Low Molecular Weight Chondroitin Sulfate Research Articles

Structural analysis and bioavailability study of low-molecular-weight chondroitin sulfate‑iron complexes prepared by photocatalysis-Fenton reaction

Increasing studies focus on depolymerization of chondroitin sulfate (CS) to enhance its biological activities. In the present study, low-molecular-weight chondroitin sulfate (LMWCS)‑iron complexes were obtained by photocatalysis-Fenton reaction. After degradation with the optimal condition of 0.25 % (w/v) TiO2, 10 mM FeSO4, and 400 mM H2O2 for 0, 15, and 60 min, the average relative molecular weights of CS were reduced to 4.77, 2.47, and 1.21 kDa, respectively. Electron paramagnetic resonance and free radical capture test identified •OH, •O2−, and h+ in the photocatalysis-Fenton system, among them h+ was the major contributor for CS degradation. The structures of degradation products were analyzed by UV, CD, XRD, SEM-EDS, and NMR, and the results indicated that CS chelated iron with its carboxyl and sulfate groups, leading to changes in conformation and microtopography. Then 10 oligosaccharides were identified in the degradation products using HPLC-MSn and the depolymerization mechanism was proposed. Furthermore, iron release was observed in simulated gastrointestinal digestion of LMWCS‑iron complexes. Notably, the everted gut sac experiment demonstrated that LMWCS‑iron complex possessed 3.75 times higher iron absorption than FeSO4 (p < 0.01) and 12.60 times higher CS absorption than original CS (p < 0.0001). In addition, LMWCS‑iron exhibited stronger in vitro antioxidant activity than CS.

Preparation of low molecular weight chondroitin sulfate from different sources by H2O2/ascorbic acid degradation and its degradation mechanism

Low molecular weight chondroitin sulfate (LMCS) has attention for enhanced bioavailability and bioactivity compared to native CS. We optimized H2O2/ ascorbic acid (Vc) degradation conditions to prepare LMCS from chicken, bovine, and shark cartilages. Degradation kinetics models and chemical composition data of LMCS showed the GlcA residues of chondroitin-4-sulfate (CSA) may be preferentially attacked. Nuclear magnetic resonance (NMR) spectroscopy and high-performance liquid chromatography-electrospray mass spectrometry (HPLC-MS) indicated that the CH of GlcA in CS was broken through a hydrogen abstraction reaction to break the β-(1 → 3) bond and form the hexendioic acid product. Standard density functional theory (DFT) calculations indicated that the energy required for the hydrogen abstraction from the C1-H bond in GlcA was lower than that of GalNAc. Molecular dynamics (MD) showed that CSA was more likely to interact with hydroxyl radicals (·OH) than non-sulfated chondroitin (CSO) and chondroitin-6-sulfate (CSC). These results provide guidance for producing LMCS.

Chemical depolymerisation of chondroitin sulfate glycosaminoglycan improves both antioxidant capacity and Fe uptake in a human intestinal Caco-2 cell model

Chondroitin sulfate (CS) is a high molecular weight sulfated polysaccharide that has been reported to improve joint health. A recent study has demonstrated the ability of enzymatically produced CS-oligosaccharides to improve non-heme iron bioavailability. In this research, CS was chemically hydrolyzed under acid, alkaline, and combined (acid & alkaline) conditions at 60 °C for different times (0–60 h) to understand if the oligosaccharides obtained can improve iron uptake in human intestinal Caco-2 cell model. The acidic hydrolyses produced a collection of desulfated and low molecular weight CS crude oligosaccharides (CS crude oligos), while under alkaline condition a high degree of deacetylation was detected with limited depolymerisation and desulfation. The combined treated CS was first degraded and desulfated in acid solution followed by alkali deacetylation. CS crude oligos produced by the combined treatment possessed the superior antioxidant capacity measured by ferric reducing and DPPH scavenging activities. All chemically hydrolyzed CS samples showed greater (p < 0.05) ferritin formation in Caco-2 cells compared to the negative control, where the greatest ferritin level was achieved by the combined treated CS. The depolymerisation of CS followed by the deacetylation created CS structural changes resulting in the increase of antioxidant and Fe-absorption enhancing capacities.

The structures and applications of microbial chondroitin AC lyase.

As an important glycosaminoglycan hydrolase, chondroitin lyases can hydrolyze chondroitin sulfate (CS) and release disaccharides and oligosaccharides. They are further divided into chondroitin AC, ABC, and B lyases according to their spatial structure and substrate specificity. Chondroitin AC lyase can hydrolyze chondroitin sulfate A (CS-A), chondroitin sulfate C (CS-C), and hyaluronic acid (HA), making it an essential biocatalyst for the preparation of low molecular weight chondroitin sulfate, analysis of the structure of the chondroitin sulfate, treatment of spinal cord injury, and purification of heparin. This paper provides an overview of reported chondroitin AC lyases, including their properties and the challenges faced in industrial applications. Up to now, although many attempts have been adopted to improve the enzyme properties, the most important factors are still the low activity and stability. The relations between the stability of the enzyme and the spatial structure were also summarized and discussed. Also perspectives for remodeling the enzymes with protein engineering are included.

A Highly Active Chondroitin Sulfate Lyase ABC for Enzymatic Depolymerization of Chondroitin Sulfate.

Enzymatic preparation of low-molecular-weight chondroitin sulfate (LMWCS) has received increasing attention. In this work, a chondroitin sulfate lyase ABC (Chon-ABC) was successfully cloned, expressed, and characterized. The Km and Vmax of the Chon-ABC were 0.54 mM and 541.3 U mg−1, respectively. The maximal activity was assayed as 500.4 U mg−1 at 37 °C in pH 8.0 phosphate buffer saline. The half-lives of the Chon-ABC were 133 d and 127 min at 4 °C and 37 °C, respectively. Enzymatic preparation of LMWCS was performed at room temperature for 30 min. The changes between the substrate and product were analyzed with mass spectrometry (MS), high-performance liquid chromatography (HPLC), gel permeation chromatography (GPC), and nuclear magnetic resonance (NMR). Overall, the Chon-ABC from Bacteroides thetaiotaomicron is competitive in large-scale enzymatic preparation of LMWCS for its high activity, stability, and substrate specificity.

A Novel Chondroitin AC Lyase With Broad Substrate Specificity From Pedobacter rhizosphaerae: Cloning, Expression, and Characterization.

Chondroitin AC lyase (ChSaseAC) is one of the essential polysaccharides lyases in low molecular chondroitin sulfate production. In this work, a novel PrChSaseAC from Pedobacter rhizosphaerae was successfully cloned, expressed in Escherichia coli. After optimizing the induction, the recombinant PrChSaseAC could be expressed efficiently at 0.1 mM IPTG, 25°C, and 12 h induction. Then, it was purified with Ni-NTA affinity chromatography. The characterization of the purified PrChSaseAC showed that it had high specific activity and good storage stability, which would favor the production of low molecular weight chondroitin sulfate. It also displayed activity toward chondroitin sulfate C and hyaluronic acid. PrChSaseAC had the highest activity at pH 7.5, 37°C, 10 mM Ca2+, and 5 mg/ml of chondroitin sulfate A. Molecular docking of substrate and enzyme showed the interactions between the enzyme and substrate; it revealed that the enzyme showed high activity to CS-A and hyaluronic acid, but lower activity to CS-C attributed to the structure of the binding pocket. The high stability and specific activity of the enzyme will benefit the industrial production or clinical treatment.

Low molecular weight chondroitin sulfate ameliorates pathological changes in 5XFAD mice by improving various functions in the brain

Our previous study found that low molecular weight chondroitin sulfate (LMWCS) had neuroprotective effects against the toxicity of amyloid-β (Aβ) peptides both in vitro and in vivo, and we speculated that the effects might be related with its anti-oxidative activities. In this study, the anti-Alzheimer′s disease (AD) activity of LMWCS was further studied in 5XFAD transgenic mice. After 4-month gavage, the levels of Aβ1-42 level, amyloid precursor protein (APP) and presenilin 1 (PS1) were significantly decreased in the brains of 5XFAD mice, indicating the alteration of APP metabolism by LMWCS. Besides, LMWCS inhibited the secretions of pro-inflammatory cytokines, including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and IL-6. Furthermore, the suppression of neuroinflammation by LMWCS was supported by the decreased expressions of glial fibrillary acidic protein (GFAP) and toll-like receptor 2 (TLR2) in the brains. LMWCS also reduced the production of reactive oxygen species (ROS) and the level of phospho-tau (Ser404) in the brains. Nevertheless, the changes in the behavior tests were moderate. In conclusion, LMWCS administration ameliorated APP metabolism, neuroinflammation, ROS production and tau protein abnormality in the brains of 5XFAD mice, displaying the potential to improve the pathological changes of AD mouse brain. LMWCS could be considered as a promising anti-AD drug candidate, nonetheless, the therapy regimen need to be optimized to improve its pharmacotherapy efficacy.

Engineering a thermostable chondroitinase for production of specifically distributed low‐molecular‐weight chondroitin sulfate

Chondroitinase ABC I (csABC I) has attracted intensive attention because of its great potential in heparin refining and the enzymatic preparation of low-molecular-weight chondroitin sulfate (LMW-CS). However, low thermal resistance(<30℃) restricts its applications. Herein, structure-guided and sequence-assisted combinatorial engineering approaches were applied to improve the thermal resistance of Proteus vulgaris csABC I. By integrating the deletion of the flexible fragment R166-L170 at the N-terminal domain and the mutation of E694P at the C-terminal domain, variant NΔ5/E694P exhibited 247-fold improvement of its half-life at 37℃ and a 2.3-fold increase in the specific activity. Through batch fermentation in a 3-L fermenter, the expression of variant NΔ5/E694P in an Escherichia coli host reached 1.7g L-1 with the activity of 1.0 × 105 U L-1 . Finally, the enzymatic approach for the preparation of LMW-CS was established. By modulating enzyme concentration and controlling depolymerization time, specifically distributed LMW-CS (7000, 3400, and 1900Da) with low polydispersity was produced, demonstrating the applicability of these processes for the industrial production of LMW-CS in a more environmentally friendly way.

Current advances in the treatment of gastroesophageal reflux disease: a focus on esophageal protection

Gastroesophageal reflux disease (GERD) is characterized by high morbidity and a significant decrease in the quality of life of patients, and is a major risk factor for esophageal adenocarcinoma. Nowadays, antisecretory therapy with proton pump inhibitors (PPI) is the "gold standard" of conservative treatment of GERD, but in some cases this therapy is unsuccessful. According to various studies, the prevalence of refractory GERD can reach 30-40%. The latest scientific data in the field of genetics and pathophysiology of GERD demonstrate that a disruption of the barrier function of the esophageal mucosa and an increase of its permeability can be the leading causes of refractoriness. Thus, the optimal therapy for patients with GERD should not only suppress the secretion of hydrochloric acid, but also restore the barrier function of the mucous membrane, providing an esophagoprotective effect. To achieve these goals, Alfasoxx was developed, which consists of a mixture of low molecular weight hyaluronic acid and low molecular weight chondroitin sulfate dissolved in a bioadhesive carrier (poloxamer 407). The clinical efficacy of this product has been confirmed by three prospective, randomized, placebo - controlled trials. Alfasoxx has a healing and restorative effect towards the esophageal epithelium and due to high ability for bioadhesion provides long - term protection of the mucous membrane of the esophagus. Combination therapy for GERD with the use of PPI and an esophagoprotector offers new perspectives for the treatment of patients with GERD.

Low-molecular-weight chondroitin sulfate attenuated injury by inhibiting oxidative stress in amyloid β-treated SH-SY5Y cells.

The neurotoxicity of aggregated amyloid β (Aβ) has been implicated as a critical cause in the pathogenesis of Alzheimer's disease. In a previous work, we have shown that low-molecular-weight chondroitin sulfate (LMWCS), a derivative of chondroitin sulfate, protected the SH-SY5Y neuroblastoma cells from Aβ25-35-induced neurotoxicity, decreased intracellular reactive oxygen species level and inhibited the cell apoptosis. However, the underlying mechanism of the antioxidative effect of LMWCS in the SH-SY5Y cells has not been well explored. In the present study, the SH-SY5Y cells were cultured and exposed to 30 μM Aβ25-35 in the absence or presence of LMWCS (50, 100 and 200 μg/ml). Results indicate that incubation of cells with LMWCS before Aβ25-35 exposure increased superoxide dismutase, glutathione peroxidase and Na/K-ATPase activities and decreased the malondialdehyde content. In addition, LMWCS inhibited the imbalance of Bcl-2 and Bax and decreased caspase-3 and caspase-9 expressions. LMWCS antagonizes Aβ25-35-induced neurotoxicity by attenuating oxidative stress, and our results suggest that LMWCS might be used as a potential compound for Alzheimer's disease prevention.

Enzymatic depolymerization – An easy approach to reduce the chondroitin sulfate molecular weight

Abstract This study reports on a new approach to a well-known method to reduce the molecular weight of chondroitin sulfate (CS) using a non-specific enzyme. Here, the enzyme hyaluronidase was utilized to depolymerize raw CS (19.2 kDa) resulting in low molecular weight CS (LMWCS) as a product. A simple ultrafiltration process utilizing circular-flat membranes with different MWCO was utilized to recover the fraction of interest (5.6 kDa). LMWCS characterized by GPC/LALLS, viscometric analyses and FTIR and NMR spectroscopies. The enzymatic depolymerization kinetics was investigated and some important parameters but poorly demonstrated in the literature, such as the rate of depolymerization for bond cleavage (0.308 mol/L h); depolymerization rate constant (2.98 × 10−3 h−1); and the relationship between Mn and Mv is provided. The present work could be a basis on which to present a suitable and original method to obtain and recover LMWCS. Furthermore, it could be a useful reference work for the investigation of how to improve the biological activity of CS in biomedical and pharmaceutical devices by reducing the CS molecular weight.

Structural characterization and in vitro antioxidant activities of chondroitin sulfate purified from Andrias davidianus cartilage

Origin and manufacturing process are key factors affecting the biological activities of chondroitin sulfate (CS), which can be utilized as a nutraceutical in dietary supplements. Herein, we extracted and purified CS from the cartilage of artificially breeding Andrias davidianus (ADCS), i.e., Chinese giant salamander (CGS), one of the prospective functional food source materials in China. Low molecular weight CS (LMWADCS) was then prepared by free radical depolymerization of ADCS. High-performance gel permeation chromatography (HPGPC) analysis showed that the average molecular weight (Mw) of ADCS was 49.2 kDa, while the Mw of LMWADCS was 6.4 kDa. After the eliminative degradation of ADCS by chondroitinase ABC, strong anion-exchange high-performance liquid chromatography (SAX-HPLC) analysis showed that the disaccharide composition of ADCS was 14.6% ΔDi0S, 60.9% ΔDi6S and 24.5% ΔDi4S. Then, in vitro antioxidant assays were performed with ADCS, LMWADCS and CS from a commercial source. Our results showed that LMWADCS exerted the highest total antioxidant activity out of the total antioxidant capacity, including the capacity of scavenging DPPH radicals, hydroxyl radicals and superoxide anion radicals. From the results of this study, we can conclude that the Mw and composition of ADCS are different from those reported for bovine and shark CS, and LMWADCS can be utilized as a valuable and potential nutraceutical for the functional food industry.

Protective effects of low molecular weight chondroitin sulfate on amyloid beta (Aβ)-induced damage in vitro and in vivo

In the present study, we investigated the effects of low molecular weight chondroitin sulfate (LMWCS) on amyloid beta (Aβ)-induced neurotoxicity in vitro and in vivo. The in vitro results showed that LMWCS blocked Aβ25–35-induced cell viability loss and apoptosis, decreased intracellular calcium concentration, reactive oxygen species (ROS) levels, the mitochondrial membrane potential (MMP) depolarization, and the protein expression of Caspase-3. During in vivo experiments, LMWCS improved the cognitive impairment induced by Aβ1–40, increased the level of choline acetyltransferase (ChAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and decreased the level of malondialdehyde (MDA) and acetylcholinesterase (AChE) in the mouse brain. Moreover, LMWCS decreased the density of pyramidal cells of CA1 regions, and suppressed the protein expression of Bax/Bcl-2 and Caspase-3, -9 in the hippocampus of mice. In conclusion, LMWCS possessed neuroprotective properties against toxic effects induced by Aβ peptides both in vitro and in vivo, which might be related to anti-apoptotic activity. LMWCS might be a useful preventive and therapeutic compound for Alzheimer’s disease.

Study the effect of His-tag on chondroitinase ABC I based on characterization of enzyme

Chondroitinase ABC I (ChSase ABC I) which could degrade chondroitin sulfate (CS) to low molecular weight CS was expressed with His-tag in Escherichia coli (E. coli) BL21(DE3). The effect of His-tag on ChSase ABC I was investigated compared with ChSase ABC I which cut His-tag for the first time. After three steps purification, the specific activity of His-ChSase ABC I was 201.9±5.4IU/mg which was two times lower than ChSase ABC I. Results of multi angle light scattering (MALS) and analytical ultracentrifugation (AUC) showed that the polymeric state of His-ChSase ABC I was not effected by His-tag and it was monomer, and ChSase ABC I was the same. The optimal temperature and pH of His-ChSase ABC I were 37°C and 7.5, and were almost same with ChSase ABC I. Vmax and kcat/Km of His-ChSase ABC I were 2.4±0.1μmol/Ls, and 22.2±0.4L/(μmols) and catalytic efficiency was lower than ChSase ABC I. Generally, His-tag had no effect on polymeric state, optimal temperature and pH, had little negative impact on specific activity, kcat/Km and secondary-structure of ChSase ABC I. This study might guide the application of ChSase ABC I in industrial production.

Enhancing the intestinal absorption of low molecular weight chondroitin sulfate by conjugation with α-linolenic acid and the transport mechanism of the conjugates

The purpose of this report was to demonstrate the effect of amphiphilic polysaccharides-based self-assembling micelles on enhancing the oral absorption of low molecular weight chondroitin sulfate (LMCS) in vitro and in vivo, and identify the transepithelial transport mechanism of LMCS micelles across the intestinal barrier. α-Linolenic acid-low molecular weight chondroitin sulfate polymers(α-LNA-LMCS) were successfully synthesized, and characterized by FTIR, (1)HNMR, TGA/DSC, TEM, laser light scattering and zeta potential. The significant oral absorption enhancement and elimination half-life (t₁/₂) extension of LNA-LMCS2 in rats were evidenced by intragastric administration in comparison with CS and LMCS. Caco-2 transport studies demonstrated that the apparent permeability coefficient (Papp) of LNA-LMCS2 was significantly higher than that of CS and LMCS (p<0.001), and no significant effects on the overall integrity of the monolayer were observed during the transport process. In addition, α-LNA-LMCS micelles accumulated around the cell membrane and intercellular space observed by confocal laser scanning microscope (CLSM). Furthermore, evident alterations in the F-actin cytoskeleton were detected by CLSM observation following the treatment of the cell monolayers with α-LNA-LMCS micelles, which further certified the capacity of α-LNA-LMCS micelles to open the intercellular tight junctions rather than disrupt the overall integrity of the monolayer. Therefore, LNA-LMCS2 with low cytotoxicity and high bioavailability might be a promising substitute for CS in clinical use, such as treating osteoarthritis, atherosclerosis, etc.

Irreversible Heavy Chain Transfer to Hyaluronan Oligosaccharides by Tumor Necrosis Factor-stimulated Gene-6

The covalent transfer of heavy chains (HCs) from inter-α-inhibitor (IαI) to hyaluronan (HA) via the protein product of tumor necrosis factor-stimulated gene-6 (TSG-6) forms the HC-HA complex, a pathological form of HA that promotes the adhesion of leukocytes to HA matrices. The transfer of HCs to high molecular weight (HMW) HA is a reversible event whereby TSG-6 can shuffle HCs from one HA molecule to another. Therefore, HMW HA can serve as both an HC acceptor and an HC donor. In the present study, we show that transfer of HCs to low molecular weight HA oligosaccharides is an irreversible event where subsequent shuffling does not occur, i.e. HA oligosaccharides from 8 to 21 monosaccharide units in length can serve as HC acceptors, but are unable to function as HC donors. We show that the HC-HA complex is present in the synovial fluid of mice subjected to systemic and monoarticular mouse models of rheumatoid arthritis. Furthermore, we demonstrate that HA oligosaccharides can be used, with TSG-6, to irreversibly shuffle HCs from pathological, HMW HC-HA to HA oligosaccharides, thereby restoring HC-HA matrices from the inflamed joint to their normal state, unmodified with HCs. This process was also effective for HC-HA in the synovial fluid of human rheumatoid arthritis patients (in vitro).

Effects of low molecular weight chondroitin sulfate on type II collagen-induced arthritis in DBA/1J mice.

In order to evaluate the improvement in the treatment of chronic arthritis, we investigated chondroitin sulfate depolymerization product (low molecular weight chondroitin sulfate, LMWCS) and intact chondroitin sulfate (CS) in vitro and in vivo. LMWCS was prepared by a chemical depolymerization process induced by hydrogen peroxide in the presence of copper salts. LMWCS (300 mg/kg) and CS (1200 mg/kg) were orally administered to DBA/1J mice once daily for 14 d prior to initial immunization with type II collagen. Their elastase activities and the production of cytokines in sera were examined on type II collagen-induced arthritis in DBA/1J mice. We also compared the paracellular transport of LMWCS and CS across Caco-2 cell monolayers and examined the inhibitory effects on elastase activities. LMWCS inhibited elastase activity slightly, but CS did not show inhibition. Hind paw edema was significantly decreased by LMWCS treatment. Levels of anti-type II collagen antibody and tumor necrosis factor-alpha (TNF-alpha) in sera were also reduced by LMWCS treatment but not in case of CS, although no significant difference was observed between LMWCS and CS on interleukin-6 (IL-6) induction. The LMWCS preparation showed preventive effects on the type II collagen-induced arthritis in DBA/1J mice and better permeability through Caco-2 cells.

Heparin Modulates Integrin-Mediated Cellular Adhesion: Specificity of Interactions with α and β Integrin Subunits

Heparin is known to influence the growth, proliferation, and migration of vascular cells, but the precise mechanisms are unknown. We previously demonstrated that unfractionated heparin (UH) binds to the platelet integrin alpha(IIb)beta(3), and enhances ligand binding. To help define the specificity and site(s) of heparin-integrin interactions, we employed the erythroleukemic K562 cell line, transfected to express specific integrins (alpha(v)beta(3), alpha(v)beta(5), and alpha(IIb)beta(3)). By comparing K562 cells expressing a common alpha subunit (Kalpha(v)beta(3), Kalpha(v)beta(5)) with cells expressing a common beta subunit (Kalpha(v)beta(3), Kalpha(IIb)beta(3)), we observed that heparin differentially modulated integrin-mediated adhesion to vitronectin. UH at 0.5-7.5 microg/ml consistently enhanced the adhesion of beta(3) expressing cells (Kalpha(v)beta(3),Kalpha(IIb)beta(3)). In contrast, UH at 0.5-7.5 microg/ml inhibited Kalpha(v)beta(5) adhesion. Experiments using integrin-blocking antibodies, appropriate control ligands, and nontransfected native K562 cells revealed that heparin's actions were mediated by the specific integrins under study. Preincubation of heparin with Kalpha(v)beta(3) cells enhanced adhesion, while preincubation of heparin with the adhesive substrate (vitronectin) had minimal effect. There was a structural specificity to heparin's effect, in that a low molecular weight heparin and chondroitin sulfate showed significantly less enhancement of adhesion. These findings suggest that heparin's modulation of integrin-ligand interactions occurs through its action on the integrin. The inhibitory or stimulatory effects of heparin depend on the beta subunit type, and the potency is dictated by structural characteristics of the glycosaminoglycan.

The bioavailability and pharmacokinetics of glucosamine hydrochloride and low molecular weight chondroitin sulfate after single and multiple doses to beagle dogs.

The purpose of this study was to determine the oral bioavailability and pharmacokinetics of a glucosamine (GL) and the disaccharides of chondroitin sulfate (CS) after single and multiple-dosing of a GL/CS combination (Cosamin, Cosequin). Male beagle dogs (n = 8, 12 kg) received the following treatments: (1) IV GL (500 mg)/CS (400 mg), (2) p.o. GL (1500 mg)/CS (1200 mg), (3) p.o. GL (2000 mg)/CS (1600 mg), (4) p.o. GL (1500 mg)/CS (1200 mg) QD for days 1-7 and p.o. GL (3000 mg)/CS (2400 mg) from days 8 to 14. Blood samples were collected over 24 h and glucosamine and the disaccharides of chondroitin sulfate were determined. Pharmacokinetic analysis was performed on glucosamine and total chondroitin sulfate disaccharides and parameters were compared across treatments using ANOVA with post hoc analysis. After the IV administration, glucosamine declined rapidly in a bi-exponential fashion with a mean (+/- S.D.) elimination t(1/2) of 0.52 (0.25) h. GL absorption was relatively fast (C(max) = 8.95 microg/ml, and T(max) 1.5 h after 1500 mg dose) and the mean bioavailability of glucosamine after single dosing was approximately 12%. The extent of absorption of chondroitin sulfate as indicated by the mean C(max) (21.5 microg/ml) and mean AUC (187 microg/ml h) of total disaccharides after dosing (1600 mg) provides evidence that chondroitin sulfate is absorbed orally. The bioavailability of CS ranged from 4.8 to 5.0% after single dosing and 200-278% upon multiple dosing. The results of this study show that both glucosamine and chondroitin sulfate (measured as total disaccharides) are bioavailable after oral dosing. In addition, the low molecular weight chondroitin sulfate used in this study displays significant accumulation upon multiple dosing.

In vivo chondroprotection and metabolic synergy of glucosamine and chondroitin sulfate.

Supplements of glucosamine hydrochloride, low molecular weight chondroitin sulfate, and manganese ascorbate were tested separately and in combination for their ability to retard progression of cartilage degeneration in a rabbit instability model of osteoarthrosis. Computerized quantitative histologic evaluation of safranin O stained sections of the medial femoral condyles measured the grade and extent of tissue involvement of lesions. Severe lesions (Mankin grade greater than 7) were absent in all animals supplemented with a dietary mixture of glucosamine, chondroitin sulfate, and manganese ascorbate. Total linear involvement (mm of lesioned surface) and total grade (mean grade x number of lesions per animal) were reduced significantly in animals given the combination compared with controls (59% and 74% respectively). Animals supplemented with glucosamine, chondroitin sulfate, or manganese ascorbate alone had less moderate and severe tissue involvement than controls but not to the extent of the combined group. In vitro, a combination of glucosamine hydrochloride and chondroitin sulfate acted synergistically in stimulating glycosaminoglycan synthesis (96.6%). Chondroitin sulfate and manganese ascorbate but not glucosamine were effective in inhibiting degradative enzyme activity. These data suggest that the disease modifying effect (the ability to retard progression of cartilage degeneration) of a mixture of glucosamine, chondroitin sulfate, and manganese ascorbate is more efficacious than either agent alone.