Human milk glycosaminoglycan composition from women of different countries: a pilot study.

Objective: In this pilot study, we report the composition, structure and properties of glycosaminoglycans (GAG) present in milk samples of various countries and ethnicities.Methods: Fifty samples of human milk were analyzed, 10 from east Europe, 10 from North Africa, 10 from Central Africa, 10 from South America and 10 from Asia. Moreover, 30 samples were obtained during the first week and 20 between 8 to 30 days of life.Results: Overall, no significant differences were observed for the qualitative composition of GAGs, mainly chondroitin sulfate, heparan sulfate and hyaluronic acid, comparing the mothers from the various countries and between the 30 milks obtained during the first week and the 20 samples collected thereafter. Moreover, no significant differences in human milk GAGs within the different groups analyzed belonging to various counties and ethnicities were observed.Conclusions: These results may be of useful, as in the case of pilot studies with infant formulas enriched with chondroitin sulfate (CS) and/or heparan sulfate (HS) necessary to verify their possible positive effects on newborns feeding in countries at high risk of infection and/or infestation.

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.

False positive screen test for mucopolysaccharidoses in healthy female newborns.

BACKGROUND: In total, 930 urine samples obtained on 2nd and 3rd day from birth have been analyzed for the early diagnosis of Mucopolysaccharidoses. METHODS: Dimethylmethylene blue (DMB) assay and one-dimensional electrophoresis were performed in all urine samples. Agarose gel electrophoresis, before and after treatment with chondroitinase ABC and heparinases, was used for a comprehensive characterization. RESULTS: Out of 930 urine samples 7 showed anomalous electrophoretic pattern; 5 of them had high GAG levels by DMB test. Atypical samples (n = 7) were analyzed by agarose gel electrophoresis. After enzymatic digestion, some slow bands were still visible. A second urine sample of the above 7 newborns was analyzed at the age of 1 month, demonstrating both a normal pattern and normal GAG levels. Additional urine and vaginal mucus samples from 10 term neonates with vaginal bleeding showed the same electrophoretic pattern observed in the 7 false positive samples. CONCLUSIONS: The altered electrophoretic pattern may be due to the presence of glycoproteins and not to specific GAGs, due to high levels of maternal hormones exposure during pregnancy. To our knowledge, this is the first time maternal estrogen hormones are proposed as a likely cause of false-positive urinary glycosaminoglycan screen test in healthy newborns.

Composition and structure of glycosaminoglycans in DBS from 2-3-day-old newborns for the diagnosis of mucopolysaccharidosis.

Dried blood spot (DBS) technology is a cheap and easy method largely applied in newborn screening. Mucopolysaccharidoses (MPS) are characterized by the deficit of enzymes that degrade glycosaminoglycans (GAGs) characterized by progressive worsening of the conditions. For a possible early diagnosis of MPS, we developed a method of uronic acid (UA)-GAGs determination in DBS of 600 healthy newborns and from a small group of MPS subjects matched for age. Spotted blood UA-GAGs of the normal newborns are composed of 67.2% chondroitin sulfate (CS), 28.6% heparan sulfate (HS) and 4.4% hyaluronic acid with a CS/HS ratio of 2.35 and a total GAGs content of 0.43 µg/DBS. A chemical evaluation of CS and HS structure was performed by measuring their disaccharide composition, sulfation and the overall charge density. The DBS of four different MPS types presented an increase of total or single UA-GAGs content and/or modifications of the CS and HS disaccharide composition as well as chemical signature also related to the MPS enzymatic defect. The modifications of the UA-GAGs composition, parameters and structure of healthy newborns determined in DBS would be useful for a possible early diagnosis of various MPS types.

Importance of the combined urinary procedure for the diagnosis of Mucopolysaccharidoses.

BACKGROUND: Mucopolysaccharidoses are characterized by the accumulation of undegraded glycosaminoglycans in lysosomes in multiple organs and by their excretion in high amounts in urine. The aim of this study is to determine if this simple, reliable and reproducible method is useful for the diagnosis of Mucopolysaccharidoses. METHODS: The study included 2154 normal urine samples and 210 samples from 73 patients affected by different types of Mucopolysaccharidoses. The glycosaminoglycans were quantified by a dimethylmethylene blue method and size-fractionated by a modified one-dimensional electrophoresis method. RESULTS: The combination of the two methods allowed to identify all the patients affected by the different types of Mucopolysaccharidosis with 100% sensitivity and specificity. CONCLUSION: This combined approach gives fast diagnostic orientation about the different types of Mucopolysaccharidoses, offering an important tool for a better understanding of diagnosis and patient management.

Total and single species of uronic acid-bearing glycosaminoglycans in urine of newborns of 2-3days of age for early diagnosis application.

BACKGROUND: Urine are easily accessible and relatively simple to process and uronic acid-bearing glycosaminoglycans (UA-GAGs) may serve as biomarkers for several diseases, like for mucopolysaccharidosis. METHODS: We report a study from a large cohort of healthy newborns of 2-3days to have a basic profile of total content of urinary UA-GAGs, their composition and structural signatures utilizing a rapid extractive method and sensitive separation of enzymatic released disaccharides by capillary electrophoresis-light induced fluorescence. Results were also compared with those obtained from normal adult subjects. RESULTS: A total of UA-GAGs content of ~35µg/mg creatinine was observed in 331 newborns versus 1.5µg/mg creatinine of adult urine composed of ~90% chondroitin sulfate (CS), ~7% heparan sulfate (HS) and ~3% hyaluronic acid (HA). No significant differences were observed with adults. Specific ratios between the main CS disaccharides were informative of a significant greater 4-sulfation and charge density for newborn compared to adults. The HS from newborn urine was mainly composed by the non-sulfated (~64%) and mono-sulfated (~28%) disaccharides. No significant differences were observed versus adult urine. CONCLUSIONS: The present method is able to measure changes in UA-GAG composition and their structure independently of the age of subjects and rapidly applicable to the newborn diagnosis without necessity to have creatinine levels. Moreover, modifications in charge density values as well as the presence of sulfate groups in specific positions may be indicative of altered conditions.

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.

Human milk glycosaminoglycans: the state of the art and future perspectives.

Recently, a complete characterization and detailed evaluation of the glycosaminoglycans of human milk were performed. The total glycosaminoglycans content in milk from healthy mothers having delivered term or preterm newborns showed a constant pattern which was essentially composed of two main polysaccharides: chondroitin sulfate (60-70%) and heparin (30-40%). Moreover, considerable variations of glycosaminoglycans concentration were found during the first month of lactation, the highest values being present in colostrum compared to mature milk. Metabolism and potential biological functions of human milk glycosaminoglycans are hypothesized and future studies are encouraged.

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.