Fabio Galeotti

Analysis of glycosaminoglycan-derived, precolumn, 2-aminoacridone–labeled disaccharides with LC-fluorescence and LC-MS detection

Glycosaminoglycans (GAGs) possess considerable heterogeneity in average molecular mass, molecular mass range, disaccharide composition and content and position of sulfo groups. Despite recent technological advances in the analysis of GAGs, the determination of GAG disaccharide composition still remains challenging and provides key information required for understanding GAG function. Analysis of GAG-derived disaccharides relies on enzymatic treatment, providing one of the most practical and quantitative approaches for compositional mapping. Tagging the reducing end of disaccharides with an aromatic fluorescent label affords stable derivatives with properties that enable improved detection and resolution. HPLC with on-line electrospray ionization mass spectrometry (ESI-MS) offers a relatively soft ionization method for detection and characterization of sulfated oligosaccharides. GAGs obtained from tissues, biological fluids or cells are treated with various enzymes to obtain disaccharides that are fluorescently labeled with 2-aminoacridone (AMAC) and resolved by different LC systems for high-sensitivity detection by fluorescence, and then they are unambiguously characterized by MS. The preparation and labeling of GAG-derived disaccharides can be performed in ∼1–2 d, and subsequent HPLC separation and on-line fluorescence detection and ESI-MS analysis takes another 1–2 h.

Online reverse phase-high-performance liquid chromatography-fluorescence detection-electrospray ionization-mass spectrometry separation and characterization of heparan sulfate, heparin, and low-molecular weight-heparin disaccharides derivatized with 2-aminoacridone.

A high-resolution online reverse-phase-high-performance liquid chromatography (RP-HPLC)-fluorescence detector (Fd)-electrospray ionization-mass spectrometry (ESI-MS) separation and structural characterization of disaccharides prepared from heparin (Hep), heparan sulfate (HS), and various low-molecular-weight (LMW)-Hep using heparin lyases and derivatization with 2-aminoacridone (AMAC) are described. A total of 12 commercially available Hep/HS-derived unsaturated disaccharides were separated and unambiguously identified on the basis of their retention times and mass spectra. The constituent disaccharides of various samples, including unfractionated Hep/HS, fast-moving and slow-moving Hep components, and several marketed products, were characterized. Furthermore, for the first time, the saturated trisulfated disaccharide belonging to the nonreducing end of Heps was detected as being approximately 2% in unfractionated samples and ~15-21% in LMW-Heps prepared by nitrous acid depolymerization. No desalting of the commercial products prior to enzymatic digestion or prepurification steps to eliminate any excess of AMAC reagent or interference from proteins, peptides, and other sample impurities before RP-HPLC-Fd-ESI-MS injection were necessary. This method has applicability for the rapid differentiation of pharmaceutical Heps and LMW-Heps prepared by means of different depolymerization processes and for compositional analysis of small amounts of samples derived from biological sources by using the highly sensitive fluorescence detector.

Novel reverse-phase ion pair-high performance liquid chromatography separation of heparin, heparan sulfate and low molecular weight-heparins disaccharides and oligosaccharides.

In this study, by using tetrabutylammonium bisulfate as ion-pairing reagent, we were able to separate all the main heparin/heparan sulfate disaccharides generated by the action of heparinases along with the main Hep tetrasaccharide possessing a 3-O-sulfate group on the sulfoglucosamine unit and resistant to enzymatic action. Moreover, this novel HPLC method was able to separate and quantify uncommon disaccharides/oligosaccharides present in low molecular weight-heparins produced by chemical treatment with nitrous acid, dalteparin, or benzylation followed by alkaline hydrolysis, enoxaparin. Additionally, this procedure yields a sensitivity ∼4-times higher compared to conventional strong-anion exchange-HPLC separation. This was obtained by a common UV detector at 232 nm avoiding the use of complex procedures capable of increasing sensitivity by post-column derivatization. Finally, it is worth mentioning that disaccharide/oligosaccharide composition by HPLC and UV detection is a common analytical approach in quality control laboratories to evaluate heparins and low molecular weight-heparins structure and quality during their extraction and production. This simple HPLC approach offers high resolution and sensitivity for the rapid differentiation of pharmaceutical native heparins and derivatives and for the compositional analysis of small amounts of samples derived from biological sources at a glycosaminoglycans level of a few hundred nanogram.

Isolation and structural characterization of chondroitin sulfate from bony fishes.

Chondroitin sulfate (CS) was purified from the bones of common fishes, monkfish, cod, spiny dogfish, salmon and tuna, and characterized in an effort to find alternative sources and new peculiar structures of this complex biomacromolecule utilized in the pharmaceutical and nutraceutical industry. Quantitative analyses yielded a CS content ranging from 0.011% for cod up to 0.34% for monkfish. The disaccharide pattern showed the presence of nonsulfated disaccharide, monosulfated species ΔDi6s and ΔDi4s, and disulfated disaccharides in different percentages. The disulfated species ΔDi2,6dis was present in all CS extracts in a range of 1.3-10.5%. The presence of these disulfated disaccharides may be a useful marker for the marine origin of CS. The newly identified sources would certainly enable the production of CS with unique disaccharide composition and properties.

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.

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.

Recent advances on separation and characterization of human milk oligosaccharides.

Free human milk oligosaccharides (HMOs) are unique due to their highly complex nature and important emerging biological and protective functions during early life such as prebiotic activity, pathogen deflection, and epithelial and immune cell modulation. Moreover, four genetically determined heterogeneous HMO secretory groups are known to be based on their structure and composition. Over the years, several analytical techniques have been applied to characterize and quantitate HMOs, including nuclear magnetic resonance spectroscopy, high‐performance liquid chromatography (HPLC), high pH anion‐exchange chromatography, off‐line and on‐line mass spectrometry (MS), and capillary electrophoresis (CE). Even if these techniques have proven to be efficient and simple, most glycans have no significant UV absorption and derivatization with fluorophore groups prior to separation usually results in higher sensitivity and an improved chromatographic/electrophoretic profile. Consequently, the analysis by HPLC/CE of derivatized milk oligosaccharides with different chromophoric active tags has been developed. However, UV or fluorescence detection does not provide specific structural information and this is a key point in particular related to the highly complex nature of the milk glycan mixtures. As a consequence, for a specific determination of complex mixtures of oligomers, analytical separation is usually required with evaluation by means of MS, which has been successfully applied to HMOs, resulting in efficient compositional analysis and profiling in various milk samples. This review aims to give an overview of the current state‐of‐the‐art techniques used in HMO analysis.

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.

Selective removal of keratan sulfate in chondroitin sulfate samples by sequential precipitation with ethanol

Keratan sulfate (KS) is present as a contaminant in chondroitin sulfate (CS) mainly extracted from shark cartilage. We report a selective removal procedure of KS in CS samples by means of sequential precipitation with ethanol. Purified shark CS containing approximately 10% to 15% KS was subjected to a precipitation procedure in the presence of increasing percentages of saturated ethanol. In contrast to other solvents, 1.0 volume of ethanol was able to selectively purify CS, with a purity of approximately 100%, from KS. The current selective and simple procedure appears to be a reliable industrial preparation of CS devoid of large amounts of the residual KS.

Determination of urinary hexosamines for diagnosis of bladder pain syndrome

Introduction and hypothesisBladder pain syndrome (BPS) is a chronic disease characterized by urgency, bladder pain, and frequency, and urinary glycosaminoglycans are thought to reflect bladder epithelial deficiency in BPS. Sensitive and specific evaluation of total urinary glycosaminoglycans may be useful for the clinical diagnosis of BPS and its treatment.MethodsA procedure for the simultaneous determination of glucosamine and galactosamine produced from urinary glycosaminoglycans has been performed in BPS patients and healthy subjects.ResultsThe total content of urinary hexosamines in BPS patients significantly increased by ~130% with the increase in glucosamine greater than galactosamine.ConclusionsA significant increase in total hexosamines content and in particular in glucosamine belonging to urinary heparan sulfate was determined in BPS patients compared with controls. We propose HS and in particular its low-molecular mass fragments and glucosamine assay as useful markers for a biochemical diagnosis of BPS and for monitoring this syndrome.