Noninvasive detection of any-stage cancer using free glycosaminoglycans.

Cancer mortality is exacerbated by late-stage diagnosis. Liquid biopsies based on genomic biomarkers can noninvasively diagnose cancers. However, validation studies have reported ~10% sensitivity to detect stage I cancer in a screening population and specific types, such as brain or genitourinary tumors, remain undetectable. We investigated urine and plasma free glycosaminoglycan profiles (GAGomes) as tumor metabolism biomarkers for multi-cancer early detection (MCED) of 14 cancer types using 2,064 samples from 1,260 cancer or healthy subjects. We observed widespread cancer-specific changes in biofluidic GAGomes recapitulated in an in vivo cancer progression model. We developed three machine learning models based on urine (Nurine = 220 cancer vs. 360 healthy) and plasma (Nplasma = 517 vs. 425) GAGomes that can detect any cancer with an area under the receiver operating characteristic curve of 0.83-0.93 with up to 62% sensitivity to stage I disease at 95% specificity. Undetected patients had a 39 to 50% lower risk of death. GAGomes predicted the putative cancer location with 89% accuracy. In a validation study on a screening-like population requiring ≥ 99% specificity, combined GAGomes predicted any cancer type with poor prognosis within 18 months with 43% sensitivity (21% in stage I; N = 121 and 49 cases). Overall, GAGomes appeared to be powerful MCED metabolic biomarkers, potentially doubling the number of stage I cancers detectable using genomic biomarkers.

Analysis of normal levels of free glycosaminoglycans in urine and plasma in adults.

Plasma and urine glycosaminoglycans (GAGs) are long, linear sulfated polysaccharides that have been proposed as potential noninvasive biomarkers for several diseases. However, owing to the analytical complexity associated with the measurement of GAG concentration and disaccharide composition (the so-called GAGome), a reference study of the normal healthy GAGome is currently missing. Here, we prospectively enrolled 308 healthy adults and analyzed their free GAGomes in urine and plasma using a standardized ultra-high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry method together with comprehensive demographic and blood chemistry biomarker data. Of 25 blood chemistry biomarkers, we mainly observed weak correlations between the free GAGome and creatinine in urine and hemoglobin or erythrocyte counts in plasma. We found a higher free GAGome concentration - but not a more diverse composition - in males. Partitioned by gender, we also established reference intervals for all detectable free GAGome features in urine and plasma. Finally, we carried out a transference analysis in healthy individuals from two distinct geographical sites, including data from the Lifelines Cohort Study, which validated the reference intervals in urine. Our study is the first large-scale determination of normal free GAGomes reference intervals in plasma and urine and represents a critical resource for future physiology and biomarker research.

Uronic acid carbazole assay and cetylpyridinium chloride titration depend on the chondroitin sulfate molecular weight.

Chondroitin sulfate (CS) of various molecular weight (MW), up to ∼3 kDa, were produced and tested for uronic acid carbazole assay and cetylpyridinium chloride (CPC) titration showing an evident decrease in the assays depending on the CS MW. The described results for uronic acid assay by carbazole reaction and CPC titration of CS poses the problem to know the MW values before their application and to use comparable standards to obtain reliable results. Otherwise, the related quantitative data can be affected by a great error and fake certificate of analysis.

Human milk glycosaminoglycans inhibit cytomegalovirus and respiratory syncytial virus infectivity by impairing cell binding.

BACKGROUND: The antiviral role of glycosaminoglycans in human milk (HM-GAGs) has been poorly investigated. They are highly sulfated polysaccharides, which were proposed to act as decoy receptors according to their structure. The aim of this study is to evaluate the antiviral potential and the mechanism of action of total and individual HM-GAGs against three pediatric clinically relevant viruses: respiratory syncytial virus (RSV), cytomegalovirus (HCMV), and rotavirus. METHODS: HM-GAGs were isolated from HM and a library of individual GAGs, structurally related to HM-GAGs, was prepared. The antiviral activity of HM-GAGs and the impact of thermal treatment were investigated in vitro by specific antiviral assays. RESULTS: We demonstrated that HM-GAGs are endowed with anti-HCMV and anti-RSV activity and that they act by altering virus attachment to cell. We clarified the contribution of individual HM-GAGs, showing a specific structure-related activity. We did not observe any alteration of HM-GAG antiviral activity after thermal treatment. CONCLUSIONS: We showed that HM-GAGs contribute to the overall antiviral activity of HM, likely exerting a synergic action with other HM antiviral agents. HM-GAGs can now be added to the list of endogenous factors that may reduce breast-milk-acquired HCMV symptomatic infections and protecting infants from respiratory tract infections by RSV. IMPACT: HM-GAGs have been poorly investigated for their antiviral action so far. We demonstrated that HM-GAGs are endowed with significant anti-HCMV and anti-RSV activity and that they are able to alter virus binding to the cell. The contribution of individual HM-GAGs is mainly exerted by the FMHep and is not based on a simple charge interaction between the virus and sulfate groups but involves a specific GAG structural configuration. Our results contribute to identifying the multiple factors synergically acting in mediating HM antiviral properties and to clarifying their specific mechanism of action.

Recent advances in capillary electrophoresis separation of monosaccharides, oligosaccharides, and polysaccharides.

This article illustrates the basis and applications of methodologies for the analysis of simple and complex carbohydrates by means of CE. After a description of the most common and novel approaches useful for the analysis and characterization of carbohydrates, this review covers the recent advances in CE separation of monosaccharides, oligosaccharides, and polysaccharides. Various CE techniques are also illustrated for the study of carbohydrates derived from complex glyco-derivatives such as glycoproteins and glycolipids, essential for biopharmaceutical and glycoproteomics applications as well as for biomarker detection. Most glycans have no significant UV absorption, and derivatization with fluorophore groups prior to separation usually results in higher sensitivity and an improved electrophoretic profile. We also discuss the recent applications and separations by CE of derivatized simple and more complex carbohydrates with different chromophoric active tags. Overall, this review aims to give an overview of the most recent state-of-the-art techniques used in carbohydrate analysis by CE.

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.

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.

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.

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.

Breast cyst fluid heparan sulphate is distinctively N-sulphated depending on apocrine or flattened type.

Breast cyst fluid (BCF) contained in gross cists is involved with its many biomolecules in different stages of breast cystic development. Type I apocrine and type II flattened cysts are classified based on biochemical, morphological and hormonal differences, and their different patterns of growth factors and active biocompounds may require different regulation. In a previous paper, hyaluronic acid in a very low content and chondroitin sulphate/dermatan sulphate were identified and characterized in BCF. In this new study, various apocrine and flattened BCFs were analyzed for HS concentration and disaccharide pattern. Apocrine HS was found specifically constituted of N-acetyl groups contrary to flattened HS richer in N-sulphate disaccharides with an overall N-acetylated/N-sulphated ratio significantly increased in apocrine compared with flattened (13.5 vs 3.7). Related to this different structural features, the charge density significantly decreased (~-30%) in apocrine versus flattened BCFs. Finally, no significant differences were observed for HS amount (~0.9-1.3 µg ml(-1) ) between the two BCF types even if a greater content was determined for flattened samples. The specifically N-sulphated sequences in flattened BCF HS can exert biologic capacity by regulating growth factors activity. On the other hand, we cannot exclude a peculiar regulation of the activity of biomolecules in apocrine BCF by HS richer in N-acetylated disaccharides. In fact, the different patterns of growth factors and active biocompounds in the two types of cysts may require different regulation by specific sequences in the HS backbone possessing specific structural characteristics and distinctive chemical groups.

Atheroprotective remodelling of vascular dermatan sulphate proteoglycans in response to hypercholesterolaemia in a rat model.

Proteoglycan accumulation within the arterial intima has been implicated in atherosclerosis progression in humans. Nevertheless, hypercholesterolaemia is unable to induce intimal thickening and atheroma plaque development in rats. The study was performed to analyse proteoglycans modifications in rats fed with a high-cholesterol diet to understand whether vascular wall remodelling protects against lesions. Sections obtained from rat aortas showed normal features, in intimal-to-media ratio and lipid accumulation. However, focal endothelial hyperplasia and neo-intima rearrangement were observed in high-cholesterol animals. Besides, hypercholesterolaemia induced an inflammatory microenviroment. We determined the expression of different proteoglycans from aortic cells by Western blot and observed a diminished production of decorin and biglycan in high-cholesterol animals compared with control (P < 0.01 and P < 0.05, respectively). Versican was increased in high-cholesterol animals (P < 0.05), whereas perlecan production showed no differences. No modification of the total content of glycosaminoglycans (GAGs) was found between the two experimental groups. In contrast, the chondroitin sulphate/dermatan sulphate ratio was increased in the high-cholesterol group as compared to the control (0.56 and 0.34, respectively). Structural alterations in the disaccharide composition of galactosaminoglycans were also detected by HPLC, as the ratio of 6-sulphate to 4-sulphate disaccharides was increased in high-cholesterol animals (P < 0.05). Our results suggest that attenuation of decorin and biglycan expression might be an effective strategy to inhibit the first step in atherogenesis, although specific GAG structural modification associated with the development of vascular disease took place. Results emphasize the potential application of therapies based on vascular matrix remodelling to treat atherosclerosis.

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.

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.

Characterization of oversulfated chondroitin sulfate rich in 4,6-O-disulfated disaccharides in breast cyst fluids collected from human breast gross cysts.

Glycosaminoglycans (GAGs) from breast cyst fluid (BCF) of gross cysts, subdivided into apocrine and flattened, directly collected from 27 gross-cystic-breast-disease (GCBD)-affected women were analysed. Heparan sulfate, not further investigated, and chondroitin sulfate were identified. This last polysaccharide, in a content of 25-27 µg ml(-1) BCF and having a high molecular mass (~20 000-22 000), was found rich in glucuronic acid (~96%-98%) and mainly sulfated in position 4 of the N-acetyl-galactosamine (~60%-64%). Moreover, the presence of ~19%-24% of uncommon 4,6-O-disulfated disaccharides CS-E inside the polysaccharide chains with a high charge density of ~1.15-1.20 was determined. No substantial differences between apocrine and flattened cysts were observed. The current study describes the first effort to examine the yield and distribution of complex macromolecules like GAGs in BCF, and the understanding of their structure may help explain some functions associated with physiological and pathological conditions.

Chondroitin sulfate from heads of corb: Recovery, structural analysis and assessment of anticoagulant activity.

In this study, glycosaminoglycans (GAGs) were extracted from corb (Sciaena umbra) heads and thoroughly examined for their structure. Through cellulose acetate electrophoresis, the GAGs were identified as chondroitin sulfate (CS), with a recovery yield of 10.35 %. The CS exhibited notable characteristics including a high sulfate content (12.4 %) and an average molecular weight of 38.32 kDa. Further analysis via 1H NMR spectroscopy and SAX-HPLC revealed that the CS primarily consisted of alternating units predominantly composed of monosulfated disaccharides at positions 6 and 4 of GalNAc (52.6 % and 38.8 %, respectively). The ratio of sulfate groups between positions 4 and 6 of GalNAc (4/6 ratio) was approximately 0.74, resulting in an overall charge density of 0.98. Thermal properties of the CS were assessed using techniques such as differential scanning calorimetry and thermogravimetric analysis. Notably, the CS demonstrated concentration-dependent prolongation of activated partial thromboplastin time (aPTT) and thrombin time (TT) while showing no effect on platelet function. At 200 µg/mL, aPTT and TT coagulation times were 1.4 and 3.7 times faster than the control, respectively. These findings suggest that CS derived from corb heads holds promise as an anticoagulant agent for therapy, although further clinical investigations are necessary to validate its efficacy.

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.

Aortic valve leaflet glycosaminoglycans composition and modification in severe chronic valve regurgitation.

BACKGROUND AND AIM OF THE STUDY: The surgical segments of aortic valve leaflets from patients with severe chronic aortic regurgitation were analyzed (by percentage and structure) for their content of complex polysaccharides and glycosaminoglycans (GAGs), and compared with control segments. METHODS: The GAG, hyaluronic acid (HA), chondroitin sulfate (CS) and dermatan sulfate (DS) and disaccharide contents were determined in segments (leaflet, root attachment region and belly) of aortic valve leaflets (non-coronary, left coronary and right coronary) using a multi-analytical approach. RESULTS: The aortic valve leaflets showed the presence of HA and CS/DS, with an overall charge density of -0.51-0.55. The CS/DS polymers showed a 4-sulfated/6-sulfated ratio of -0.70-0.77 in the belly, and -1.60-1.72 in commissure parts (-/+124%). The total amount of GAGs was -1.60-2.40 microg/mg of tissue. A significant increase in sulfated GAGs was observed in all valve parts in patients suffering from severe aortic insufficiency, as well as an increase in the 4-sulfated/6-sulfated ratio in the leaflet belly (-/+102%). CONCLUSION: It is speculated that differences in 4-sulfated/6-sulfated ratio determined in the belly and leaflet attachment region-commissure parts of the leaflets may correlate with the tensile or compressive loading of normal aortic valve regions. At the same time, it may be assumed that the increase in sulfated GAGs and 4-sulfated/6-sulfated ratio in the leaflet belly of valves taken from patients suffering severe aortic insufficiency was consistent with an altered matrix microstructure capable of influencing the hydration of these pathological tissues, and of conditioning their mechanical weakness.

Plasma and Urine Free Glycosaminoglycans as Monitoring and Predictive Biomarkers in Metastatic Renal Cell Carcinoma: A Prospective Cohort Study.

PURPOSE: No liquid biomarkers are approved in metastatic renal cell carcinoma (mRCC) despite the need to predict and monitor response noninvasively to tailor treatment choices. Urine and plasma free glycosaminoglycan profiles (GAGomes) are promising metabolic biomarkers in mRCC. The objective of this study was to explore if GAGomes could predict and monitor response in mRCC. PATIENTS AND METHODS: We enrolled a single-center prospective cohort of patients with mRCC elected for first-line therapy (ClinicalTrials.gov identifier: NCT02732665) plus three retrospective cohorts (ClinicalTrials.gov identifiers: NCT00715442 and NCT00126594) for external validation. Response was dichotomized as progressive disease (PD) versus non-PD every 8-12 weeks. GAGomes were measured at treatment start, after 6-8 weeks, and every third month in a blinded laboratory. We correlated GAGomes with response and developed scores to classify PD versus non-PD, which were used to predict response at treatment start or after 6-8 weeks. RESULTS: Fifty patients with mRCC were prospectively included, and all received tyrosine kinase inhibitors (TKIs). PD correlated with alterations in 40% of GAGome features. We developed plasma, urine, and combined glycosaminoglycan progression scores that monitored PD at each response evaluation visit with the area under the receiving operating characteristic curve (AUC) of 0.93, 0.97, and 0.98, respectively. For internal validation, the scores predicted PD at treatment start with the AUC of 0.66, 0.68, and 0.74 and after 6-8 weeks with the AUC of 0.76, 0.66, and 0.75. For external validation, 70 patients with mRCC were retrospectively included and all received TKI-containing regimens. The plasma score predicted PD at treatment start with the AUC of 0.90 and at 6-8 weeks with the AUC of 0.89. The pooled sensitivity and specificity were 58% and 79% at treatment start. Limitations include the exploratory study design. CONCLUSION: GAGomes changed in association with mRCC response to TKIs and may provide biologic insights into mRCC mechanisms of response.

Composition and Anticoagulant Potential of Chondroitin Sulfate and Dermatan Sulfate from Inedible Parts of Garfish (Belone belone).

Glycosaminoglycans (GAGs) play a crucial role due to their significant biomedical functions. Chondroitin sulfate (CS) and dermatan sulfate (DS), the main representative family of GAGs, were extracted and purified from garfish (Belone belone) by-products, i.e., skin (GSB), bones (GCB), and heads (GHB), and their composition and anticoagulant activity were investigated. CS/DS were purified by ion-exchange chromatography with yields of 8.1% for heads, 3.7% for skin, and 1.4% for bones. Cellulose acetate electrophoresis was also explored for analyzing the extracted CS/DS. Interestingly, GHB, GSB, and GCB possessed sulfate contents of 21 ± 2%, 20 ± 1%, and 20 ± 1.5%, respectively. Physico-chemical analysis showed that there were no significant differences (p > 0.05) between the variances for sulfate, uronic acid, and total sugars in the GAGs extracted from the different parts of fish. Disaccharide analysis by SAX-HPLC showed that the GSB and GCB were predominately composed of ΔDi-4S [ΔUA-GalNAc 6S] (74.78% and 69.22%, respectively) and ΔDi-2,4S [ΔUA2S-GalNAc 4S] (10.92% and 6.55%, respectively). However, the GHB consisted of 25.55% ΔDi-6S [ΔUA-GalNAc 6S] and 6.28% ΔDi-2,6S [ΔUA2S-GalNAc 4S]. Moreover, classical anticoagulation tests were also used to measure their anticoagulant properties in vitro, which included the activated partial thromboplastin time, prothrombin time, and thrombin time. The CS/DS isolated from garfish by-products exhibited potent anticoagulant effects. The purified CS/DS showed exceptional anticoagulant properties according to this research and can be considered as a new agent with anticoagulant properties.

Electrophoresis for the analysis of heparin purity and quality.

The adulteration of raw heparin with oversulfated chondroitin sulfate (OSCS) in 2007-2008 produced a global crisis resulting in extensive revisions to the pharmacopeia monographs and prompting the FDA to recommend the development of additional methods for the analysis of heparin purity. As a consequence, a wide variety of innovative analytical approaches have been developed for the quality assurance and purity of unfractionated and low-molecular-weight heparins. This review discusses recent developments in electrophoresis techniques available for the sensitive separation, detection, and partial structural characterization of heparin contaminants. In particular, this review summarizes recent publications on heparin quality and related impurity analysis using electrophoretic separations such as capillary electrophoresis (CE) of intact polysaccharides and hexosamines derived from their acidic hydrolysis, and polyacrylamide gel electrophoresis (PAGE) for the separation of heparin samples without and in the presence of its relatively specific depolymerization process with nitrous acid treatment.