Labaratoire de Cinètique des Xènobiotiques. Facultè des Sceniences Pharmaceutiques. 35. chemin des Maraichers, 31062 Toulouse Cedex. France. Laboratore de Toxicologie et Pharmacocinetique Clinique, C:H:U Rangueil, 31403 Toulouse Cedex4. France. Institut de Recherche Pierre Fabre. Rue Jean Rostand, B.P. 687, 31319 Labège Innopole, France.

Corresponding author. Tel. +33 (0)5.61.32.22.51

E-mail: houin.g@chu-toulouse.fr

Abstract

The glycosaminoglycan chondrotin sulfate, is an oral drug (Structum) used as a chondroprotective agent in several European countries. In vivo studies have produced controversia data concerning the absorption and metabolism of the drug. Using a dimethylmethylene blue assay and FPLC we have studied the degradation by the contents or tissues in vitro in rat. There was no degradition by the contents or tisue of the small intestine or with colon tissue. However, the chondroitin sulfate was rapidly degraded in the contents of the caecum and to a lesser extend in the colon contents. FPLC analysis indicated that the degradation was down to the disaccharide unit. Studies with sacs from the different parts of the intestine showed that the degradation products crossed the wall of the caecum and that the small anounts of the parent molecule could also cross mucosal wall in all parts of the intestine.

Key-words:

Chondroitin sulfate; glycosaminoglycan; intestinal contests; instentinal tissues; intestinal sacs.

Introduction:

Chondroitin sulfate (CS) is an acidic mucopolysaccharide which is found naturally in cartilaginous tissues. It is a polymer of a disaccharide consisting of glucuronic acid and sulfated N-acetyl galactosamine. This anionic endogenous compound is also used therapeutically in humans as a symptomatic slow acting drug in osteoarthritis and as a potential chondroprotective drug. It is administrated by the oral route (Charlot et al., 1992). STRUCTUM is an oral drug marketed in several European countries, which contains two chondroitin sulfate isomers: the 4 and 6-sulfate in a ratio of approximately 2/3 and 1/3. Although this compound seems to give good clinical results in arthrictic patients (pain and inflammation reduction, movement improvement, cartilage metabolism and improvement of immunological parameters) little is known about ist stability and absorption in the gastrointestinal tract. The bioavailibility and metabolic fate or orally administrated chondroitin sulfate in man and animals are the subject of much debate in the literature. Some studies seem to show an elevation of plasmatic, urinary and synovial chondroitin sulfate concentrations after oral administration (Conte et al., 1991a and 1991b and 1995, Gross, 1983, Paroli et al., 1991). Conversely, other authors failed to show an absorption of chondroitin sulfate following oral administration (Konador and Kawiak, 1997: Baici et al., 1992: Anderman and Dietz, 1982). Some studies have also shown the presence of low molecular weight fragments derived from the chondroitin sulfate in urine or plasma following oral dosing (Conte et al., 1991a; 1995 and Palmieri et al., 1990). It is not clear at which site in the body the degradation of CS might have occurred. Liau and Horowitz (1974) showed depolymerisation and desulfation of CS by enzymes in the stomach and intestinal tissues. This enzymatic activity was probably lysosomal, given that the pH used was 4.0. Thus there may degradation of the chondroitin sulfate macromolecule during absorption across the gastrointestinal barrier, if endocytosis of the molecule was taking place. Although there are no intestinal, pancreatic or border brush enzymes known to degradate polymers such as CS, the activity of the bacterial flora could also be involved, especially in the large intestine (Rubinstein et al., 1992 and 1997; Salyers and Kotarski, 1980; Salyers O`Brien 1980). Given these conflicting results, the diversity of administrated doses and of methods used to measure CS, our objective was to determine, in vitro, the stability (ie potential depolymerisation and metabolism) of STRUCTUM in the contents of rat stomach, small intestine, solon and ceacum, and in the intestinal tissue, usin a simple colorimetric reaction (dimethylmethylene blue) and also by Fast Performance Liquid Chromatography (FPLC). The tissue degradation was measured at pH 4.0 to detect any potential lysosomal degradation of CS. The colorimetric reaction had been shown tomeasure the intact polymer (Farndale et al., 1982) and therefore could be used to measure possible degradation of chondroitin sulfate without characterization of the degradation products. When degradation was observed using the colorimetric method, experiments with radiolabelled chondroitin sulfate were also carried out to separate and identify the possible metabolic products of the drug by FPLC coupled to a Flow Scinillation Analyser (separation of different molecular weight products with the labelled CS). This technique was also used to measure labelled CS and ist metabolities that were transported across the different gastrointestinal sites when incubated in intestinal sacs in vitro.

2. Experimental procedure

2.1. Materials

CS of molecular weights: 10,15,17, and 18 kDa were supplied by Institut de Recherche Pierre FABRE (IRPF) and (Acetyl-1-C)-chondroitin sulfate (specific activity: 2.37 Mbq/mg) was prepared and purified by Amersham International plc (Buckinghamshire, England) using the 17 kDa product. Tissue culture medium, TC-199, chondroitinase AC, dimethylmethylene-blue, chondroitin disaccharides *4S and *6S were obtained from Sigma-Aldrich Chemie (S Quentin Fallavier, France). FPLC reagents were obtained from Sulpeco (S Quentin Fallavier, France). Superose 12 RH 10/30 column and Sephadex G-25 columns (PD-10) were obtained from Pharmacia Biotech (Uppsala, Sweden). Adult male Sprague-Dawley rats were supplied by Centre d`élevage Dépré (ST Doulchard, France). Ultima Flo AP Scintillation fluid was obtained from Packard (Rungis, France).

2.2. Preparation of the gastrointestinal fluids and tissues

A pool of gastrointestinal fluids and tissues from several male Sprague Dawley rats was prepared at the beginning of the study and the same pool used throughout the validation and the experimental study. The rats were starved overnight and sacrified by cervical dislocation.

Stomach contents. The stomach was removed and the contents were washed out with 10 ml (w/v) of 0,9% NaCl/0,01 M HCI, ph 2.0. The stomach contents were briefly homogenised using an Ultra-Turrax homogeniser, and coarse particles removed by filtration through glass wool.

Small intestine, colon and caecum contents. The small intestine, the colon and the caecum were removed and flushed through with 2 x 10 ml of Phosphate-buffered saline (PBS) (pH 7.0). The contents were briefly homogenised in PBS and coarse particles removed by filtration through glass wool.

Small intestine and colon tissues. After washing out the contents, the small intestine and colon were cut open lengthwise and flattened onto a glass plate on ice. The epithelium was scraped off the tissue with a glass microscope slide, and weighed. Each tissue was homogenised for 1 minute in 20 ml of PBS (pH 7.0) using an Ultra-Turrax homogeniser. All the preparations were aliquoted into 0.5 ml aliquots and stored at &emdash;20 °C.

2.3. Spectrophotometric method

Preparation of the colour reagent and buffers. The colour reagent was prepared by dissolving 16 mg dimethylmethylene blue in 1 litre of distalled water containing 3.04 g glycine, 2.37 g NaCI and 95 ml 0.1 M HCI, to give a solution at pH 3.0. The reagent was stable in a brown bottle at romm temperature for at least 3 months.

Colour reaction. 100 ul of the test gastrointestinal medium (or buffer) was addet to 400 ul of buffer (HCI/NaCI 0.2M: pH 2.0; PBS: pH 7.0 or Na-acetate/acetic acid 0.25M: pH 4.0) containig calibration points or quality controls of CS solution. The 0.5 ml sample was treated immediately after mixing by heating at 100 °C for 3 min to inactive the enzymes and denature proteins, followed by spinning for 30 min in a centrifuge (4000 g). 200 ul of the supernatant were placed in a glass hemolysis tube and 2.5 ml dimethylene blue colour reagent added. Mixing was completed by pouring the solution into disposable spectophotometer cuvettes, and the absorbance at 525 nm was read immediately (approx 15 s). The reference was destilled water. For each experiment, calibration curves were prepared in the test medium/ tissue and in the appropriate buffer. 3 concentrations of quality control (QCs) were also tested. The dimethylmethylene blue assey was validated in every gastrointestinal medium, with a calibration curve from 0 to 100 ug/ml of CS. The calibration curves consisted of eight points plus zero (0, 5, 10, 15, 20, 25, 20, 75, 100 ug/ml). 3 levels of quality controls (20, 50, and 80 ug/ml) were tested. The repeatability was tested as follows: each quality control sample was assayed five times in one day and compared with the calibration curve. The reproducibility was tested as follows: a calibration curve was constructed each day for five days and each quality control sample assayed in duplicate. The following validation criteria and limits expressed were used: precision : Standard deviation/ Mean calculated concentration, expressed as a percentage: >15% for each point of the calibration curve and for the quality control samples (QC), except at the limit of detection where >20% was acceptable; accuracy:

(calculated concentration &emdash; theoritical concentration) / theorictical concentration, expressed as a positive or a negative percentage: <15% for each point of the calibration curve, and for the quality control sample, except at the limit of detection (<20%); calibration curves: the least squares regression coefficient should be: r > 0.95.

2.4. Incubation tests

Incubation with commercial chondroitinase. Three different molecular weight CS (10, 15, and 18 kDa) were each dissolved in 4 ml of PBS buffer (pH 7.0) at a concentration of 125 ug/ml (to give a final concentration of 100 ug/ml). 1 ml of chondroitinase solution (activity 0.02 U) was added and the mixture incubated at

37 °C. 0.6 ml samples were removed at 0, and every 20 min up to 2 h and 5 h. Samples were heated to 100 °C for 3 min to inactivate the enzyme and centrifuged at 4000 g for 30 min. 200 ul of the supernatants were analysed by the spectrophotometric method using dimethylmethylene blue. For each molecular weight CS, a control test without chondroitinase was treated under the same conditions and a calibration curve in PBS (from 0 to 100 ug/ml) was constructed.

Incubation with the gastrointestinal fluids and tissues.

Small intestine, colon and stomach. Three different molecular weight CS (10, 15, and 18 kDa) were dissolved in appropriate buffer (HCI/NaCI 0.2M, pH 2.0 for stomach; PBS, pH 7.0 for intestinal and colon contents and tissues, 0.25M Na-acetate, pH 4.0 for colon and intestinal tissue) to give final concentrations of 0.1, 1.0 and 10 mg/ml after addition of medium. 1 ml of each medium was added to 4 ml of each CS solution in triplicate and incubated at 37 °C, and in some cases 3 ml of the medium/tissue was added to 2 ml of each CS solution. 0.6 ml samples were removed at 0 and every 20 min up to 2 h. In the case of incubation colon contents (pH 7.0) additional samples were removed at 4, 6, 8 and 23 h.

Caecum contents. CS (17 kDa) was dissolved in PBS (pH 7.0) at the appropriate concentration to give a final concentration of 100 ug/ml after addition of medium (total final volume = 5 ml). 1 ml and 3 ml of the caecum contents in chondroitin sulfate solution (in duplicate) were incubated at 37 °C. In the both cases, 0.6 ml were removed at 0 and every 30 min up to 2h and at 4h, 6h and 24h. In all cases, samples were heatet to 100° for 3 min to inactivity the enzymes and centrifuged at 4000 g for 30 min. 200 ul of the supernantants were analysed by the spectrophotometric method using dimethylmethylene blue. For the higher concentrations of CS (1 and 10 mg/ml), dilutions of the samples (respectively 1/10 and 1/100) with appropriate buffer were required to be within the useful range of the colour reaction. At the same time, control tests: CS without gastrointestinal media and gastroinentinal media without CS were carried out with each CS solution and treated under the same conditions.

2.5. Fast Performance Liquid Chromatography

This chromatography was performed on a Fast performance Liquid Chromatography system (FPLC) using a Superose 12 RH 10/30 column. The mobile phase was sodium sulfate 0.5M and the flow rate was 0.8 ml/min. The absorbance of the column eluate was monitored at 205 nm. The UV detector was coupled to a Flow Scintillation Analyser (Flo-One) which detected the radiolabelled products.

2.6. Incubation of C-labelled CS in caecum contents

CS (17 kDa) was dissolved in PBS (pH 7.0) at the appropriate concentration to give a final concentration of 100 ug/ml after addition of the medium, and labelled with 5 ul of C-chondroitin sulfate. 1 ml and 3 ml of the caecum contents was added to 4 ml or 2 ml of the labelled chondroitin sulfate and incubated at 37 °C. In the both cases, 0.2 ml were removed at 0 and every 30 min up to 2h and at 4h, 6h and 24h. Each sample was treated immediately by heating at 100 °C for 3 min to inactivate the enzymes and denature proteins, followed by spinning for 30 min in a centrifuge (4000 g). 20 ul of the supernatant were injected int the FPLC system and the radiolebelled peaks detected with a Flow Scintillation Analyser.

2.7.Rapid chromatography on G25 Sephadex columns

500 ul of the analysed labelled sample were diluted with 500 ul PBS (pH 7.0) and loaded onto a G-25 Sephadex PD-10 column and eluted with 20 x 1 ml of PBS. 500 ul of each fraction were counted for radioactivity.

2.8. Sac preparation and incubation

A male Wistar rat was starved overnight, sacrified, and the ceacum, the intestine and the colon were quickly removed. The whole caecum and its contents and section of the small intestine and the colon (4-6 cm) were filled with 1 or 2 ml of the 17 kDa (33 mg/ml) labelled C-chondroitin sulfate and tied tightly at each and with silk suture and incubated in 20 ml of oxygenated TC-199 medium at 37 °C with shaking. 500 ul samples (in duplicate) of the incubation medium were removed at different incubation times. (T0, T15, T30, T45, T60, T75, T90, T120, and T210 min). After each sample, 1 ml of fresh oxygenated medium was added to the flosk. One of these 500 ul sample was counted for radioactivity. In the order to separate C-chondroitin sulfate and its possible degradation products, the second 500 ul sample (T15 and T60 min) was analysed by rapid chromatography on a G25 Sephadex column. The initial solution introduced into the sacs was also analysed by this method. 20 ul of the second duplicate 500 ul sample (T30, T45, T60, T75, T90, T120, and T210 min)were injected into the FPLC chromographic system with UV detection and Flow Scintillation Analyser (Flo-One).

3. Results

3.1. Validation of the spectrophotometric method using dimethylmethylene blue

This simple colorimetric method, developed by Ferndale et al. (1982 and 1986), described the determination of chondroitin sulfate by the binding of the anonic chondroitin sulfate with the dye dimethylmethylene blue and then reading the absorbance at 525 nm. This method was suitable for analyzing chondroitin sulfate with a molecular size greater than or equal to that of the hexasccharide (Baici et al., 1992). The validation results obtained in our study are described in Table 1. All the calibration curves were linear except in instentinal and colon contents (pH 7.0) and in colon tissue (pH 4.0) where the calibration curves where adjust with a second degree polynomial equation. The method was specific, with good precision and accuracy in the tested range of concentrations. It should be noted than in the case of the validation in the colon tissue (pH 4.0), 80 ul of 5M NaOH were added before the heating step to improved the method`s sensitivity. The calibration curve in the intestinal tissue at pH 4.0 was not determined as in this medium the sensitivity of the method was too low.

3.2. In vitro degradation study using the spectrophotometric method

Incubation with chondroitinase. Fig. 1 shows that for all these CS tested, the concentration of the product decreased with the incubation time. After 2 h incubation, the concentration of CS in the incubation medium (PBS, pH 7.0) represented 20 to 25% of the initial concentration (i.e. 75% to 80% degradation). This results of the incubation of CS with commercial chondroitinase confirmed that the colorimetric method using dimethylmethylene blue was sufficiently sensitive to accurately measure the degradation of CS. It also showed that the rate of degradation was apparently independant of the molecular weight of the CS substrate.

Incubation with the gastrointestinal media. All the used gastrointestinal liquids and tissue were assayed for specific marker enzymes. The comparison of these activities with the initial values at preparation confirmed that there was no biochemical degradation of the sample during the storage at &emdash;20 °C. In vitro, no degradation of CS was observed with rat stomach contents or small intestine contents or tissue. Increasing the ratio of the enzymes, to increase the metabolism, did not increase degradation of CS in these preparation. A degradation of CS (10 and 18 kDa) was observed in colon contents (Fig. 2), but it was a slight degradation (15 to 20 %) during the first 2 h. The degradation was more apparent after 23 h incubation (75%). This suggested degradation of CS by the colonic bacteria. Fig. 3 shows the case of incubation of CS with the caecum contents. It can be seen that with the both 1 ml and 3 ml of caecum contents, there was a rapid degradation of CS (17 kDa). With 1 ml the degradation was complete in 4 h (50 % in 90 min). With 3 ml the degradation was complete in 1 hour (50% degradation in 30 min). The rate of degradation was approximately proportional to the quantity of caecum contents. Thus degradation of CS in the rat gastrointestinal tract was not observed in the stomach or small intestine, and only a after prolonged time in the colon. By contrast, there was rapid degradation of chondroitin sulfate in the caecum.

3.4. In vitro degradation study of C-Chondroitin sulfate in caecum contents

Fig. 4 shows the determination of the retention time of different molecular weight CS and the chondroitin disaccharide *4S by Fast Performance Liquid Chromatography with UV detection at 205 nm enabling a calibration curve to be constructed. The molecular weights of the possible labelled degradation products found in the caecum contents were estimated from this calibration curve after detection with the Flow Scintillation Analyseer (Flo-One).

A peak at 24 min appeared after 90 min incubation. Reference to the calibration curve showed that this peak corresponded to a low molecular weight degradation product. The appearance of this product increased with time as shown in Fig. 5, which plots the area of this peak at the different incubation times. There was also a broad peak between 16 and 22 min probably corresponding to the initial ACS and/or ist first degradation products. The ratio between this two peaks changed during the incubation time, the peak corresponding to the dissacharide increasing with the incubation time. After 24 h incubation of CS in the caecum contents, the low molecular weight product peak was in the majority in the caecum contents; the initial CS being being probably totally degraded. These results were in good agreement with the results which showed a continous degradation of CS in the caecum contents with the colorimetric method using dimethylmethylene blue. The results obtained after incubation with 1 ml caecum were similar to the results obtained after incubation in 3 ml caecum contents, but the appearance of the low molecular weight degradation product peak was slower, confirming that the rate of ACS degradation was proportional to the quantity of caecum contents.

3.5. In vitro determination of absorption of C-Chondroitin sulfate and/or ist metabolities across the rat intestine, colon and caecum

Fig. 6 shows that there was a continous release of radioactivity into the medium from the caecum, the colon or the small intestine sacs containing CS. With the caecum, after 3.5 h approximately 47 % of the starting radioactivity of the CS had crossed the tissue and been released into the incubation medium. In both cases of colon and small intestine sac, after 4 h incubation respectively 17% and 19% of the starting activity of CS had been released into the incubation medium. Fig. 7 shows the caecum results with Sephadex G-25 column analysis of the incubation medium at T15 and T60 min. In the both cases, two peaks of radioactive material present. The first peak of radioactivity eluted at the same time as the reference labelled CS solution and the second peak corresponding to a degradation product of CS. With the aim of identifying the molecular weight of the transported T210 min material, 20 ul of incubation medium samples removed T30, T45, T75, T90, T120 and were injected in a FPLC chromotographic system with UV detection and Flow Scintillation Analyser (Fol-Oe). A peak at 25 min appeared after 30 min incubation in the medium. Reference to the calibration curve with UV detection showed that this peak corresponded to a low molecular weight CS degradation product. Fig. 8 shows that the peak increased with the incubation time. There was also a broad peak between 16 and 22 min probably corresponding to the initial CS and/or ist first degradation products. The ratio between these two peaks changed during the incubation time: the peak corresponding to the disaccharide increasing with the incubation time. There was an increasing production of disaccharide in the caecum with the incubation time, which crossed the wall of the caecum. The initial CS or ist first degradation products also crossed the caecum mucosa, but given the continous degradation of CS in the caecum, their absorption was decreased with the incubation time. Fig. 9 and Fig. 10 show the colon and the intestine results with Sephadex G-25 column analysis of the incubation medium at T15 and T60 min. In both cases, there was only one peak or radioactive material present, eluting at the same time as the reference CS. With this technique, no degradation of CS was observed. To confirm these results, 20 ul differents time samples of the incubation medium were analysed with the FPLC method. In both cases of absorption of C-CS across the colon and the intestine barrier, after 4 h incubation, no peak corresponding to a low molecular weight degradation product was detected in the incubation medium. Only a broad peak corresponding to the initial labelled CS was observed. The area of this peak increased with the incubation time.

4. Discussion

in the in vitro degradation study of CS in the rat gastrointestinal tract, using the spectrophotometric method, no degradation of CS was observed in rat stomach and intestine tissue or contents. The results showed an appreciable degradation in the caecum contents, ant to a slight extent, and only after a prolonged time, in the colon. In the caecum, the rate of CS degradation was proportional to the quality of caecum contents. The colorimetric dimethylmethylene blue method described by Ferndale et al., (1986) was validated in several gastrointestinal media t different pH, and the method was tested by using a commercial preparation of chondroitinase AC. Chondroitinase AC splits the glycosaminoglycan chain with the release of disaccharide units. The pattern of degradation showed that the rate was not influenced by the molecular weight of the CS used. However, the lower limit of quantifikation obtained was 5 ug/ml which is twice the limit of quantification in plasma (2 ug/ml) described by original the authors. With the aim of better characterizing this degradation in caecum, labelled (Acetyl-1-C) CS was used. The results of in vitro degradation study of C-CS in caecum contents, were in good agreement with the colorimetric method using dimethylmethylene blue. The chromographic analysis showed that CS could be degraded down to disaccharides. These results support the observations of Rubinstein et al., (1992, 1995) who showed the presence in rat caceum of chondroitin induced degradative enzymes, probably of bacterial origin. Salyers, (1979); Salyers and Kotarski, 1980, Salyers and O`Brien, 1980 showed that human colonic anaerobic bacterioides (Bacterioides thetaiotaomicron and B. Ovantus) could use CS as a substrate. These microorganisms utilize CS as an energy source (Salyers, 1979). They metabolize CS through the sequential action of three enzymes: a periplasmatic CS-chondroitinase first releases unsaturated disaccharides from the CS polymer. The disaccharide is then sulfated by an intracellular sulfatase, followed by the action of an intracellular glucuronidase, which splits the unsulfated disaccharide into the monomers. *4,5-glucuronic acid and N-acetyl galactosamine. The monomers are then used as an energy source by Bacteriodes ssp. No evidence was found for the production of CS fragments larger then the disaccharide (Salyers and Kotarski, 1980). Our in vitro determination of the absorption of C-chondroitin sulfate along the gastrointestinal tract showed that the radioactivity could cross small intestinal, colon and caecum epithelia. The The greatest transport was observed across caecum. The chromotographie analysis on Sephadex G-25 column of material transported revealed that two products are crossing the caecum to a degradation product. The FPLC analysis confirmed these results and showed that the degradation product is down to the disaccharide. In the case of the transport across both small intestine and colon, experiments with a Sephadex G-25 column and the FPLC analsis showed that only the initial CS crossed the epithelium after 4 h incubation. Simple in vitro screening methods to quantify intestinal uptake can be valuable tools in any development program. Some years ago, Bridges et al., (1978) turned to using a preparation of sac of intestine, but incubated in a tissue in the simple salt media (Levine et al., 1970). The intestine is a very active tissue metabolically and abundant energy sources are required to maintain full viability and active funktion. The tissue culture medium contains amino acids, vitamins, cofactors etc, and notably glutamine, which is a major energy source for intestinal tissue, as well as glucose, another energy source. Thus the viability of the tissue is well maintained during the incubation, and the method has clearly demonstrated the ability of the intestinal mucosa to transport macromolecules (Pato et al., 1994; Naisbett and Woodley, 1994). There are no other reports of in vitro absorption studies with CS. The results obtained by several authors after oral administration of CS are much debated (Lualdi, 1993; Baici and Wagenhauser, 1993). Some observed no or only negligable absorption (Baici et al., 1992: Kondor and Kawiak, 1977). By contrast, other researchers have described a marked absorption of CS by the oral route (Dawes et al., 1989; Conte et al., 1991a, 1991b, 1995; Palmieri et al., 1990). Some of the differences found by the various authors can probably be ascribed to the animal species and to the method for the determination of the absorbed material. Dawes et al., (1989) showed absorption of dermatan sulfate, a high molecular weight glycosaminoglycan, after oral administration in humans. Conte et al., (1991b; 1995) using respectively non-labelled and H-labeled chondroistin sulfate demonstrated in man an intestinal absorption of a fraction of exogenous chondroitin sulfate together with high molecular weight polysaccharides resulting from a partial depolymerization and/or desulfation of the drug. An even more consistent fraction was absorbed as a low molecular weight compound resulting from a more marked depolymerization and desulfation of the drug. Palmieri et al., (1990) and Conte et al., (1991a), showed high absorption of radioactivity from H-CS, after giving the product orally to rats and dogs. Considering the total radioactivity present in the plasma, urine and tissue, more then 70 % of the orally administered radioactivity appeared to be obsorbed. Analysis showed that the radioactivity material present in the physiological fluid was heterogenous with the respect to molecular weight (CS, poly-oligo saccharides, monomers). However the labeling method used, by reduction with H-borohydride, where only terminal sugar was labelled, is open to criticism. This labelling was not totally representative of the intact molecule and measuring the radioactivity of absorbed material was actually following the metabolic fate of only the last residue in the polymer chain. Moreover, this kind of labelling can give problems of tritiated water by exchange. Indeed, effect previous experiments using H-ACS, in vitro and in vivo showed that more than 90 % of the radioactivity recovered was tritiated water (IRPF, internal report: unpublished results). Therefore, for the current study, C-acetyl CS was synthesised on the basis of a deacetylation process (Nadkarni et al., 1996) and reacetylation using C-acetic anhydride. This means that the label is chemically stable and is distributed along the whole of the polymer chain, and as it is a carbon label, exchanges with water are eliminated, thus ensuring accurate measurements of CS and ist degradation products. By contrasts, following oral treatment of human volunteers with chondroitin sulfate, and analysis of serum glycosaminoglycans with the dimethylmethylene blue colorimetric method, Baici et al., (1992) considered that CS administrated orally to man was neither absorbed in an intact form, nor as a sulfated oligosaccharide fragment with a molecular mass exceeding about 1500. However, the possibility that low MW desulfated oligomers and monomers may be produced and absorbed could not be ruled out on the basis of that study. Several attempt at the optimisation of CS assays in the presence of rat plasma using dimethymethylene blue, were carried out in our labaratory, but it was not possible to reduce the sensitivity limit of 2 ug/ml obtained by Ferndale et al., (1986). Preliminary experiments showed a poor reproducibility of the method, problems with the quality controls and a limit of quantification of about 20 ug/ml. None of the parameters (linearity, accuracy and precision) attained to the accepted validation criteria of our labaratory. Konador and Kawiak, (1977), observed no increase in the level of the bloodplasma glycosaminoglycans, in terms of uronic acid, after intragrastic introduction of CS to rabbits at the dose of 5g/kg. They were able to measure endogenous concentration of CS in plasma of about 7 ug/ml. An improvement method was developed and optimized in our labaratory with the aim of measuring CS in rat plasma. It consisted of a colorimetric determination of uronic acid, following extraction of CS on a strong anion exchange (SAX column), based on the methods described by Conte et al., (1991b), Blumenkrantz and Absoe-Hansen, (1997) and Calatroni and Vinci, (1987). Preliminary experiments again showed a poor reproducibility of the method, problems with quality controls and a limit of quantification of about 20 ug/ml and again none of the parameters (linearity, accuracy and precision) reached the accepted validation criteria of our laboratory. Moreover this method is not specific for chondroitin sulfate because uronic acids are also present in other glycosaminoglycans such as hyaluronic acid, heparin, or dermatan sulfate, exculting the use of this method for measuring specifically absorptin of CS. In general, macromolecules are very poorly absorbed. Baici et al., (1992), considered that the mammalian intestinal epithelium is a highly effective barrier which hinders the diffusion of charged compounds or those having a high molecular mass. However, CS is an anionic macromolecule, and Pato et al., (1994) showed that in vitro, anionic polymers were more likely to cross the small intestine epithelium than neutral or cationic polymers, albeit in small quantities. Recently, Wiwattanapatapee et al., (1998) have shown that anionic dendrimers are able to cross the small intestine epithelium in vitro. Our study clearly shows that CS can be degraded in vitro by intestinal microorganisms and that the released degradation products can traverse the intestinal wall. Evidence was also obtained showing that small amounts of the parent molecule were able to cross the epithelium in all regions of the intestine, though it should be noted that the concentration of Cs inside the sacs was relatively high (mg/ml). These in vitro results will need to be complemented by an in vivo study in rat using the same labeled (acetyl-1-C)-CS, to confirm the passage of radioactivity across the gastrointestinal tract. Given that CS in naturally occuring dietary polymer, it might be imagined that it would be degraded in the in the gastrointestinal tract.

Acknowledgements: Our thanks to S. Delassen, for providing the Superose column and the FPLC technique and to Plantes et Industrie (Gaillac) for the synthesis of various CS products.

Fig.1. Degradation of 3 different molecular weight CS (100 ug/ml) after incubation with 1 ml Chondroitinase (activity 0.02U) in atotal volume of 5 ml PBS (pH 7.9). 600 ul were removed at 0 and every 20 min up to 2 and at 5 h, and CS assayed with the dimethylmethylenen blue colorimetric method. (n) CS 10 kDa; (+) CS 15 kDa, (s) CS 18 kDa.

Fig.2. Degradation of CS 10 kDa (100 ug/ml) after incubation with 3 ml colon contents (pH 7.0) in a total volume of 5 ml. 600 ul were removed at 0 and every 20 min up to 2 h and at 4, 6, 8 and 23 h, and CS assayed with the dimethylmethylene blue colorimetric method. (*) CS 10 kDa in colon contents, (n)control: CS 10 kDa in PBS (pH 7.0).

Fig.3. Degradation of CS 17 kDa (100 ug/ml) after incubation in caecum contents at 37 °C. 600 ul were removed every 30 min and CS assayed with the colorimetric method using dimethylmethylene blue. (n) after addition of 3 ml caecum contents. (u) after addition of 1 ml caecum contents in total volume of 5 ml.

Fig.4. Calibration curve: retention time of different molecular weight CS and chondroitin disaccharide *4S by Fast Performance Liquid Chromatography with UV detection at 205 nm.

Fig.5. Appearance of a low molecular weight product after incubation of labelled CS in the colon contents. Injection of 20 ul incubation medium samples onto Sepharose 12 column and with radioactivity detection (Flo- One).

Fig.6. Percentage of the administared radioactivity released into the medium after incubation of C-CS in caecum, colon and small intestine. Rat caecum, colon and intestine sacs were filled with 1 or 2 ml of C-chondroitin sulfate (33 mg/ml) and incubated in 20 ml oxygenated TC-199 medium at 37 °C. 500 ul of the incubation medium were removed at different incubation times and counted for the radioactivity. (l) caecum; (s), small intestine; (X), colon.

Fig.7. Separation with a Sephadex G-25 column of CS and ist degradation products after crossing the caecum sac. 500 ul of incubation medium were diluted with 500 ul PBS, loaded onto the column and eluted with 20 x 1 ml of PBS. Each fraction was counted. (j) T15 min (l) T60 min. Ð CS=elution of starting material.

Fig.8. Appearance of a low molecular weight product in the incubation medium in vitro study after crossing the caecum sac. Injection of 20 ul incubation medium samples onto Sepharose 12 column and with radioactivity detection (Flo-One).

Fig.9. Separation with a Sephadex G-25 column of CS and ist degradation products after crossing the colon sac. 500 ul of incubation medium were diluted with 500 ul PBS, loaded onto the column and eluted with 20 x 1 ml of PBS. Each fraction was counted. (j) T15 min (l) T60 min. Ð CS=elution of starting material.

Fig.10. Separation with a Sephadex G-25 column of CS and ist degradation products after crossing the small intestine sac. 500 ul of incubation medium were diluted with 500 ul PBS, loaded onto the column and eluted with 20 x 1 ml of PBS. Each fraction was counted. (j) T15 min (l) T60 min. Ð CS=elution of starting material.

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