2 Laboratoire de Recherche sur la Croissance, la Réparation et la Régénération Tissulaires (CRRET), CNRS FRE-2412, Université Paris 12-Val de Marne, 61 Avenue du Général de Gaulle, 94010 Créteil cedex, France
3 Société OTR3 SARL, 33, avenue Pierre Brossolette, 94000 Créteil cedex, France
Received on February 21, 2003; revised on April 14, 2003; accepted on May 8, 2003
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Abstract |
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Key words: cell culture / 1,9-dimethylmethylene blue / glycosaminoglycans / skeletal muscle / skin
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Introduction |
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Several spectrophotometric assays are available in literature to measure levels of sulfated GAGs in biological samples. Many of these assays are based on changes in the absorption spectrum of the dye 1,9-dimethylmethylene blue (DMMB) when bound to GAGs (Whitley et al., 1989; Chandrasekhar et al., 1987
; Farndale et al., 1982
). Although recent studies have demonstrated the efficiency of DMMB over other assays (toluidine blue, Alcian blue, etc.), we have found that reported DMMB methods suffer from multiple drawbacks. The reagent used is unstable, interferences with DNA or other negatively charged molecules seriously modify the response, and, in addition, the GAGdye complex is not stable in solution, and GAG measurements are not accurate because of this instability. Indeed, variations of the time between sample measurements may modify results (Farndale et al., 1982
; Panin et al., 1986
; Stone et al., 1994
). Furthermore, accuracy, precision, and linearity are poor and even unacceptable at levels of less than 5 µg/ml of GAGs.
In this article we report a modified procedure for the use of DMMB that overcomes these problems. Our assay is based on the generation of an insoluble precipitate of the GAGDMMB complex. We present conditions allowing the formation of a solid GAGdye complex that was stable for at least 1 h, which allows its isolation from other sample constituents, including other biological molecules and excess of DMMB. Linearity, precision, and accuracy of the assay were validated at concentrations of GAGs ranging from 0.025 to 10 µg/ml in biological samples. We have applied this method to the study of the total sulfated GAG content in cultured cells and tissue extracts. The method was also used to discriminate the contents of heparan sulfate (HS) from chondroitin sulfate (CS) in these biological materials after nitrous acid treatment. Our results showed relevant modifications in the amounts of total sulfated GAG and in their composition during skeletal muscle repair, in differentiating myogenic cell cultures, and in ulcerated skin.
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Results and discussion |
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To overcome these drawbacks, we decided to isolate the GAGDMMB complex from soluble materials, including DMMB excess. Therefore we developed conditions that increased complexation of dye to sulfated GAGs by using a DMMB solution prepared in a formate buffer pH 3.0 containing 5% ethanol and 0.2 M guanidine hydrochloride (GuHCl). The use of ethanol at low pH allowed exhaustive complex precipitation within 30 min when vigorous shaking was applied during this time. The presence of GuHCl has already been reported by others to avoid interactions with negatively charged macromolecules other than GAG during the complexation process. The GAGDMMB complex was obtained as a stable pellet after centrifugation of the treated sample, and its isolation was particularly easy by simple decantation of soluble materials.
The next step consisted of the dissociation of the complex to render DMMB soluble. The UV absorption of the decomplexated DMMB is then proportional to the GAG amount complexed from the original sample. This step was achieved by using a decomplexation 4 M GuHCl solution at pH 6.8 containing 10% propan-1-ol. Dissociation was favored at neutral pH because the cationic state of the dye, which induced complexation to polyanionic GAGs, was highly reduced at this pH. The high GuHCl concentration allowed GAG solubility favoring its dissociation from the complex. We included 10% porpan-1-ol in the decomplexation solution to enhance DMMB signal at 656 nm. This wavelength became the most sensitive to changes in dye concentration and was thus selected for quantification after decomplexation (Figure 1a and 1b). As an example, when different amounts of CS were treated by the DMMB complexation/decomplexation protocol, two peaks of absorption, at about 610 nm and 656 nm, were detected in samples (Figure 1d). The absorbance of the highest peak (656 nm) was proportional to the amount of GAG present in the original sample (see method validation). It must be kept in mind that in some classical methods, GAGs are evaluated by ratio determinations of the absorbance at about 525 nm (complex signal) versus 595 or 652 nm (noncomplexed DMMB signal). When vigorous agitation and complex isolation steps were omitted from our complexation/decomplexation protocol, the UV spectrum of the complexed sample showed an additional peak (shoulder) at 525 nm resulting from the absorption of the GAG/dye complex (Figure 1c).
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Condition for minimizing DNADMMB interaction in DNA-containing samples
Biological samples are supposed to contain DNA. Samples were presently treated by proteinase K before the complexation step as described in Materials and methods. This treatment dissociated GAGs from protein cores and at the same time liberated DNA from nuclear proteins. It has been reported that, like sulfated GAGs, DNA readily forms complexes with the dye, absorbing at nearly the same wavelength as the GAGdye complex. Some authors suggested the use of GuHCl (up to 0.24 M) in the complexation solution to decrease these DNA/dye interactions (Chandrasekhar et al., 1987). Accordingly, our results show that low GuHCl concentration (0.08 M) did not avoid interaction of DNA with DMMB and thus disturbed GAG determination (Figure 2). Another way to overcome DNAdye interaction was to eliminate or highly reduce DNA by filtration of samples after proteinase K treatment. Filtration of the preheated proteinase Ktreated sample proved to eliminate most of the interfering DNA, which was no longer detected by complexation with DMMB even in the absence of GuHCl. Our results show that GuHCl concentration may be increased to 0.20 M without affecting GAGDMMB complexation (Figure 2) as recommended by Chandrasekhar et al. (1987)
. The proteinase K treatment and the filtration procedure did not affect CS concentration of standard solutions.
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Linearity was ascertained by calibration curves. Because extraction recoveries from GAG-spiked tissue extracts and extraction recoveries from standard solutions were comparable, calibration curves were included into each assay performed with appropriate diluted standard solutions. Figure 3 shows the linear regression curves obtained by plotting the absorbance values of DMMB in solution after decomplexation from CS versus its nominal ng or µg/ml concentration. For submicrogram quantities of GAGs (0.0251.1 µg/ml), the calibration curve (a) was used (Figure 3a). For samples containing microgram quantities of GAG (0.55 µg/ml), the calibration curve (b) was applied (Figure 3b). Linearity at both low and high quantities of GAGs was excellent (R2 = 0.9919 and R2 = 0.9931, respectively) and was demonstrated up to 10 µg/ml (data not shown).
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Analysis of sulfated GAGs in different biological samples
The DMMB complexation/decomplexation protocol allowed quantification of sulfated GAGs in cell and in tissue extracts. Tables II and III show amounts of GAGs in myogenic cell cultures and in intact or injured tissues. The method also allowed GAG quantification after nitrous acid treatment, which discriminated between HS and CS in the biological samples. Standard deviations were found to be optimal (at most 13%) when three determinations were performed on the same tissue extract. Results have shown that total sulfated GAG amount increased when myoblasts differentiated into myotubes. During in vivo muscle regeneration, we found that total sulfated GAG content highly decreased during the myolysis phase (day 1). It increased from day 3 after the crush, when newly formed myotubes appeared. This increase might in part be due to GAG synthesis by myogenic cells, which regenerate myofibers. Indeed, GAG produced by myoblasts increased on myogenic differentiation in vitro (Table II).
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In the skin, although the proportion of sulfated GAGs (HS and CS) was not changed, a decrease in the amount of total sulfated GAG was observed when the skin reached a maximum of ulceration. To our knowledge, there is no available data on GAG content in this model. However, GAG content has been reported to be decreased in the skin of diabetic rats (Cechowska-Pasko et al., 1999).
Altogether, we believe that the present method based on the isolation of sulfated GAGDMMB complex will contribute to more efficient studies concerning the physiological evolution of the different GAGs in biological processes, particularly during tissue repair. We are currently working in this area.
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Materials and methods |
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Preparation of DMMB solution
Preparation of the DMMB solution was derived from that reported by Farndale et al. (1982) with some modifications. In brief, 16 mg DMMB were dissolved in 25 ml ethanol and filtered through filter paper. One hundred milliliters of 1 M GuHCl, 1g sodium formate, and 1 ml 98% formic acid were then added to the DMMB ethanolic solution, and the final volume was completed to 500 ml with distilled water. This solution was unstable and needed to be rapidly diluted (1:1) with the formate solution prepared as described but without DMMB. This DMMB solution was stable for at least up to 4 months when stored at room temperature in the darkness.
DMMB decomplexation solution
A 50 mM sodium acetate solution buffer (pH 6.8) containing 10% propan-1-ol was prepared and used to solubilize powdered GuHCl to a final concentration of 4 M. This solution was stable for at least 4 months at room temperature.
Biological materials
Cell culture
Mouse myoblast cell line C2.7 was maintained as subconfluent monolayers in Dulbecco modified Eagle medium (DMEM) containing 1 g/L glucose and 4 mM L-glutamine supplemented with 20% fetal bovine serum (FBS), 100 U/ml penicillin, and 10 µg/ml streptomycin. Cell cultures were incubated at 37°C in 12% CO2. Samples of proliferating cells (myoblasts) were taken at day 3 after plating. To induce differentiation, the medium was changed at day 4 after plating to DMEM supplemented with 0.25% FBS and 0.25% horse serum. Samples of differentiated cells (referred to as myotubes) were taken 24 h after.
Muscle
Regenerating skeletal muscle (extensor digitorum longus muscle) of adult male Wistar rats (age 2 months) were obtained after crush according to Bassaglia and Gautron (1995). The muscle crushing protocol was performed after the rats had been anesthetized with pentobarbital (0.2 ml/100 g). At days 1, 3, and 9 after crush, animals were terminally anesthetized with pentobarbital, and regenerating muscles were removed and weighed. Intact muscles were used as a control. The muscles were frozen in liquid nitrogen and stored at -80°C until use.
Skin
Swiss mice were depilated with depilatory cream 2 days before ulcer generation by intradermal injection of doxorubicin in the back (Balsari et al., 1989; Rudolph et al., 1979
). Mouse skin was at its maximum of ulceration 11 days after injection. Healthy or ulcerated skin fragments were then taken on the back of each animal, which were terminally anesthetized with pentobarbital. Samples were stored at -80°C until use.
Housing of animals and anesthesia were performed following the guidelines established by the Institutional Animal Welfare with the European guide for care and use of laboratory animals.
Preparation of biological samples for the DMMB assay
Myogenic cell cultures, skeletal muscles, and skin were used as biological materials. Whatever its origin, material was digested in a solution of 50 µg/ml proteinase K in 100 mM K2HPO4 pH 8.0 at 56°C overnight. Proteinase K was then inactivated by heating the preparation 10 min at 90°C (Calabro et al., 2000). After centrifugation, digested tissue was filtered through an Ultrafree filter to eliminate DNA and tissue debris from the extract. This preparation was used for sulfated GAG quantification. The amount of DNA in samples was determined by diaminophenyl indole (DAPI) assay using salmon sperm DNA as standard (Brunk et al., 1979
).
Spectroscopic determination of sulfated GAG: GAGDMMB complexation/decomplexation
The content of sulfated GAGs was determined using the DMMB solution as follows. 1 ml working DMMB solution was added to 100 µl proteinase Ktreated sample, and the mixture was vigorously vortexed for 30 min to promote complete complexation of the GAG with DMMB. The insoluble GAGDMMB complex was then separated from the soluble materials, including DMMB excess, by centrifugation (12,000 x g, 10 min). The supernatant was discarded, and the pellet was dissolved with the decomplexation solution. The added volume of this solution was adjusted according to the quantity of GAGs. For samples containing GAGs at the microgram level, 1 ml decomplexation solution was added. For lower quantities (<1.0 µg) 500 µl was added. Decomplexation was achieved by shaking the mixture for 30 min. Absorbance was measured at 656 nm. Sulfated GAG quantities in biological samples were determined by comparison with a calibration curve of CS solutions used as standard and treated as described. For submicrogram and microgram quantities of GAGs, calibration curves were used from 0.025 to 1 µg/ml and from 1.0 to 5 µg/ml of CS, respectively. HS was also used as standard, giving similar curves than CS (less than 5% difference).
Quantification of HS
HS was eliminated from the original sample according to Bosworth and Scott (1994) with some modifications. In brief, 100 µl proteinase Ktreated sample was mixed with 100 µl sodium nitrite (5%) and 100 µl acetic acid (33%). Samples were gently shaken and kept at room temperature for 1 h. To stop the reaction, 100 µl ammonium sulfamate (12.5%) was added, and the mixture was shaken for a further 5 min. Remaining sulfated GAGs were determined in 100 µl of this nitrous acid reaction mixture by following the DMMB protocol as described. The GAG remaining in the sample represented O-sulfated GAGs, including CS. The N-sulfated GAGs (HS) content was then calculated as the difference between the total GAGs and the O-sulfated GAGs in each sample. A calibration curve was constructed as shown by preparing mixtures of known amounts of CS treated in the same way.
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Acknowledgements |
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1 To whom correspondence should be addressed; e-mail: papy{at}univ-paris12.fr
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Abbreviations |
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References |
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