

Plasmatic kinetics of dermatan sulfate during enzyme replacement therapy with iduronate-2-sulfatase in a mucopolysaccharidosis II patient.

Enzyme replacement therapy (ERT) is the worldwide standard of care for a number of mucopolysaccharidosis (MPS) diseases. We report a kinetic study of plasmatic dermatan sulfate (DS) in a 3-year-old subject affected by a severe form of MPS II during the first 10 months of ERT with Idursulfase. A strong increase in the DS plasmatic concentration was measured immediately after the first enzyme infusion, with a maximum after 3 h, followed by a continuous decrease in the 8-15 days following the beginning of treatment. After this, a constant plasmatic content of DS concentration was observed. Overall, during the 10-month treatment period, ERT reduced the plasmatic concentration of DS up to ~80-85 %, but it was unable to totally remove it from the blood. We can suppose that immediately after the first enzyme administrations, a large amount of abnormal DS is removed from tissues reaching the blood compartment and eliminated via the urine, and thereafter only minimal changes are observed. The persistency of the residual amounts of DS with the actually recommended dosage in our Patient may suggest the opportunity to promote further studies with increased enzyme dosages to completely remove the accumulation of lysosomal DS.

Mild mental retardation and low levels of urinary heparan sulfate in a patient with the attenuated phenotype of mucopolysaccharidosis type IIIA.

OBJECTIVES: We report the case of a 28-year-old female subject affected by the attenuated phenotype of mucopolysaccharidosis type IIIA characterized by moderate slowly evolving mental retardation in which the urinary content of heparan sulfate was demonstrated as being substantially low compared to that found in patients with the severe phenotype. DESIGN AND METHODS: The specific evaluation of macromolecular heparan sulfate by electrophoresis and the determination of related glucosamine in the urine were performed. RESULTS: In our patient, the urinary macromolecular heparan sulfate content (4.2µg/mg creatinine) was ~7.5-times higher than in healthy subjects (0.56µg/mg creatinine±0.9 SD) while it was ~28-times lower compared to the severe mucopolysaccharidosis IIIA group (117µg/mg creatinine±44.8 SD). Furthermore, the urinary glucosamine (86.4µg/mg creatinine) was ~2.4-times greater than in healthy subjects (36.0µg/mg creatinine±18.2 SD) but ~2.4-times lower than in severe subjects (208.1µg/mg creatinine±55.0 SD). CONCLUSIONS: The above data could reflect the reduced heparan sulfate storage in her tissues and organs, and in particular in the brain, consequently explaining her moderate mental retardation. Furthermore, the clinical presentation of patients with an attenuated form of MPS III confirms the need for a specific evaluation of urinary GAGs in all young and adult subjects showing a not well-defined or not particularly severe mental retardation, along with an early MPS diagnosis. Such investigation should also be associated with a more specific characterization of heparan sulfate.

On-line high-performance liquid chromatography-fluorescence detection-electrospray ionization-mass spectrometry profiling of human milk oligosaccharides derivatized with 2-aminoacridone.

A high-resolution normal-phase high-performance liquid chromatography-fluorescence detection-electrospray ionization-mass spectrometry separation and structural characterization of the main oligosaccharides along with lactose from human milk samples is described. A total of 22 commercially available oligosaccharides were fluorotagged with 2-aminoacridone and separated on an amide column and identified on the basis of their retention times and mass spectra. Derivatized species having mass lower than approximately 800 to 900 exhibited mainly [M-H](-1) anions, oligomers with mass up to approximately 1000 to 1100 were represented by both [M-H](-1) and [M-2H](-2) anions, and oligomers greater than approximately 1200 to 1300 were characterized by a charge state of -3. Furthermore, the retention times were directly related to the glycans' molecular mass. Human milk samples from the four groups of donors (Se±/Le±) were analyzed for their composition and amount of free oligosaccharides after rapid and simple prepurification and derivatization steps also in the presence of lactose in high content. This analytical approach enabled us to perform the determination of species not detected by traditional techniques, such as sialic acid, as well as of species present in low content easily mistaken with other peaks. Finally, labeled human milk oligosaccharides were analyzed without any interference from excess fluorophore or interference from proteins, peptides, salts, and other impurities normally present in this complex biological fluid.

Glycosaminoglycan content in term and preterm milk during the first month of lactation.

BACKGROUND: In a recent study, we performed a complete structural characterization of glycosaminoglycans (GAGs) in human mature milk. However, no data are available on the total content of GAGs in human milk from healthy mothers having delivered term or preterm newborns. OBJECTIVES: In this study, we evaluated the total content of GAGs in pooled milk from healthy mothers having delivered term or preterm newborns during the first month of lactation. METHODS: Highly specific and sensitive analytical approaches were used to quantify human milk total GAGs. RESULTS: Highest GAG values are present at day 4 (9.3 and 3.8 g/l in preterm and term milk, respectively), followed by a progressive decrease up to day 30 (4.3 and 0.4 g/l). The more remarkable differences are related to the first phases of lactation in which a strong decrease in GAGs was observed between days 4 and 10 (about -73% in term and -50% in preterm newborns). CONCLUSIONS: During the first month of lactation, the absolute amount of polysaccharides was constantly and significantly higher in preterm than in term milk, with a similar behavior in the decrease. These data further indicate that human milk GAGs may have an active role in protecting newborns during the first phases of lactation.

Plasmatic and Urinary Glycosaminoglycans Characterization in Mucopolysaccharidosis II Patient Treated with Enzyme-Replacement Therapy with Idursulfase.

We report the structural characterization of plasmatic and urinary GAGs in a patient affected by MPS II (Hunter syndrome) before and during the first 10 months of enzyme-replacement therapy (ERT). Plasmatic GAGs before ERT were rich in pathological DS consisting of iduronic acid (IdoA) and composed of ~90% ΔDi4s and trace amounts of disulfated disaccharides. DS was also characterized as the main (~90%) urinary GAG mainly composed of ~90% ΔDi4s with minor percentages of monosulfated and disulfated disaccharides, in particular ΔDi2,4dis. After 300 days of ERT, plasmatic DS strongly decreased but ~14% of IdoA-rich ΔDi4s was still detected. Similarly, urinary galactosaminoglycans were mainly composed of 78% ΔDi4s, ~11% ΔDi6s and ~4% ΔDi0s with the persistence of ΔDi2,4dis (~4%). About 40% of IdoA-formed ΔDi4s were also calculated, thus confirming that pathological DS is still present in excreted urinary GAGs during ERT. By considering the % of IdoA, we observed rather similar kinetics of excretion in fluids from the beginning of the treatment. Immediately after the first enzyme infusion, a large amount of abnormal DS is removed from tissues reaching the blood compartment and eliminated via the urine, and this process lasts for about 2 weeks. After this, the percentage of IdoA-rich material present in biological fluids remains fairly constant over the following 9 months of treatment. To date, these are the first data regarding plasmatic and urinary kinetics directly measured on products released by the activity of the recombinant enzyme Idursulfase, iduronate-2-sulfatase, evaluated using specific and sensitive analytical procedures.

Preterm milk oligosaccharides during the first month of lactation.

OBJECTIVE: Oligosaccharides represent one of the main components of human milk, and they have been assigned important biological functions for newborns. Qualitatively and quantitatively, their presence in milk is strictly related to the expression of the mother's Se and/or Le genes, on the basis of which 4 different milk groups have been described. The aim of the study was to provide new data on the oligosaccharide composition of preterm milk in relation to the 4 groups. METHODS: High-pH anion-exchange chromatography was used to quantify levels of 23 oligosaccharides and lactose in 252 milk samples collected from 63 mothers during the first month of lactation and to identify the 4 milk groups. RESULTS: Substantial differences in oligosaccharide contents were found within the groups and were strictly related to the presence or absence of specific fucosyl-oligosaccharides. The highest concentration was found in group 1 (>20 g/L), the lowest level was found in group 4 (∼10 g/L), and intermediate values were observed in groups 2 and 3. No statistically significant differences in lactose concentrations were observed among the groups. CONCLUSIONS: Our data confirm lower lactose concentrations in preterm milk, compared with term milk, and they provide the first detailed characterization of oligosaccharides in preterm milk, demonstrating important differences in oligosaccharide contents in the 4 groups. These differences might exert an influence on several biological functions that are particularly important for preterm infants and currently are attributed to milk oligosaccharides.

Oligosaccharides in 4 different milk groups, Bifidobacteria, and Ruminococcus obeum.

OBJECTIVES: The aim of this study was to identify a link between the total amount of breast milk oligosaccharides and faecal microbiota composition of newborns at the end of the first month of life, with special attention paid to bifidobacteria, and establish the role, if any, of the different oligosaccharides in determining the gut microbiota composition. SUBJECTS AND METHODS: Milk oligosaccharide groups were identified by high-performance anion exchange chromatography analysis. DPCRNA from newborns' faecal samples at 30 days of life was isolated and processed by polymerase chain reaction analyses that allow the identification of 6 species of bifidobacteria (adolescentis, bifidum, breve, catenulatum, longum, infantis) and Ruminococcus spp; denaturing gradient gel electrophoresis analysis was also performed. RESULTS: No substantial differences in bifidobacteria species composition within milk groups 1, 2, and 3 were observed; however, infants fed with group 4 milk show a microbiota characterised by a greater frequency of Bifidobacteria adolescentis and the absence of Bifidobacteria catenulatum. For the first time, a high percentage of the Ruminococcus genus in infants fed with all milk groups was found. CONCLUSIONS: Our data show that milk groups 1, 2, and 3, containing an amount of oligosaccharides ranging within 10 to 15 g/L, share a substantially identical composition of the intestinal microbiota in breast-fed infants, despite quali-quantitative difference in oligosaccharides content. Newborns taking milk with only 5 g/L of oligosaccharides (group 4) harbour a different intestinal microbiota.

High-throughput determination of urinary hexosamines for diagnosis of mucopolysaccharidoses by capillary electrophoresis and high-performance liquid chromatography.

Mucopolysaccharidoses (MPS) diagnosis is often delayed and irreversible organ damage can occur, making possible therapies less effective. This highlights the importance of early and accurate diagnosis. A high-throughput procedure for the simultaneous determination of glucosamine and galactosamine produced from urinary galactosaminoglycans and glucosaminoglycans by capillary electrophoresis (CE) and HPLC has been performed and validated in subjects affected by various MPS including their mild and severe forms, Hurler and Hurler-Scheie, Hunter, Sanfilippo, Morquio, and Maroteaux-Lamy. Contrary to other analytical approaches, the present single analytical procedure, which is able to measure total abnormal amounts of urinary GAGs, high molecular mass, and related fragments, as well as specific hexosamines belonging to a group of GAGs, would be useful for possible application in their early diagnosis. After a rapid urine pretreatment, free hexosamines are generated by acidic hydrolysis, derivatized with 2-aminobenzoic acid and separated by CE/UV in ∼10min and reverse-phase (RP)-HPLC in fluorescence in ∼21min. The total content of hexosamines was found to be indicative of abnormal urinary excretion of GAGs in patients compared to the controls, and the galactosamine/glucosamine ratio was observed to be related to specific MPS syndromes in regard to both their mild and severe forms. As a consequence, important correlations between analytical response and clinical diagnosis and the severity of the disorders were observed. Furthermore, we can assume that the severity of the syndrome may be ascribed to the quantity of total GAGs, as high-molecular-mass polymers and fragments, accumulated in cells and directly excreted in the urine. Finally, due to the high-throughput nature of this approach and to the equipment commonly available in laboratories, this method is suitable for newborn screening in preventive public health programs for early detection of MPS disorders, diagnosis, and their treatment.

Composition and structure elucidation of human milk glycosaminoglycans.

To date, there is no complete structural characterization of human milk glycosaminoglycans (GAGs) available nor do any data exist on their composition in bovine milk. Total GAGs were determined on extracts from human and bovine milk. Samples were subjected to digestion with specific enzymes, treated with nitrous acid, and analyzed by agarose-gel electrophoresis and high-performance liquid chromatography for their structural characterization. Quantitative analyses yielded ∼7 times more GAGs in human milk than in bovine milk. In particular, galactosaminoglycans, chondroitin sulfate (CS) and dermatan sulfate (DS), were found to differ considerably from one type of milk to the other. In fact, hardly any DS was observed in human milk, but a low-sulfated CS having a very low charge density of 0.36 was found. On the contrary, bovine milk galactosaminoglycans were demonstrated to be composed of ∼66% DS and 34% CS for a total charge density of 0.94. Structural analysis performed by heparinases showed a prevalence of fast-moving heparin over heparan sulfate, accounting for ∼30-40% of total GAGs in both milk samples and showing lower sulfation in human (2.03) compared with bovine (2.28). Hyaluronic acid was found in minor amounts. This study offers the first full characterization of the GAGs in human milk, providing useful data to gain a better understanding of their physiological role, as well as of their fundamental contribution to the health of the newborn.