Glycosaminoglycans of the rat incisor pulp

446

BIOCHIMICA ET BIOPHYSICA* ACTA

BBA 26940

GLYCOSAMINOGLYCANS OF T H E RAT INCISOR PULP

ANDERS LINDE

Department of Histology, University of G6teborg, Fack, S- 4 o o 33, G6teborg 33 (Sweden) (Received May 29th, 1972)

SUMMARY

I. The continuously growing, dentinogenically active rat incisor is a suitable model for the biochemical as well as morphological study of the process of hard tissue deposition. Thus the glycosaminoglyeans (acid mucopolysaccharides) of the rat incisor dental pulp have been isolated, separated, quantitated and characterized by means of a cetylpyridinium chloride (CPC)-cellulose micro-column technique. 2. The total amount of hexosamine was found to be 4.8 Fg/mg tissue dry weight. The dry weight of this loose connective tissue was lO% of the tissue wet weight. 3. Four hexosamine-containing fractions were isolated from the CPC-cellulose columns: one glycoprotein- and keratan sulphate-containing fraction, one byaluronate fraction, one fraction containing predominantly 4-sulphated chondroitin sulphate, and one fraction with predominantly 6-sulphated chondroitin sulphate. In maxillary incisor dental pulps the combined chondroitin sulphate fractions accounted for 75 %, and the hyaluronate fraction for I2%, of the total hexosamine. This is consistent with earlier results obtained using electrophoretic methods. 4. By means of epichlorohydrin triethanolamine (ECTEOLA)-cellulose chromatography, a minor keratan sulphate fraction could b~ found. 5. The hexosamines in the hyaluronate and combined chrondroitin sulphate fractions were separated on Dowex 5oW-X8 columns. In the former only glucosamine was found. In the latter galactosamine dominated, but a small amount of glucosamine (less than lO%) was als~ found. 6. A "neutral MgC12 elution profile" indicated that the molecular weight range of the chondroitin sulphates was 4" Io4-7 . lO4. 7. An "acid MgC12 elution profile" for the combined chondroitin sulphate fractions was also obtained. This suggested a variation in the sulphate content of the chondroitin sulphate.

INTRODUCTION

It has been known for several years that there are large amounts, and a rapid synthesis, of glycosaminoglycans (acid mucopolysaccharides) at or near calcification Abbreviations: CPC, cetylpyridinium chloride; ECTEOLA, epichlorohydrin triethanolamine.

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RAT INCISOR GLYCOSAMINOGLYCANS

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loci. Thus, several histochemical investigations have shown the presence of these polysaccharides in the ground substance of the loose connective tissue of dental pulps, and in the odontoblast--predentine layer, from several species 1-~. In two previous investigations~°, n the glycosaminoglycans in dental pulps from different species, and from teeth with different degrees of mineralization activity, have been separated and semi-quantitated by biochemical methods. When the rate of dentinogenesis is high, the chondroitin sulphate fraction accounts for most of the glycosaminoglycans present. The content of this glycosaminoglycan, as well as the total glycosaminoglycan amount, decreases considerably upon completion of hard tissue deposition. On the contrary, the relative amount of hyaluronate increases. As the dental pulp of the continuously growing rat incisor is a suitable model for the biochemical study of loose connective tissue and of the calcification process, and is used as such in this laboratory, it was felt that a more detailed investigation of its pulpal glycosaminoglycan content by means of more sophisticated methods was necessary. MATERIALS AND METHODS

Animals

Dental pulps from a total of 2oo male white rats (Sprague-Dawley strain; body weight 20o-230 g; Anticimex, Sollentuna) were used. The animals were anaesthetized with ether and bled to death. In preliminary experiments the maxillary incisor pulps from 32 rats were dissected out by a technique previously described~L and these were combined in groups of four. Subsequently, pulpal connective tissue from both maxillary and mandibular incisors, obtained by the same technique, was pooled from 30-5 ° rats at a time. All samples were weighed wet. Glycosaminoglycan isolation

The minced tissue was dehydrated for 72 h on a shaker with several changes of acetone, and lipids were extracted in the same way for 48 h with chloroform-methanol ( I : I , v/v). The material was dried under reduced pressure and weighed. For liberation of the glycosaminoglycans from the tissue, digestion with papain TM (twice crystallized; Sigma Chemical Co) was performed for 8 h on a shaking water bath at 65 °C. In the first series, i ml of digestion buffer was used per sample. For the pooled material, 4 ° ml digestion buffer were used per g dry weight of the sample. After digestion, trichloroacetic acid was added to a concentration of IO%, and after precipitation and centrifugation at 4 o o o x g for 30 rain, the supernatant was dialysed agaiflst deionized water for 24 h. To the dialysate were added 4 vol. of 95 % ethanol containing 1% potassium acetate (w/v) and 1% acetic acid (v/v). The glycosaminoglycans were allowed to precipitate for 48 h and were spun down at 4000 x g for I h. The pellet was washed once with ethanol, and the material was dried under reduced pressure overnight. Hexosamine determination

The glycosaminoglycan amount was measured as hexosamine by the ElsonMorgan reaction as described by Antonopoulos et al. 14. The samples were hydrolysed in 6 M HC1 in stoppered Quickfit tubes for 8 h in a boiling water bath. The colour Biochim. Biophys. Acta, 279 (1972) 446.-455

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was measured in a standard spectrophotometer (PMQ I I ; Carl Zeiss) using cells with a 5-cm light path (MT 4; Carl Zeiss). Glucosamine.HC1 (Sigma Chem. Co.) was used as a standard.

Micro-column glycos~minoglycanfractionation Separation of different glycosaminoglycans was performed on cellulose microcolumns with a length of 60 m m and an inner diameter of 3 ram. These were prepared with W h a t m a n CF I I cellulose 14 so that I ml of eluant ran through in 12-15 min. Before application of the sample the columns were washed with 5 ml 1% aqueous cetylpyridinium chloride (CPC)(w/v; Sigma Chem. Co). The sample was dissolved in 50 #1 distilled water. The different glycosaminoglycan fractions were successively eluted from the columns at 27 °C with I-ml portions of the solutions shown in Table II. I t should be noted that the columns were washed with a 0.05 % aqueous CPC solution after Fractions 3, 4 and 5. To the first two fractions I ml conc. HC1 was added. The MgCl~-containing fractions were mixed with 3 ml water and 0.5 ml 1% CPC, and the glycosaminoglycancetylpyridinium complexes were allowed to precipitate overnight and were centrifuged 4000 × g for I h. The supernatant was carefully decanted and discarded, and 0.5 ml 6 M HC1 was added to the precipitate. The propanol-methanol fraction was evaporated on a Rotavapor, after which 0.5 ml 6 M HC1 was added. The last fraction already containing 6 M HC1, and the others fractions, were hydrolysed for 8 h as above. The hydrolysates were completely dried over NaOH pellets in an exsiccator connected to a trap (cooled with ethanol-solid COs) and a vacuum pump 14. The glycosaminoglycan content of the fractions was measured as hexosamine as above. The washing solutions were always completely devoid of hexosamine.

Macro-column glycosaminoglycansfractionation In order to obtain enough material for the Dowex and epichlorohydrin triethanolamine (ECTEOLA) separations (vide infra), glycosaminoglycans from pooled dental pulps were separated on a cellulose column, 20 cm long and having an inner diameter of I cm. This was jacketed at 27 °C. Before application of the sample, the column was washed with 200 ml 1% CPC. The different fractions were eluted with 4o-ml portions of the following solutions: 1% CPC, 0.3 M NaC1 containing 0.05 % CPC, o.7 M MgCI~ containing 0.05% CPC, and 6 M HC1. The glycosaminoglycans were isolated by precipitation with 5 vol. ethanol as above. The 6 HC1 fraction was assayed for he×osamine content.

Hexosamine separation Hexosamine identification in the different fractions from both micro- and macro-cellulose columns was performed by the method of Gardel115, as applied on the micro-scale by Antonopoulos 1~. Glycosaminoglycan samples from pooled material were applied to CPC micro-columns and were eluted with i-ml portions of the solutions used for the macro-columns. The 6 M HC1 fractions were assayed for hexosamine content as a control for complete elution of the glycosaminoglycans. Several NaC1 fractions, as well as MgC12 fractions, had to be combined. The glycosaminoglycans were precipitated with ethanol, hydrolyzed in 6 M HC1 and evaporated as above. In the same way, glycosaminoglycans from the NaC1 and MgCI~ fractions from macrocolumns were hydrolyzed and evaporated. Biochim. Biophys. Acta, 279 (1972) 446-455

RAT INCISOR GLYCOSAMINOGLYCANS

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Dowex 5oW-X8 (200-400 mesh) resin was washed several times with 4 M HC1, and 32-cm columns, with an inner diameter of 3 mm, were prepared. The hydrolyzed glycosaminoglycans were dissolved in IOO/A distilled water and applied to the column, and the hexosamines were eluted with 0.3 M HC1 at a speed of 0.4-0.5 ml/h. Two fractions were taken every hour, and after evaporation the hexosamine content was determined by the Elson-Morgan method as above.

ECTEOLA separation As the 1% CPC fraction from the CPC-cellulose columns may contain both keratan sulphate and glycoproteins, an attempt was made to separate these on ECTEOLA-cellulose columns. ECTEOLA-cellulose (0.55-0.74 mequiv/g), prepared according to the method of Anseth and LaurenW, was used in 6o-mm columns with an inner diameter of 3 mm. Tile sample, dissolved in distilled water, was applied to the column; elution was carried out with I-ml portions of distilled water, 0.02 M HC1 and 6 M HC1, at a rate of 0.7-0.8 ml/h TM.Hexosamine content was measured as above.

Neutral MgCl, elution profile To obtain a picture of the molecular weight polydispersity of the chondroitin sulphate fraction, CPC micro-columns with pooled material were run as above but were eluted with I-ml portions of 1% CPC, 0.3 M NaC1 with 0.05% CPC, MgC1, solutions of increasing molarity all containing 0.05% CPC, and 6 M HC1 as a control. These were treated as above and assayed for hexosamine.

Acid MgCl, elution profile Similarly, glycosaminoglycans from pooled incisor pulps were applied to CPC micro-columns. These were eluted with I-ml portions of 10/0 CPC, 0.3 M NaC1 containing 0.05% CPC, MgC1, solutions of increasing molarity containing o.I M acetic acid and 0.05% CPC, and 6 M HC1.

Standards For standardization of the CPC-cellulose technique and the ECTEOLA-cellulose separation, the following glycosaminoglycan standards (generously donated by Dr L.-A. Fransson, University of Lund, and Dr L. Roddn, University of Chicago) were used: hyaluronate, chondroitin-4-sulphate , chondroitin-6-sulphate, dermatan sulphate, heparan sulphate from beef lung and keratan sulphate from human costal cartilage. For calibrating the Dowex separations, glucosamine-HC1 and galactosamine. HC1 (Sigma Chem. Co) were used. RESULTS

As described eariierlL the material analyzed consisted of the basal two-thirds of the rat incisor dental pulp, thereby avoiding the degenerative changes OCCUlTing in the incisal end. In Table I the average amount of tissue obtained per rat is shown. As can be seen, the dry weight accounts for only one-tenth of the wet weight. The total amount of glycosaminoglycan, measured as hexosamine, is also shown. Table II shows the results obtained from sixteen CPC-cellulose micro-columns. Maxillary incisor pulps from two rats were combined in each sample. The 10/0 CPC Biochim. Biophys. Acta, 279 (I972) 446-455

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fraction m a y contain both keratan sulphate and glycoproteins. Three other glycosaminoglycan fractions were obtained; these were identified as hyaluronate, and predominantly 4-sulphated and 6-sulphated chondroitin sulphate by the solvents in which they appeared, and by the identity of the hexosamines present (Table II). No dermatan sulphate or heparin could be found. The results of these separations are summarized in Fig. i. The results of the hexosamine characterization of different fractions by means of Dowex columns were the same whether micro-column fractions were combined or material from the cellulose macro-columns was used. In the "hyaluronate fraction" only glucosamine could be found. In the "chondroitin sulphate fraction" galactosamine was found to be the major hexosamine. However, when using the larger amounts of material from the macro-columns, a small but definite amount of glucosamine could be detected (Fig. 2). This was always less than lO% of the total hexosamine. From four ECTEOLA-cellulose columns separations a mean of 23% of the applied material appeared in the 6 M HC1 fraction. However, this value is somewhat uncertain, as the amount of material applied was small, and the variation between different glycosaminoglycan preparations was great. However, there does appear to be some keratan sulphate among the glycosaminoglycans of the rat incisor pulp. This is consistent with earlier electrophoretic results n. The results of six separations on CPC-columns, to obtain a "neutral MgCI~ elution profile", are described in Table I I I . No chondroitin sulphate appears in the MgC12 fractions until the concentration reaches 0.45 M. As before, the lack of hexosamines TABLE I TISSUE WEIGHTS

A N D A M O U N T OF H E X O S A M I N ] ~

Pooled dental p u l p s from rat maxillary and m a n d i b u l a r incisors. Average a m o u n t tissue obtained per r a t (wet wt) D r y w t per wet w t H e x o s a m i n e per wet w t H e x o s a m i n e per dry w t

35.6 mg 9-7% 0.48/~g/mg 4-9/zg/mg

TABLE II MICRO-COLUMN SEPARATION OF GLYCOSAMINOGLYCANSFROM MAXILLARY INCISOR PULPS A m o u n t of glycosaminoglycan, measured as hexosamine, recovered in different fractions from CPC-cellulose micro-columns.

Eluting solutions

Glycosaminoglycan content as hexosamine of total recovered =k S.D.

1% CPC 0. 3 M N a C l + o . o 5 % CPC 0.275 M MgC12+o.o5% CPC Propanol-methanol-acetic acid-water* + 0 . 4 % CPC o.75 M M g C l , + o . i M acetic a c i d + o . o 5 % CPC 0.75 M MgC12+o.o5% CPC 6 M HC1

I3 ± 5 12 ~- i

Total n u m b e r of columns A m o u n t of hexosamine recovered *

4o:2o: 1.5:38.5, b y vol.

Biochim. Biophys. Acta, 279 (1972) 446-455

Hexosamine identity

GIc-NH 2

65 + 6 Gal-NH 2 io :k 3

16 12 - - 22 #g

451

RAT INCISOR GLYCOSAMINOGLYCANS

I

2

4

3

5

6

7.

Fig. i. E l u t i o n p a t t e r n of t h e g l y c o s a m i n o g l y c a n s f r o m r a t m a x i l l a r y incisor pulps. M e a n v a l u e s ± S.D. of h e x o s a m i n e c o n t e n t (in % of t h e total) in t h e different fractions o b t a i n e d from s i x t e e n CPC-cellulose m i c r o - c o l u m n s (see T a b l e II).

~Jg 5;

I

1

35

l

, 40 /

number

Fig. 2. H e x o s a m i n e c o n t e n t in different 3 o - m i n fractions of a r e p r e s e n t a t i v e h e x o s a m i n e separation on D o w e x 5 o W - X 8 f r o m t h e c h o n d r o i t i n s u l p h a t e f r a c t i o n " . G a l a c t o s a m i n e (to t h e right) is t h e m a j o r c o n s t i t u e n t . Glucosamine, in t h i s case 8~o, is also f o u n d (left).

Biochim. Biophys. Acta, 279 (I972) 446-455

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in the 6 M HC1 indicates that there is total elution in the earlier fractions. The mean values for the different fractions are shown in Fig. 3. Table IV shows the results of six CPC-cellulose micro-column fractionations to obtain an "acid MgC12 elution profile". This profile is illustrated in Fig. 4. TABLE III MICRO-COLUMN SEPARATION FOR A "NEUTRAL MgCA2 ELUTION PROFILE" H e x o s a m i n e a m o u n t s in different f r a c t i o n s in /~g, a n d % of t h e t ot a l , from a s e p a r a t i o n on s i x CPC-cellulose c o l u m n s (A-F).

Eluting solutions

Hexosamine (tzg and %) A

1% CPC 0.3 M N a C l + o . o 5 % CPC 0.3 ° M MgCl~+o.o5°/0 CPC 0.35 M M g C l z + o . o 5 % CPC 0.40 M M g C l , + o . o 5 % CPC 0.45 M M g C 1 2 + o . o 5 % CPC 0.5 ° M MgC12+ 0.05°/0 CPC 0.55 M M g C 1 2 + o . o 5 % C P C 0.60 M M g C l , + o . o 5 % CPC 0.65 M MgC12+o.o5% CPC 0.70 M MgCI~+ 0.05% CPC 6 M HC1

B

C

D

E

F

4.2 2. 4

3° 15

4.0 1.8

27 12

3.8 2.1

29 16

4 .0 2.2

3° 16

4.1 1.9

31 14

3 .8 2.1

32 18

3.2 2. 4 1.8 0-5

22 17 13 4

3-3 2.6 2.1 i.i

22 17 14 7

2.9 2. 3 1. 5 0.4

22 18 12 3

3.3 2.1 i.i 0.6

25 i6 8 5

2.6 2. 4 1.6 0.9

19 18 12 7

2.1 2.1 i.i 0.6

18 18 io 5

A v e r a g e r e c o v e r y (%) R e c o v e r y r a n g e (%)

93 82-1o2

T A B L E IV MICRO-COLUMN SEPARATION FOR A "NEUTRAL MgCI~ ELUTION PROFILE" H e x o s a m i n e a m o u n t s in different f r a c t i o n s in t~g, a n d % of t h e t ot a l , from a s e p a r a t i o n on six CPCcellulose c o l u m n s (A-F).

Eluting solutions

Hexosamine (#g and %)

1% CPC o.3 M N a C l + o . o 5 ° / 0 CPC o.25 M M g C l , + o . i M acetic 0. 05% CPC o.3o M MgC12+o.i M acetic 0.05% CPC 0.35 M MgC12+o.I M acetic 0.05% CPC 0.40 M M g C l ~ + o . I M acetic o.050/0 CPC 0.45 M M g C l ~ + o . i M a c e t i c 0.050/0 CPC 0.50 M M g C l ~ + o . I M acetic 0.o5°/0 CPC 0.55 M M g C l ~ + o . I M acetic 0.05% CPC 0.60 M M g C l ~ + o . i M a c e t i c 0.05% CPC 0.65 M MgC12+o.I M acetic 0.05°/0 CPC o.7o M MgCI~ + o. I M acetic 0.05% CPC 6 M HC1 A v e r a g e r e c o v e r y (%) R e c o v e r y r a n g e (%)

5.o 1. 5

34 io

4.5 1.8

29 ii

4.7 1.6

32 ii

5 .o 1. 7

33 ii

4 .8 1.8

35 13

4.9 1.6

32 ii

1. 7

ii

1.8

12

1.6

ii

1.3

9

1.8

13

1.6

II

1. 5

io

1. 5

ii

0.8

5

A

B

C

D

E

F

acid+ acid+ acid+ acid+ acid+ 1.4

9

2.8

18

i.i

8

1. 5

io

1.2

8

1.6

ii

I.i

7

1. 3

io

1. 9

13

1. 5

IO

1. 7

1. 9

13

1.8

12

0. 5

4

1.8

I2

1.3

9

i.o

6

i.i

7

1-3

9

1.3

9

1.8

12

0. 9

6

0.9

6

0.9

6

1. 3

9

0.7

5

0.8

acid+ acid+ ii

acid+ acid+ acid +

Biochim. Biophys. Acta, 279 (1972) 446-455

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7-

1% CPC

0.3M NaCI

0.3

0.4

0.5 M MgCI2

0.6

J

0.7

6M HCl

Fig. 3. CPC-cellulose column elution profile ("neutral MgCI=elution profile") of the chondroitin sulphate fraction from pooled rat maxillary and mandibular incisor pulps. As a comparison the 1% CPC fraction and the 0. 3 M NaC1fraction are included. Mean values of hexosamine in different fractions from six columns in % of total recovered (see Table III).

1% CPC

0.3M NaCI

0.3

0.4 0.5 0.6 M IvlgCl2 4-0.1M acetic ocid

0.7

6M HCI

Fig. 4- CPC-cellulose column elution profile ("acid MgCI~elution profile") of the chondroitin sulphate fraction from pooled rat maxillary and mandibular incisor pulps. Mean values of hexosamine in different fractions from six columns in % of total recovered (see Table IV). DISCUSSION I n two earlier studies1°, 11 the glycosaminoglycans in dental pulps from teeth at different stages of development, and from different species, were examined. F r o m these and other investigations the rat incisor was shown to be a suitable model for the s t u d y of mineralization biochemistry. Thus, for example, the odontoblastic layer can be harvested for the purpose of enzyme assays b y biochemical methods 1.. As expected, the water content of the rat incisor pulp is very high, about 90%. The total hexosamine content of this tissue is quite consistent with t h a t of unerupted porcine tooth pulps ~°. However, the molar ratio of hexosamine to uronic acid (the latter obtained from Linde 11) is high in the rat pulp (i : o.7), and this m a y be explained b y the considerable a m o u n t of hexosamine present in the 1% CPC fraction, which contains virtually no uronic acid. The separation of glycosaminoglycans b y means of electrophoresis, which has been done earlier for dental pulp glycosaminoglycans from different species n, is a rapid and reliable method. However, the identification of the different glycosaminoBiochim, Biophys. Acta, 279 (1972) 446-455

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glycans is based on similarities in migration to reference samples. Glycoproteins do not take the Alcian blue stain, and quantification of keratan sulphate is doubtful. No subdivision of the chondroitin sulphate fraction is effected, and, furthermore, only semi-quantitative results are obtainable. The CPC-cellulose micro-column technique of Antonopoulos et al. 14, with modificationstL seems to be the most exact and reproducible method available, and is widely used for the separation and quantitation of small amounts of glycosaminoglycans from tissue samples. By the hexosamine characterization on Dowex ion-exchange resin le, the identification of the glycosaminoglycans present is made much more reliable. From Table II it can be seen that the variation in the glycosaminoglycan fractions eluted from sixteen CPC micro-columns is very slight from column to column. The variation is greatest in the 1% CPC fraction. No heparan sulphate or dermatan sulphate was detected, although they were found in porcine pulps 10. However, minor quantities may be present, as the error of the method increases with smaller amounts of hexosamine. As hybrid forms of chondroitin sulphate with both 4- and 6-sulphation exist, the conclusion that the chondroitin sulphate fractions obtained consist only of chondroitin-4-sulphate or chondroitin-6-sulphate, respectively, cannot be drawn. Judging from infrared spectra, both 4- and 6-sulphated chondroitin sulphate are present in porcine dental pulps 1°.The contamination by glucosamine of the chondroitin sulphate fraction could be due to"impurities" in the chondroitin sulphate chains, or to heparan sulphate that might appear in this fraction. The amount of material recovered in the 1% CPC fraction was less when using the samples from maxillary incisors (Fig. I) than when pooled pulps from both maxillary and mandibular incisors were used. In addition to the difference in tissue material, a more effective digestion and dialysis in the former case may be the reason for this. The present glycosaminoglycan pattern in the rat incisor pulp (Fig. I) is very similar to that obtained by electrophoresis n. Chondroitin sulphate is the main fraction, and its predominance is even more pronounced when it is quantitated as hexosamine. When the material included tissue samples from mandibular incisors (Figs 3 and 4), the hyaluronate amount was slightly higher relative to the chondroitin sulphate fraction. Thus, with this kind of material, cellulose acetate membrane electrophoresis in copper (II) acetate buffer at pH 3.6 is a valuable separation technique, if one considers the drawbacks mentioned above. Laurent and Scott 2° showed that there exists a linear relationship between the logarithm of the critical electrolyte concentration (i.e. the salt concentration at which 50% of the total polysaccharide is solubilized) and the reciprocal value of the glycosaminoglycan molecular weight. Thus "neutral MgCl~ elution profiles" have been obtained for chondroitin sulphate2°, ~1 and dermatan sulphate ~2. Fig. 3 thus illustrates the molecular weight polydispersity of the chondroitin sulphate fraction. This is consistent with the electrophoretic pattern obtained for this fraction n. If the values for the critical electrolyte concentration obtained are compared with those given for chondroitin sulphate by Szirmai et al. (ref. 21, Fig. II), the molecular weights of the present chondroitin sulphate fraction would range from 4'1o4-7 •IO4. However, the degree of sulphation may also have a slight influence on this profile. It has been found by Fransson et al. 2~ that the "acid MgC12 elution profiles" for dermatan sulphate principally reflect the uronic acid composition as well as the sulphate content of different preparations, and that differences in molecular weight have Biochim. Biophys. Acta, 279 (1972) 446-455

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o n l y a m i n o r influence. A l t h o u g h such studies have n o t been performed for chond r o i t i n sulphate, it is t e m p t i n g to speculate t h a t the wide range of a p p a r e n t critical electrolyte c o n c e n t r a t i o n s o b t a i n e d (Fig. 4) reflects a v a r i a t i o n of s u l p h a t i o n of these polysaccharides, as no m a j o r variations in uronic acid composition are to be expected. This view is also supported b y the " n e u t r a l elution profile". The exact role of the g l y c o s a m i n o g l y c a n s in the d e n t a l pulp d u r i n g d e n t i n o genesis remains to be clarified. For a discussion of possible functions, see L i n d e n. I t would appear t h a t valuable i n f o r m a t i o n can be o b t a i n e d from studies on the proteoglycan level. ACKNOWLEDGEMENTS I a m i n d e b t e d to D r L - f i t , F r a n s s o n for s t i m u l a t i n g discussion a n d to D r I. L e v i t a n for reading the m a n u s c r i p t . I wish to t h a n k Miss E v a Peterson for her skilful technical assistance. The i n v e s t i g a t i o n was supported b y the Swedish Medical Research Council grants No. B72-24X-2789-o3 a n d B72-24P-3592-oI. REFERENCES I 2 3 4 5 6 7 8 9 IO ii IZ 13 14 15 16 I7 18 19 zo 21 22

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