Reflectance near-infrared spectroscopic method with a chemometric technique using partial least squares multivariate calibration for simultaneous determination of chondroitin, glucosamine, and ascorbic acid.

A reflectance near-infrared (RNIR) spectroscopy method was developed for simultaneous determination of chondroitin (CH), glucosamine (GO), and ascorbic acid (AS) in capsule powder. A simple preparation of the sample was done by grinding, sieving, and compression of the powder sample for improving RNIR spectra. Partial least squares (PLS-1 and PLS-2) was successfully applied to quantify the three components in the studied mixture using information included in RNIR spectra in the 4240-9780 cm(-1) range. The calibration model was developed with the three drug concentrations ranging from 50 to 150% of the labeled amount. The calibration models using pure standards were evaluated by internal validation, cross-validation, and external validation using synthetic and pharmaceutical preparations. The proposed method was applied for analysis of two pharmaceutical products. Both pharmaceutical products had the same active principle and similar excipients, but with different nominal concentration values. The results of the proposed method were compared with the results of a pharmacopoeial method for the same pharmaceutical products. No significant differences between the results were found. The standard error of prediction was 0.004 for CH, 0.003 for GO, and 0.005 for AS. The correlation coefficient was 0.9998 for CH, 0.9999 for GO, and 0.9997 for AS. The highly accurate and precise RNIR method can be used for QC of pharmaceutical products.

Reflectance near-infrared spectroscopic method with Chemometric techniques for simultaneous determination of Chondroitin, glucosamine, and methyl sulfonyl methane.

Reflectance near-IR (RNIR) spectroscopy was used for the simultaneous determination of chondroitin (CH), glucosamine (GO), and methyl sulfonyl methane (MSM) in tablets. Simple sample preparation was done by grinding, sieving, and compression of the tablets for improving RNIR spectra. Principal component regression and partial least squares (PLS-1 and PLS-2) were successfully applied to quantify the three components in the studied mixture using information included in RNIR spectra in the range of 4350-9100 cm(-1). The calibration model was developed with drug concentration ranges of 14.5-44.2% (w/w) for CH, 18.4-55.3% (w/w) for GO, and 6-18.6% (w/w) for MSM with addition of tablet excipients to the calibration set in the same ratio as in the tested tablets. The calibration models were evaluated by internal validation, cross-validation, and external validation using synthetic and pharmaceutical preparations. The proposed method was applied for analysis of six batches of the pharmaceutical product. The results of the proposed method were compared with the results of the pharmacopoeial method for the same batch of the pharmaceutical product. No significant differences between the results were found. The RNIR method is accurate and precise, and can be used for QC of pharmaceutical products.

Detection of specific glycosaminoglycans and glycan epitopes by in vitro sulfation using recombinant sulfotransferases.

Sulfated glycans play critical roles during the development, differentiation and growth of various organisms. The most well-studied sulfated molecules are sulfated glycosaminoglycans (GAGs). Recent incidents of heparin drug contamination convey the importance of having a convenient and sensitive method for detecting different GAGs. Here, we describe a molecular method to detect GAGs in biological and biomedical samples. Because the sulfation of GAGs is generally not saturated in vivo, it is possible to introduce the radioisotope (35)S in vitro using recombinant sulfotransferases, thereby allowing detection of minute quantities of these molecules. This strategy was also successfully applied in the detection of other glycans. As examples, we detected contaminant GAGs in commercial heparin, heparan sulfate and chondroitin samples. The identities of the contaminant GAGs were further confirmed by lyase digestion. Oversulfated chondroitin sulfate was detectable only following a simple desulfation step. Additionally, in vitro sulfation by sulfotransferases allowed us to map glycan epitopes in biological samples. This was illustrated using mouse embryo and rat organ tissue sections labeled with the following carbohydrate sulfotransferases: CHST3, CHST15, HS3ST1, CHST4 and CHST10.

Cellular differentiation and the aging process in cartilaginous tissues. Mucopolysaccharide synthesis in cell cultures of chondrocytes.

Primary cell cultures of differentiated chondrocytes were shown to produce chondroitin-4-sulfate as the predominant mucopolysaccharide, with suggestive evidence for the synthesis of keratan sulfate and possibly chondroitin-6-sulfate. Chicken embryonic cartilage was shown to be composed mainly of chondroitin-4-sulfate, with a small amount of chondroitin-6-sulfate, but essentially no keratan sulfate. These findings were compared to the data of others, and a hypothesis explaining the aging process in cartilage in terms of cellular differentiation was presented.

The Hurler syndrome: a study of cultured lymphoid cell lines.

Lymphoid suspension lines have been established from three patients with the Hurler syndrome and four normals. The Hurler lines can be distinguished from normals by (a) staining characteristics, (b) increase in total cellular mucopolysaccharide content, and (c) increase in dermatan sulfate. Hyaluronic acid is absent in cultured lymphoid cells from normal persons and patients with the Hurler syndrome. The availability of biochemically marked suspension cultures should prove useful for enzymatic studies as well as for further elucidation of this clinical syndrome.

Properties of extracellular adhesion-mediating particles in myoblast clone and its adhesion-deficient variant.

Both the skeletal muscle myoblast cell line L6 and an adhesion-deficient variant of L6 released glycoprotein complexes, termed adherons, into their culture medium. The adherons from the variant, M3A, differed from those of L6 in a number of properties. M3A adherons were much less effective in promoting the cell-substratum and cell-cell adhesion of myoblasts than L6 particles. The adherons from the two cell lines also differed in their relative sedimentation velocities in sucrose gradients and had different chemical compositions. The M3A particle lacked chondroitin and contained relatively less collagen and fibronectin than the L6 adheron. Both L6 and M3A particles adhered to plastic surfaces and cells equally well in the absence of calcium ions. Neither cell-cell adhesion nor particle aggregation occurred in calcium-free medium. However, in the presence of calcium, the L6 adherons aggregated completely and M3A particles aggregated poorly. These data suggest that at least two sets of interactions are required for adheron-mediated adhesion: a calcium-independent binding of the adheron to the cell, and a calcium-dependent interaction between particles that is directly responsible for adhesion. The M3A variant is blocked at the calcium-dependent step, resulting in an adhesion deficiency.

Basal lamina of embryonic salivary epithelia. Nature of glycosaminoglycan and organization of extracellular materials.

The ultrastructural organization and the composition of newly synthesized glycosaminoglycan (GAG) in the epithelial basal lamina of mouse embryo submandibular glands were assessed. The labeled GAG accumulating in the lamina is distinct from that in its tissue of origin, the epithelium, or from that in the surrounding mesenchyme. In the lamina, hyaluronic acid accounts for approximately 50% of the labeled GAG, chondroitin-4-sulfate is twice the chondroitin-6-sulfate, and there is a low proportion of chondroitin. This composition is constant regardless of whether the lamina is labeled by whole glands or, in the absence of mesenchyme, by isolated epithelia retaining a lamina and by isolated epithelia generating a lamina de novo. The results andicate that the labeled GAG are bona fide components of the lamina, and suggest that laminar GAG is deposited in units of constant composition. Ultrastructural observations following ruthenium red staining or tannic acid fixation extablish that the lamina is a highly ordered specialization of the basal cell surface. Discrete structures in macroperiodic arrays apparently attached to the plasmalemma are visualized. This organization is seen in intact glands and in the laminae produced by epithelia in the absence of mesenchyme or biological substrate. The data are interpreted as indicating that the basal lamina contains supramolecular complexes of hyaluronic acid and proteoglycan which are organized into an extracellular scaffolding which imposes structural form on the epithelium.

Biosynthesis of Chondroitin in Engineered Corynebacterium glutamicum.

Chondroitin, the precursor of chondroitin sulfate, which is an important polysaccharide, has drawn significant attention due to its applications in many fields. In the present study, a heterologous biosynthesis pathway of chondroitin was designed in a GRAS (generally recognized as safe) strain C. glutamicum. CgkfoC and CgkfoA genes with host codon preference were synthesized and driven by promoter Ptac, which was confirmed as a strong promoter via GFPuv reporter assessment. In a lactate dehydrogenase (ldh) deficient host, intracellular chondroitin titer increased from 0.25 to 0.88 g/l compared with that in a wild-type host. Moreover, precursor enhancement via overexpressing precursor synthesizing gene ugdA further improved chondroitin titers to 1.09 g/l. Chondroitin production reached 1.91 g/l with the engineered strain C. glutamicum ΔL-CgCAU in a 5-L fed-batch fermentation with a single distribution Mw of 186 kDa. This work provides an alternative, safe and novel means of producing chondroitin for industrial applications.

Identification of unknown intraocular material after cataract surgery: evaluation of a potential cause of toxic anterior segment syndrome.

PURPOSE: To describe and identify unknown opaque material between the optic of an AR40 intraocular lens (IOL) injected with the Emerald Series implantation system (both AMO, Inc.) and the posterior capsule at the conclusion of routine phacoemulsification to prevent an outbreak of toxic anterior segment syndrome (TASS). SETTING: Ambulatory care center operating room, University of North Carolina Hospitals and Department of Ophthalmology, University of North Carolina School of Medicine at Chapel Hill, Chapel Hill, North Carolina, USA. METHODS: After coaxial phacoemulsification in multiple patients, opaque material was present between the optic of a posterior chamber IOL and the posterior capsule. Although there was no TASS, the material was removed from 2 eyes and analyzed with scanning electron microscopy (SEM) and x-ray microanalysis (XRM). Similarly, crystalline lens, Klenzyme (Steris Corp.), Viscoat (sodium hyaluronate 3.0%-chondroitin sulfate 4.0%), and Provisc (sodium hyaluronate 1.0%) were analyzed. RESULTS: On SEM, the material had an irregular undulating surface similar to that of Provisc. Viscoat and the crystalline lens had smoother surfaces. On XRM, the material contained sodium, chlorine, and calcium, like Viscoat and Provisc, and phosphorous and sulfur, like Viscoat. The material also contained silicone, magnesium, aluminum, titanium, iron, and zinc. Klenzyme had smaller peaks of sodium, chlorine, and calcium and a higher carbon background than the unknown material. CONCLUSIONS: The material was likely ophthalmic viscosurgical device that was chemically and structurally altered by the cleaning and sterilization process. The silicone and metallic elements were probably from the Emerald Series implantation system as the disposable cartridge is coated with silicone and the reusable injector is metal.

Regional distribution of acid mucopolysaccharides in the kidney.

Kidneys from 20 dogs were dissected into cortical and medullary components and analysed for acid mucopolysaccharide content. Heparitin sulfate accounted for approximately 80% of cortical acid mucopolysaccharide, 10% was chondroitin sulfate B, and 10% was low molecular weight hyaluronic acid. Medullary tissue exhibited a 4- to 5-fold higher concentration of acid mucopolysaccharide than did cortical tissue, and the dominant compound was moderately highly polymerized hyaluronic acid. While chondroitin sulfates A and (or) C were not detected in this study, the presence of minor amounts of these substances could not be excluded. A model experiment indicated that hyaluronic acid retards sodium diffusion, apparently due to its viscous properties rather than its electronegativity.

The glycosaminoglycans of normal and arthritic cartilage.

The cartilages from the hip joints of 13 normal and 15 osteoarthritic humans were analyzed for glycosaminoglycan content and distribution. The GAGs were separated by elution with CPC on a short cellulose column by the technique of Svejcar and Robertson after digestion of the tissue with pronase and papain. The eluates were identified by a variety of methods including determination of molar ratios, N-acetyl-hexosamine determinations after hyaluronidase treatment and thin-layer chromatography of unhydrolyzed and hydrolyzed GAGs. From the data obtained, it was demonstrated that cartilage from arthritic patients showed a significant increase in the concentration of chondroitin 4-sulfate and a significant decrease in keratan sulfate, with only slight changes in the total amount of GAG present. Calculations of the molar ratios showed variation in the sulfation with chondroitin 4-sulfate appearing in the "supersulfated" state in the arthritic cartilage. The data lead to speculation regarding the process of osteoarthritis, and it is concluded that the changes seen are more likely to represent an altered pattern of synthesis rather than selective degradation. Since the changes suggest a younger cartilage, a theory is advanced that the chondrocyte responds to the chronic stress of osteoarthritis by modulation to a chondroblastic phase.

Commercial extracellular matrix scaffolds for rotator cuff tendon repair. Biomechanical, biochemical, and cellular properties.

BACKGROUND: We are not aware of any in vitro study comparing the biomechanical, biochemical, and cellular properties of commercial extracellular matrix materials marketed for rotator cuff tendon repair. In this study, the properties of GraftJacket, TissueMend, Restore, and CuffPatch were quantified and compared with each other. The elastic moduli were also compared with that of normal canine infraspinatus tendon. METHODS: Samples were tested from different manufacturing lots of four materials: GraftJacket (ten lots), TissueMend (six), Restore (ten), and CuffPatch (six). The Kruskal-Wallis test was used to compare thickness, stiffness, and modulus as well as hydroxyproline, chondroitin/dermatan sulfate glycosaminoglycan, hyaluronan, and DNA contents among these matrices. The moduli of the extracellular matrices were also compared with those of normal canine infraspinatus tendon. RESULTS: All four extracellular matrices required 10% to 30% stretch before they began to carry substantial load. Their maximum moduli were realized in their linear region at 30% to 80% strain. The elastic moduli of all four commercial matrices were an order of magnitude lower than that of canine infraspinatus tendon. TissueMend had significantly higher DNA content than the other three matrices (p<0.0001), although both Restore and GraftJacket also had measurable amounts of DNA. CONCLUSIONS: Our data demonstrate chemical and mechanical differences among the four commercial extracellular matrices that we evaluated. Probably, the source (dermis or small intestine submucosa), species (human, porcine, or bovine), age of the donor (fetal or adult), and processing of these matrices all contribute to the unique biophysical properties of the delivered product. The biochemical composition of commercial extracellular matrices is similar to that of tendon. However, the elastic moduli of these materials are an order of magnitude lower than that of tendon, suggesting a limited mechanical role in augmentation of tendon repair.

[Protective effect on the corneal endothelium and remaining effect at the anterior chamber for three different kinds of viscoelastic devices].

PURPOSE: The present study was performed to evaluate the corneal endothelium protection and anterior chamber stagnation abilities of three different types of viscoelastic substances (Healon, Viscoat, HealonV). METHODS: Viscoelastic substances were selected at random for 120 eyes with cataracts, and the postoperative reduction rates of the corneal endothelium cells were compared. The residual viscoelastic substances after filling of the anterior chamber of pig eyes and aspiration with a handpiece were measured by an anterior eye segment image analysis system. The same procedures were performed in rabbit eyes and the residual levels of viscoelastic substances on the corneal endothelium were photographed histologically. RESULTS: The reduction rate of endothelium corneal cells tended to decrease with Viscoat three months after surgery. The results obtained with the anterior eye segment image analysis system showed that the residual level in the anterior chamber was higher with Healon. Histological analyses demonstrated residual Viscoat at the center of the corneal endothelium after perfusion. CONCLUSION: HealonV was superior in terms of spatial retention and Viscoat had corneal endothelium protection potential.

The glycosaminoglycans in human hepatic cancer.

A method is proposed for the analysis of glycosaminoglycans that were isolated from human liver, combining cellulose acetate electrophoresis and enzymatic digestion with mucopolysaccharidases. The major constituent of glycossaminoglycans in the healthy liver is heparin sulfate and/or heparin (about 65%), with approximately equal quantities of dermatan sulfate and hyalauronic acid (about 13.5 and 13%, respectively) and a small amount of chondroitin sulfate. These components, especially chondroitin sulfate and hyaluronic acid, are markedly increased in hepatic carcinomas.

Structural characterization of fucosylated chondroitin sulfates from sea cucumbers Apostichopus japonicus and Actinopyga mauritiana.

Two samples of fucosylated chondroitin sulfate (FCS), AJ and AM, were isolated from holothurian species Apostichopus japonicus and Actinopyga mauritiana, respectively. Purification of FCS was performed by ion exchange chromatography followed by gel filtration. Structure of the biopolymers was elucidated using chemical and NMR spectroscopic methods. Both polysaccharides were shown to contain a typical chondroitin core built up of repeating disaccharide units →3)-ß-d-GalNAc-(1→4)-ß-d-GlcA-(1→ and decorated by sulfate groups and α-l-Fuc branches. Two polysaccharides were different in pattern of sulfation of GalNAc and fucosyl branches connected to O-3 of GlcA. The ratio of GalNAc4S6S:GalNAc4S for AJ was about 2:1, whereas for AM this value was approximately 1:1. AJ contained Fucp2S4S and Fucp3S4S residues linked to O-3 of GlcA in a ratio of 3:1, while for AM this ratio was 1:4. Small portions of Fucp4S units attached to O-3 of GlcA were also found in both polysaccharides. Moreover, in a structure of AM the presence of Fucp3S residues linked to O-6 of GalNAc were determined using the data of NMR spectra.

The hydration of proteoglycans of bovine cornea.

The water sorptive and retentive capacities of three corneal proteoglycans with different keratan sulfate/chondroitin-4-sulfate compositions were investigated. The calcium salt of a predominantly keratan sulfate containing proteoglycan had hydration properties similar to that of calcium keratan sulfate. The proteoglycan containing predominantly calcium chondroitin-4-sulfate side chains sorbed water to a greater extent than pure calcium chondroitin-4-sulfate but its retentive power was somewhat less. The proteoglycan containing about twice as much keratan sulfate as chondroitin-4-sulfate, on a dissaccharidic molar basis and had hydration properties which were closer to the behavior of chondroitin-4-sulfate than keratan sulfate. The results are discussed in terms of structure and polymer interaction in the proteoglycan matrices.

Chondroitin sulphate of calf knee-joint cartilage.

Chondroitin sulphate from different layers of calf knee-joint cartilage has been isolated and purified. Analysis for hexosamine, uronic acid, sulphate and relative proportions of the 4- and 6-isomers revealed no differences between the layers. However, an increase in the average molecular weight of the chondroitin sulphate was found to correspond with distance from the articular surface. In particular, the average molecular weight in the epiphyseal cartilage was significantly higher than that in the articular cartilage. The chondroitin sulphate of the 40-mu-mthick articular surface layer was found to have a higher molecular weight than the rest of the articular part of the cartilage and also to be more polydisperse. These results, regarding molecular size, are in agreement with the cetylpyridinium chloride cellulose microcolumn fractionation patterns.

Lipoprotein-acid mucopolysaccharide complexes of human atherosclerotic lesions.

Lipoprotein-acid mucopolysaccharide complexes occurring in different types of human atherosclerotic lesions were isolated and partially characterized. Complexes from fatty streaks and fibrous plaques were extracted from pooled tissues with 0.15 M NaCl, purified by gel filtration, and fractionated by ultracentrifugation. Both low and very low density lipoproteins were present; low density lipoprotein was predominant. The complexes were analyzed for uronic acid, cholesterol, phospholipid, and Ca-2+ contents; there was no significant difference in the relative molar ratios between complexes from fatty streaks and fibrous plaques. Acid mucopolysaccharides were isolated from the complexes and identified by electrophoresis and enzymatic studies. Chondroitin sulfate C and hyaluronic acid were found in complexes from fatty streaks and fibrous plaques. Heparin was detected only in fibrous plaques.

Distribution of proteoglycans during the hair growth cycle in human skin.

The involvement of proteoglycans in hair growth has been recognized through the observation of increased hair growth in diseases such as the mucopolysaccharidoses and pre-tibial myxedema, which involve an increase in skin proteoglycan content. In an attempt to understand this, we have examined the distribution of chondroitin 6 sulphate (C6S), unsulphated chondroitin (COS), dermatan sulphate (DS), and heparan sulphate proteoglycans (HSPG) in frozen tissue sections of normal scalp by immunostaining. Results show that during anagen, the thick connective tissue sheath around the follicle strains strongly for C6S, COS, and DS. COS is uniquely associated with this region and is not found beneath the epidermis or infundibular epithelium. HSPG is, however, localized in the basement membrane zone adjacent to the outer root sheath. In addition, all of these proteoglycans are localized in the dermal papilla. In mid-catagen, we observed significant loss of C6S and COS staining from both the dermal papilla and the connective tissue sheath, but no decrease in staining for HSPG. In late catagen, very little staining of C6S and COS was observed. In early anagen, we observed that C6S was again present in the connective tissue sheath and dermal papilla; however, COS staining appeared to be weaker and less closely associated with the follicle. HSPG staining was observed in early anagen in a pattern very similar to that found for other basement membrane components. Results for DS were not obtained for catagen or early anagen. These results provide further evidence that hair growth is associated with the presence of chondroitin proteoglycans in the follicle environment and that the cessation of growth is associated with their removal. Further studies are underway to characterize the relationship between hair growth and proteoglycans.

Composition of acidic glycosaminoglycans in human cerebral arteries.

(1) The composition of acidic glycosaminoglycans (AGAG) in the pooled human cerebral arteries was investigated by electrophoretic characterization before and after digestion with chondroitinases. Constitution of the chondroitin sulfate (CS) family was determined qualitatively on the basis of the disaccharide subunits of the CS chains after depolymerization with the enzymes. (2) The data obtained indicated that the AGAG in cerebral arteries consisted of, in order of amount, heparan sulfates, chondroitin-6-sulfate, dermatan sulfate, chondroitin-4-sulfate and hyaluronic acid. (3) The existence of oversulfated CS and undersulfated CS in the cerebral AGAG was supported by the detection of unsaturated di-sulfated and non-sulfated disaccharides on paper chromatography. In addition, the presence of hyaluronic acid was indicated by electrophoretic and enzymatic separation. (4) The distribution of the individual AGAG in cerebral arteries was also examined on the basis of molecular weight by gel filtration on a Sephadex G-100 column.