Construction of mouse cochlin mutants with different GAG-binding specificities and their use for immunohistochemistry.

Glycosaminoglycan (GAG) is a polysaccharide present on the cell surface as an extracellular matrix component, and is composed of repeating disaccharide units consisting of an amino sugar and uronic acid except in the case of the keratan sulfate. Sulfated GAGs, such as heparan sulfate, heparin, and chondroitin sulfate mediate signal transduction of growth factors, and their functions vary with the type and degree of sulfated modification. We have previously identified human and mouse cochlins as proteins that bind to sulfated GAGs. Here, we prepared a recombinant cochlin fused to human IgG-Fc or Protein A at the C-terminus as a detection and purification tag and investigated the ligand specificity of cochlin. We found that cochlin can be used as a specific probe for highly sulfated heparan sulfate and chondroitin sulfate E. We then used mutant analysis to identify the mechanism by which cochlin recognizes GAGs and developed a GAG detection system using cochlin. Interestingly, a mutant lacking the vWA2 domain bound to various types of GAGs. The N-terminal amino acid residues of cochlin contributed to its binding to heparin. Pathological specimens from human myocarditis patients were stained with a cochlin-Fc mutant. The results showed that both tryptase-positive and tryptase-negative mast cells were stained with this mutant. The identification of detailed modification patterns of GAGs is an important method to elucidate the molecular mechanisms of various diseases. The method developed for evaluating the expression of highly sulfated GAGs will help understand the biological and pathological importance of sulfated GAGs in the future.

Adherence of Candida albicans and Malassezia Species to Skin Cells Induces Changes in the Expression of Genes Responsible for Heparan and Chondroitin Sulfate Chain Synthesis. / La unión de Candida albicans y Malassezia spp. a células de piel promueve cambios de expresión en los genes responsables de la síntesis de las cadenas de heparán y condroitín sulfato.

Superficial fungal infections are common in dermatology and are often caused by opportunistic species in the Candida and Malassezia genera. The aim of this study was to analyze changes in the expression of genes coding for enzymes involved in the biosynthesis of glycosaminoglycans (GAGs) chains following the adherence of Candida and Malassezia yeasts to skin cell lines. Gene expression was analyzed using reverse transcriptase-quantitative polymerase chain reaction assays. Interactions between the yeasts and the skin cells induced the following changes in genes involved in the biosynthesis of heparan sulfate and chondroitin sulfate: downregulation of CHPF in keratinocytes and downregulation of EXT1, EXT2, CHSY3, and CHPF in fibroblasts. Adherence to fibroblasts had an even greater effect on GAG biosynthetic enzymes, inducing the downregulation of 13 genes and the upregulation of two (CHST15 and CHST7). Interactions between yeasts and skin cells might affect the binding affinity of GAG chains, possibly changing their ability to function as receptors for pathogens and interfering with a key stage at the start of infection.

Reliable and sensitive detection of glycosaminoglycan chains with immunoblots.

Complex glycans play vital roles in many biological processes, ranging from intracellular signaling and organ development to tumor growth. Glycan expression is routinely assessed by the application of glycan-specific antibodies to cells and tissues. However, glycan-specific antibodies quite often show a large number of bands on immunoblots and it is hard to interpret the data when reliable controls are lacking. This limits the scope of glycobiology studies and poses challenges for replication. We sought to resolve this issue by developing a novel strategy that utilizes an immunoreaction enhancing technology to vastly improve the speed and quality of glycan-based immunoblots. As a representative case study, we used chondroitin sulfate glycosaminoglycan (CS-GAG) chains as the carbohydrate target and a monoclonal antibody, CS-56, as the probe. We discovered that preincubation of the antibody with its antigenic CS-GAG chain distinguishes true-positive signals from false-positive ones. We successfully applied this strategy to 10E4, a monoclonal anti heparan sulfate GAGs (HS-GAGs) antibody, where true-positive signals were confirmed by chemical HS-GAG depolymerization on the membrane. This evidence that glycan-specific antibodies can generate clear and convincing data on immunoblot with highly replicable results opens new opportunities for many facets of life science research in glycobiology.

Structural analysis of glycosaminoglycans from Oviductus ranae.

Oviductus ranae (O.ran.) has been widely used as a tonic and a traditional animal-based Chinese medicine. O.ran. extracts have been reported to have numerous biological activities, including activities that are often associated with mammalian glycosaminoglycans such as anti-inflammatory, antiosteoperotic, and anti-asthmatic. Glycosaminoglycans are complex linear polysaccharides ubiquitous in mammals that possess a wide range of biological activities. However, their presence and possible structural characteristics within O.ran. were previously unknown. In this study, glycosaminoglycans were isolated from O.ran. and their disaccharide compositions were analyzed by liquid chromatography-ion trap/time-of-flight mass spectrometry (LC-MS-ITTOF). Heparan sulfate (HS)/heparin (HP), chondroitin sulfate (CS)/dermatan sulfate (DS) and hyaluronic acid (HA) were detected in O.ran. with varied disaccharide compositions. HS species contain highly acetylated disaccharides, and have various structures in their constituent chains. CS/DS chains also possess a heterogeneous structure with different sulfation patterns and densities. This novel structural information could help clarify the possible involvement of these polysaccharides in the biological activities of O.ran..

Characterization of Glycosaminoglycan Disaccharide Composition in Astrocyte Primary Cultures and the Cortex of Neonatal Rats.

Astrocytes are major producers of the extracellular matrix (ECM), which is involved in the plasticity of the developing brain. In utero alcohol exposure alters neuronal plasticity. Glycosaminoglycans (GAGs) are a family of polysaccharides present in the extracellular space; chondroitin sulfate (CS)- and heparan sulfate (HS)-GAGs are covalently bound to core proteins to form proteoglycans (PGs). Hyaluronic acid (HA)-GAGs are not bound to core proteins. In this study we investigated the contribution of astrocytes to CS-, HS-, and HA-GAG production by comparing the makeup of these GAGs in cortical astrocyte cultures and the neonatal rat cortex. We also explored alterations induced by ethanol in GAG and core protein levels in astrocytes. Finally, we investigated the relative expression in astrocytes of CS-PGs of the lectican family of proteins, major components of the brain ECM, in vivo using translating ribosome affinity purification (TRAP) (in Aldh1l1-EGFP-Rpl10a mice. Cortical astrocytes produce low levels of HA and show low expression of genes involved in HA biosynthesis compared to the whole developing cortex. Astrocytes have high levels of chondroitin-0-sulfate (C0S)-GAGs (possibly because of a higher sulfatase enzyme expression) and HS-GAGs. Ethanol upregulates C4S-GAGs as well as brain-specific lecticans neurocan and brevican, which are highly enriched in astrocytes of the developing cortex in vivo. These results begin to elucidate the role of astrocytes in the biosynthesis of CS- HS- and HA-GAGs, and suggest that ethanol-induced alterations of neuronal development may be in part mediated by increased astrocyte GAG levels and neurocan and brevican expression.

Structural analysis of glycosaminoglycans from Colla corii asini by liquid chromatography-electrospray ion trap mass spectrometry.

Colla corii asini (CCA) made from donkey-hide has been widely used as a traditional animal-based Chinese medicine. Chondroitin sulfate (CS), dermatan sulfate (DS) and hyaluronic acid (HA) are structurally complex classes of glycosaminoglycans (GAGs) that have been implicated in a wide range of biological activities. However, their possible structural characteristics in CCA are not clear. In this study, GAG fractions containing CS/DS and HA were isolated from CCA and their disaccharide compositions were analyzed by high sensitivity liquid chromatography-ion trap/time-of-flight mass spectrometry (LC-MS-ITTOF). The result showed that CS/DS/HA disaccharides were detected in the three lower salt fractions from anion-exchange chromatography. The sulfation patterns and densities of CS/DS chains in these fractions differed greatly, while HA chains varied in their chain lengths. The quantitative analysis first revealed that the amount of GAGs in CCA varied significantly in total and in each fraction. This novel structural information could help clarify the possible involvement of these polysaccharides in the biological activities of CCA.

Bottom-up analysis using liquid chromatography-Fourier transform mass spectrometry to characterize fucosylated chondroitin sulfates from sea cucumbers.

Fucosylated chondroitin sulfates (FCSs) from sea cucumbers have repetitive structures that exhibit minor structural differences based on the organism from which they are recovered. A detailed characterization of FCSs and their derivatives is important to establish their structure-activity relationship in the development of new anticoagulant drugs. In the current study, online hydrophilic interaction chromatography-Fourier transform mass spectrometry (FTMS) was applied to analyze the FCS oligosaccharides generated by selective degradation from four species of sea cucumbers, Isostichopus badionotus, Pearsonothuria graeffei, Holothuria mexicana and Acaudina molpadioides. These depolymerized FCS fragments were quantified and compared using the glycomics software package, GlycReSoft. The quantified fragments mainly had trisaccharide-repeating compositions and showed significant differences in fucosylation (including its sulfation) among different species of sea cucumbers. Detailed analysis of FTMS ion peaks and top-down nuclear magnetic resonance spectroscopy of native FCS polysaccharides verified the accuracy of this method. Thus, a new structural model for FCS chains from these different sea cucumbers was defined. This bottom-up approach provides rich detailed structural analysis and provides quantitative information with high accuracy and reproducibility and should be suitable for the quality control in FCSs as well as their oligosaccharides.

Quantitative analysis of chondroitin sulfate disaccharides from human and rodent fixed brain tissue by electrospray ionization-tandem mass spectrometry.

Chondroitin sulfates (CS) are long, negatively charged, unbranched glycosaminoglycan (GAG) chains attached to CS-proteoglycan (CSPG) core proteins that comprise the glycan component in both loose interstitial extracellular matrices (ECMs) and in rigid, structured perineuronal net (PNN) scaffolds within the brain. As aberrant CS-PNN formations have been linked to a range of pathological states, including Alzheimer's disease (AD) and schizophrenia, the analysis of CS-GAGs in brain tissue at the disaccharide level has great potential to enhance disease diagnosis and prognosis. Two mass-spectrometry (MS)-based approaches were adapted to detect CS disaccharides from minute fixed tissue samples with low picomolar sensitivity and high reproducibility. The first approach employed a straightforward, quantitative direct infusion (DI)-tandem mass spectrometry (MS/MS) technique to determine the percentages of Δ4S- and Δ6S-CS disaccharides within the 4S/6S-CS ratio, while the second used a comprehensive liquid chromatography (LC)-MS/MS technique to determine the relative percentages of Δ0S-, Δ4S-, Δ6S-, Δ4S6S-CS and Δ2S6S-CS disaccharides, with internal validation by full chondroitin lyase activity. The quantitative accuracy of the five primary biologically relevant CS disaccharides was validated using a developmental time course series in fixed rodent brain tissue. We then analyzed the CS disaccharide composition in formalin-fixed human brain tissue, thus providing the first quantitative report of CS sulfation patterns in the human brain. The ability to comprehensively analyze the CS disaccharide composition from fixed brain tissue provides a means with which to identify alterations in the CS-GAG composition in relation to the onset and/or progression of neurological diseases.

Negative-Ion Mode Capillary Isoelectric Focusing Mass Spectrometry for Charge-Based Separation of Acidic Oligosaccharides.

Glycosaminoglycans (GAGs) are biologically and pharmacologically important linear, anionic polysaccharides containing various repeating disaccharides sequences. The analysis of these polysaccharides generally relies on their chemical or enzymatic breakdown to disaccharide units that are separated, by chromatography or electrophoresis, and detected, by UV, fluorescence, or mass spectrometry (MS). Isoelectric focusing (IEF) is an important analytical technique with high resolving power for the separation of analytes exhibiting differences in isoelectric points. One format of IEF, the capillary isoelectric focusing (cIEF), is an attractive approach in that it can be coupled with mass spectrometry (cIEF-MS) to provide online focusing and detection of complex mixtures. In the past three decades, numerous studies have applied cIEF-MS methods to the analysis of protein and peptide mixtures by positive-ion mode mass spectrometry. However, polysaccharide chemists largely rely on negative-ion mode mass spectrometry for the analysis of highly sulfated GAGs. The current study reports a negative-ion mode cIEF-MS method using an electrokinetically pumped sheath liquid nanospray capillary electrophoresis-mass spectrometry (CE-MS) coupling technology. The feasibility of this negative-ion cIEF-MS method and its potential applications are demonstrated using chondroitin sulfate and heparan sulfate oligosaccharides mixtures.

Glycosaminoglycans compositional analysis of Urodele axolotl (Ambystoma mexicanum) and Porcine Retina.

Retinal degenerative diseases, such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP), are major causes of blindness worldwide. Humans cannot regenerate retina, however, axolotl (Ambystoma mexicanum), a laboratory-bred salamander, can regenerate retinal tissue throughout adulthood. Classic signaling pathways, including fibroblast growth factor (FGF), are involved in axolotl regeneration. Glycosaminoglycan (GAG) interaction with FGF is required for signal transduction in this pathway. GAGs are anionic polysaccharides in extracellular matrix (ECM) that have been implicated in limb and lens regeneration of amphibians, however, GAGs have not been investigated in the context of retinal regeneration. GAG composition is characterized native and decellularized axolotl and porcine retina using liquid chromatography mass spectrometry. Pig was used as a mammalian vertebrate model without the ability to regenerate retina. Chondroitin sulfate (CS) was the main retinal GAG, followed by heparan sulfate (HS), hyaluronic acid, and keratan sulfate in both native and decellularized axolotl and porcine retina. Axolotl retina exhibited a distinctive GAG composition pattern in comparison with porcine retina, including a higher content of hyaluronic acid. In CS, higher levels of 4- and 6- O-sulfation were observed in axolotl retina. The HS composition was greater in decellularized tissues in both axolotl and porcine retina by 7.1% and 15.4%, respectively, and different sulfation patterns were detected in axolotl. Our findings suggest a distinctive GAG composition profile of the axolotl retina set foundation for role of GAGs in homeostatic and regenerative conditions of the axolotl retina and may further our understanding of retinal regenerative models.

Online preconcentration and determination of chondroitin sulfate, dermatan sulfate and hyaluronic acid in biological and cosmetic samples using capillary electrophoresis.

Capillary electrophoresis with large-volume sample stacking using an electroosmotic flow pump was developed for the determination of chondroitin sulfate, dermatan sulfate, and hyaluronic acid. Central composite design was used to simultaneously optimize the parameters for capillary electrophoresis separation. The optimized capillary electrophoresis conditions were 200 mM sodium dihydrogen phosphate, 200 mM butylamine, and 0.5% w/v polyethylene glycol as a background electrolyte, pH 4 and -16 kV. Exploiting large-volume sample stacking using an electroosmotic flow pump, the sensitivity of the proposed capillary electrophoresis system coupled with UV detection was significantly improved with limits of detection of 3, 5, 1 mg/L for chondroitin sulfate, dermatan sulfate, and hyaluronic acid, respectively. The developed method was applied to the determination of chondroitin sulfate and hyaluronic acid in cell culture media, cerebrospinal fluid, cosmetic products, and supplementary samples with highly acceptable accuracy and precision. Therefore, the proposed capillary electrophoresis approach was found to be simple, rapid, and reliable for the determination of chondroitin sulfate, dermatan sulfate, and hyaluronic acid in cell culture media, cerebrospinal fluid, cosmetic, and supplementary samples without sample pretreatment.

Chondroitin Sulfate Safety and Quality.

The industrial production of chondroitin sulfate (CS) uses animal tissue sources as raw material derived from different terrestrial or marine species of animals. CS possesses a heterogeneous structure and physical-chemical profile in different species and tissues, responsible for the various and more specialized functions of these macromolecules. Moreover, mixes of different animal tissues and sources are possible, producing a CS final product having varied characteristics and not well identified profile, influencing oral absorption and activity. Finally, different extraction and purification processes may introduce further modifications of the CS structural characteristics and properties and may lead to extracts having a variable grade of purity, limited biological effects, presence of contaminants causing problems of safety and reproducibility along with not surely identified origin. These aspects pose a serious problem for the final consumers of the pharmaceutical or nutraceutical products mainly related to the traceability of CS and to the declaration of the real origin of the active ingredient and its content. In this review, specific, sensitive and validated analytical quality controls such as electrophoresis, eHPLC (enzymatic HPLC) and HPSEC (high-performance size-exclusion chromatography) able to assure CS quality and origin are illustrated and discussed.

Tools for the Quality Control of Pharmaceutical Heparin.

Heparin is a vital pharmaceutical anticoagulant drug and remains one of the few naturally sourced pharmaceutical agents used clinically. Heparin possesses a structural order with up to four levels of complexity. These levels are subject to change based on the animal or even tissue sources that they are extracted from, while higher levels are believed to be entirely dynamic and a product of their surrounding environments, including bound proteins and associated cations. In 2008, heparin sources were subject to a major contamination with a deadly compound-an over-sulphated chondroitin sulphate polysaccharide-that resulted in excess of 100 deaths within North America alone. In consideration of this, an arsenal of methods to screen for heparin contamination have been applied, based primarily on the detection of over-sulphated chondroitin sulphate. The targeted nature of these screening methods, for this specific contaminant, may leave contamination by other entities poorly protected against, but novel approaches, including library-based chemometric analysis in concert with a variety of spectroscopic methods, could be of great importance in combating future, potential threats.

Coupled X-ray Fluorescence and X-ray Absorption Spectroscopy for Microscale Imaging and Identification of Sulfur Species within Tissues and Skeletons of Scleractinian Corals.

Identifying and mapping the wide range of sulfur species within complex matrices presents a challenge for understanding the distribution of these important biomolecules within environmental and biological systems. Here, we present a coupled micro X-ray fluorescence (µXRF) and X-ray absorption near-edge structure (XANES) spectroscopy method for determining the presence of specific sulfur species in coral tissues and skeletons at high spatial resolution. By using multiple energy stacks and principal component analysis of a large spectral database, we were able to more accurately identify sulfur species components and distinguish different species and distributions of sulfur formerly unresolved by previous studies. Specifically, coral tissues were dominated by more reduced sulfur species, such as glutathione disulfide, cysteine, and sulfoxide, as well as organic sulfate as represented by chondroitin sulfate. Sulfoxide distributions were visually correlated with the presence of zooxanthellae endosymbionts. Coral skeletons were composed primarily of carbonate-associated sulfate (CAS) along with minor contributions from organic sulfate and a separate inorganic sulfate likely in the form of adsorbed sulfate. This coupled XRF-XANES approach allows for a more accurate and informative view of sulfur within biological systems in situ and holds great promise for pairing with other techniques to allow for a more encompassing understanding of elemental distributions within the environment.

One-pot analysis of sulfated glycosaminoglycans.

Routine isolation, estimation, and characterization of glycosaminoglycans (GAGs) is quite challenging. This is compounded by the fact that the analysis is technique-intensive and more often there will be a limitation on the quantity of GAGs available for various structural, functional and biological studies. In such a scenario, the sample which can be made available for estimation and elucidation of disaccharide composition and species composition as well remains a challenge. In the present study, we have determined the feasibility where isolated sulfated GAGs (sGAG) that is estimated by metachromasia is recovered for further analysis. sGAG-DMMB complex formed after estimation of sGAG by DMMB dye-binding assay was decomplexed and sGAGs were recovered. Recovered sGAGs were analysed by cellulose acetate membrane electrophoresis and taken up for disaccharide composition analysis by HPLC after fluorescent labelling. Good recovery of sGAGs after metachromasia was observed in all samples of varying levels of purity by this protocol. Further analysis using cellulose acetate membrane electrophoresis showed good separation between species of sGAGs namely chondroitin/dermatan sulfate and heparan sulfate, with comparatively lesser interference from hyaluronic acid, a non-sulfated GAG. Analysis of recovered sGAGs, specifically heparan sulfate by HPLC showed characteristic disaccharide composition akin to that of GAG obtained by the conventional protocol. Thus, in the present paper, we show that sGAG can be recovered in comparatively purer form after routine estimation and can be used for further analysis thus saving up on the precious sample.

Glycosaminoglycans from bovine eye vitreous humour and interaction with collagen type II.

Glycosaminoglycans (GAGs) play an important role in stabilizing the gel state of eye vitreous humour. In this study, the composition of GAGs present in bovine eye vitreous was characterized through disaccharide analysis by liquid chromatography-mass spectrometry. The interaction of GAGs with collagen type II was assessed using surface plasmon resonance (SPR). The percentage of hyaluronic acid (HA), chondroitin sulfate (CS) and heparan sulfate (HS), of total GAG, were 96.2%, 3.5% and 0.3%, respectively. The disaccharide composition of CS consisted of 4S (49%), 0S (38%) 6S (12%), 2S6S (1.5%) and 2S4S (0.3%). The disaccharide composition of HS consisted of 0S (80%), NS2S (7%), NS (7%), 6S (4%), NS6S (2%), and TriS, 2S and 4S6S (each at 0.1%). The average molecular weights of CS and HS were 148 kDa and 204 kDa, respectively. SPR reveals that collagen type II binds to heparin (primarily composed of TriS) with a binding affinity (K D) of 755 nM and interacts with other GAGs, including CSB and CSE. Both bovine vitreous CS and HS interact with collagen type II, with vitreous HS showing a higher binding affinity.

Glycosaminoglycans from fish swim bladder: isolation, structural characterization and bioactive potential.

The swim bladder of fish is an internal gas-filled organ that allows fish to control their buoyancy and swimming depth. Fish maws (the dried swim bladders of fish) have been used over many centuries as traditional medicines, tonics and a luxurious gourmet food in China and Southeast Asia. Little is known about the structural information of polysaccharides comprising this important functional material of fish tissue. In the present study, the total glycosaminoglycan (GAG) from fish maw was characterized. Two GAGs were identified, chondroitin sulfate (CS, having a molecular weight of 18-40 kDa) and heparan sulfate (HS), corresponding to 95% and 5% of the total GAG, respectively. Chondroitinase digestion showed that the major CS GAG was composed of ΔUA-1 â†’ 3-GalNAc4S (59.7%), ΔUA-1 â†’ 3-GalNAc4,6S (36.5%), ΔUA-1 â†’ 3-GalNAc6S (2.2%) and ΔUA-1 â†’ 3-GalNAc (1.6%) disaccharide units. 1H-NMR analysis and degradation with specific chondroitinases, both CS-type A/C and CS-type B were present in a ratio of 1.4:1. Analysis using surface plasmon resonance showed that fibroblast growth factor (FGF)-2 bound to the CS fraction (KD = 136 nM). These results suggest that this CS may be involved in FGF-signal pathway, mediating tissue repair, regeneration and wound healing. The CS, as the major GAG in fish maw, may have potential pharmacological activity in accelerating wound healing.

The adolescent idiopathic scoliotic IVD displays advanced aggrecanolysis and a glycosaminoglycan composition similar to that of aged human and ovine IVDs.

PURPOSE: The present study was designed to ascertain how altered biomechanics in adolescent idiopathic scoliotic (AIS) intervertebral discs (IVDs) affected tissue compositions and aggrecan processing compared to age matched and aged human IVDs. Newborn, 2- and 10-year-old ovine IVDs were also examined. METHODS: Aggrecan populations were separated by Sepharose CL2B chromatography, composite agarose polyacrylamide gel electrophoresis (CAPAGE) and identified by immunoblotting. The KS and CS content of IVD tissue extracts from AIS IVDs were compared with age-matched normal adolescent IVDs and with old human IVDs. Extracts from newborn, 2- and 10-year-old ovine IVDs were also examined in a similar manner. RESULTS: Adolescent idiopathic scoliotic IVD Aggrecan populations shared similar levels of polydispersity and aggregatability with hyaluronan as old IVD proteoglycans. CAPAGE demonstrated three aggrecan populations in AIS, aged human and ovine IVDs increased polydispersity and mobility in CAPAGE. AIS IVDs had GAG compositions similar to aged human and ovine IVDs. Sulphated KS (5-D-4) and chondroitin-6-sulphate, 3-B-3(+) were markers of tissue maturation, and chondroitin-4-sulphate, 2-B-6(+) was prominent in immature IVDs but its levels were lower in mature IVDs. DISCUSSION: Sulphated KS and 3-B-3(+) CS were prominently associated with IVD maturation and AIS IVDs, while the 2-B-6(+) CS isomer was associated with immature IVD tissues. The polydispersity of aggrecan in AIS IVDs, which was similar to in old human and ovine IVDs, reflected altered processing in the AIS IVDs in response to the biomechanical microenvironments the disc cells were exposed to in AIS IVDs. These slides can be retrieved under Electronic Supplementary Material.

A sensitive surface-enhanced Raman scattering method for chondroitin sulfate with Victoria blue 4R molecular probes in nanogold sol substrate.

Using silver nanoparticles (AgNPs) as the nanocatalyst, l-cysteine rapidly reduced HAuCl4 to make a stable gold nanoparticle sol (Ag/AuNP) that had a high surface-enhanced Raman scattering (SERS) activity in the presence of Victoria blue 4R (VB4r) molecular probes. Under the selected conditions, chondroitin sulfate (Chs) reacted with the VB4r probes to form associated complexes that caused the SERS effect to decrease to 1618 cm-1 . The decreased SERS intensity was linear to the Chs concentration in the range 3.1-500 ng/ml, with a detection limit of 1.0 ng/ml Chs. Accordingly, we established a simple and sensitive SERS quantitative analysis method to determine Chs in real samples, with a relative standard deviation of 1.47-3.16% and a recovery rate of 97.6-104.2%.

[Analysis of chondroitin sulfate content of Cervi Cornu Pantotrichum with different processing methods and different parts].

The differences and the variations of chondroitin sulfate content in different parts of Cervi Cornu Pantotrichum(CCP) with different processing methods were investigated. The chondroitin sulfate from velvet was extracted by dilute alkali-concentrated salt method. Next， the chondroitin sulfate was digested by chondroitinase ABC.The contents of total chondroitin sulfate and chondroitin sulfate A， B and C in the samples were determined by high performance liquid chromatography(HPLC).The content of chondroitin sulfate in wax，powder，gauze，bone slices of CCP with freeze-drying processing is 14.13，11.99，1.74，0.32 g·kg⁻¹ï¼Œ respectively. The content of chondroitin sulfate in wax，powder，gauze，bone slices of CCP with boiling processing is 10.71，8.97，2.21，1.40 g·kg⁻¹ï¼Œ respectively. The content of chondroitin sulfate in wax，powder，gauze，bone slices of CCP without blood is 12.47，9.47，2.64，0.07 g·kg⁻¹ï¼Œ respectively. And the content of chondroitin sulfate in wax，powder，gauze，bone slices of CCP with blood is 8.22，4.39，0.87，0.28 g·kg⁻¹ respectively. The results indicated that the chondroitin sulfate content in different processing methods was significantly different.The content of chondroitin sulfate in CCP with freeze-drying is higher than that in CCP with boiling processing.The content of chondroitin sulfate in CCP without blood is higher than that in CCP with blood. The chondroitin sulfate content in differerent paris of the velvet with the same processing methods was arranged from high to low as： wax slices， powder， gauze slices， bone slices.