THE MUCOPOLYSACCHARIDES OF BONE

KARL MEYER Department of Medicine, Columbia University, hrem York

OUR group has been studying the types and quantities of acid mucopolysaccharides which can be isolated from various sources of connective tissue. It is generally assumed that these acid mucopolysaccharides as protein complexes are components of the so-called ground substances. While this appears to be true for the non-sulphated polysaccharides, the sulphated polysaccharides might be considered more appro- priately as components of structural elements. As far as I know, there is not very much information on the chemistry of the mucopolysaccharides of bone. I shall not discuss the histochemical data on bone, because I believe that no state- ment can be made on the nature of the polysaccharides on the basis of available histochemical methods. Rogers (1951) reported the presence in ox-shaft bone of 0.3-0 - 4 per cent of a sulphated polysaccharide, composed of about equimolar concentrations of hexosamine, uronic acid and sulphate, presumably a chondroitin sulphate. However, only about 10 per cent of this amount was isolated. Eastoe and Eastoe (1954) reported the isolation of a mucopolysaccharide-protein complex obtained by lime-water extraction of air-dried bone powder (0 - 24 per cent by weight). On hydrolysis, they demon- strated both chondrosamine, 7 - 67 per cent, and glucosamine, 1 .23 per cent, in this complex, and 1 . 6 3 per cent sulphate-S. On paper chromatography, they reported the presence of galactose, mannose and xylose.

Our own work on bone is unfortunately still unfinished. We wanted to answer the following questions :

(1) What is the nature and the quantity of mucopolysac- charides in adult and growing bone and how does the

COSE 65 4

Bone Structure and Metabolism G. E. W. Wolstenholme.Cecil,ia M. O'Con,ner

Copyright 0 1956 Ciba Foundation Symposium

66 KARL MEYER

pattern differ from cartilage and other connective tissue?

(2) Is there any indication of a phosphorylated polysac- charide as suggested by DiStefano, Neuman and Rouser (1953) in growing bone?

(3) What rnucopolysaccharides are synthesized by fibro- blasts of bone in tissue culture?

The answer to the second question is the simplest. We have searched in vain for a phosphorylated polysaccharide in growing calf bone.

L

FIG. 1. Structure of Hyaluronic Acid.

The first two polysaccharides of Table I, hyaluronic acid and chondroitin, are sulphate-free. The two are isomers and their structures have now been established. They contain repeating disaccharides, in the one case hyalobiu- ronic acid, in the other chondrosin of the structure of a p-glucuronido-1 : 3-hexosamine. They are polymerized without any demonstrable branching via p-1: 4 linkages (Fig. 1).

Next we come to the three chondroitin sulphates which we distinguish on the basis of their solubilities, their optical rotation, their digestion by hyaluronidase and their colour reactions with carbazole and orcinole. In our problem of bone and cartilage, chondroitin sulphate A and C are of interest. B does not occur in cartilage or bone. The structure of the repeating units of A and C are identical and known. The

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68 KARL &IEYER

sulphate group is presumably attached to carbon 6 of the chondrosamine and they are presumably polymerized through p-1 : 4 linkages (Fig. 2). We do not know the cause of the difference between A and C.

The next sulphated polysaccharide was named by us keratosulphate. It is a polymer of a galactose, N-acetyl- glucosamine and sulphate. I ts structure is unknown. We isolated i t from cornea where i t represents one-half of the total mucopolysaccharides. We now have isolated i t or a fraction similar to keratosulphate from calf bone.

‘ 0 k;-.;2<i=;21 H OH U NHAc ( X

FIG. 2. Repeating Unit of Cartilage Chondroitin Sulphate.

The next fractions are highly interesting, though not in relation to bone. These have been found by us in aorta. They have in common a positive optical rotation, they contain glucosamine and a uronic acid which gives colour reactions different from the others and they are resistant to testicular hyaluronidase. I n all probability, they are related to heparin and they may originate from mast cells.

Table I1 gives a summary of the distribution of the muco- polysaccharides from different sources.

Our data on bone are summarized in Tables I11 and IV. As I have already mentioned, we hoped to find out whether growing bone contains polysaccharides which are absent in other connective tissues and absent in adult bone. Let us take adult bone first. From decalcified ox-shaft bone powder (Table 111) we isolated, as the only component, a polysac-

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so4 [@I, Heaosamine Uronic acid carbazole

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Group IV.

Group V.

Group VI.

Group VII.

Group VIII

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T m MUCOPOLYSACCHARIDES OF BONE 69

Tab le I1

DISTRIBUTION OF MUCOPOLYSACCHARIDES

Hyaluronic acid. Vitreous humour, synovial fluid, tumours such as mesothe- lioma, liposarcoma, fowl leucosis, Rous sarcoma, Fujinami tumour.

Chondroitin sulphate A and/or C. Hyaline cartilage (trachea), chondrosarcoma, rhordoma.

Hyaluronic acid plus chondroitin sulphate C. Primitive mesothelial tissue such as umbilical cord, loose connective tissue of electric organ of eel.

Chondroitin sulphate B plus hyaluronate. Skin.

Chondroitin sulphate B plus C plus hyaluronate. Tendon, heart valves, ligamentum nuchae.

Keratosulphate plus chondroitin sulphate A plus chondroitin. Cornea.

Mucoitin sulphate of unknown structure. Amyloid.

Chondroitin sulphate A plus keratosulphate plus hyaluronate plus unidentified sulphated fractions.

Hyaluronate plus chondroitin sulphate A or C plus B plus mucoitin sulphate of unknown structure.

Bovine aorta.

Bone.

charide in a yield of 250 mg. per cent, which has the character- istics of chondroitin sulphate A in solubility, rotation, analysis, and enzymatic hydrolysis. No fraction other than this one was evident.

Tab le I11

O x SHAFT BONE

DECALCIFIED POWDER ANALYSIS (AFTER ADSORPTION ON DOTVEX 1 x 1 COLCMN)

TOTAL MUCOPOLYSACCHARIDE 250 MG. PER CENT OF ACETONE-DRIED

Digested by testicular, resistant to pneumococcal hyaluronidase.

70 KARL MEYER

I\i

Table IV FRACTIONS ISOLATED FROM CALF BONE

Hexos- amine Fraclioiz

2 .67 2 .85 2 .81 2 .86 2 .88 2 .87 3 . 6 6

75 A1 75 A11 75 BI 75 BIII 83 AII* 83 A111 D V26.RII

2 6 . 1 28 .1 2 6 . 3 26 .4 30 .4 25 .8 26 .3

* ! M V I I O A i 81 1'1 IOB

3.27 39.1 2.39 26.4

Uronic acid I

33 .6 33 .8 - -

33 .6 32 .5 -

- -

29.0 13 .5 33 .3 10 .5 32 .0 10.1 32 .0 9.0 31 .9 12 .0 31.0 13 .8

1 . 5 12 .9

43.2 10.6 28.0 11.6

- 30

- 44t -36

- 70 -36

* * Hexose (anthrone, calculated as galactose) 25.6 per cent. t Paper chromatography: chondrovamine 3 +, glucosamine 2 +, hycirolysed by both

hyali~ronidases.

Our calf bone (Table IV) consisted of the distal ends of the long bones, frozen in the deep freeze, cut longitudinally and then by an electric jig saw trimmed to cz thickness of 5-lOmm. The material consisted of approximately 20 per cent epiphysis, the rest being cancellous and shaft bone. From a histological point of view, the starting material was quite a mixture. The material was first ground in a chickenfeed mill and then in a power meat grinder and dehydrated with acetone and cther. The dry weight was 35 per cent of the wet weight.

We used two procedures. I n the first procedure, we brought the p H to 1 - 5 with HC1 and digested with pepsin and trypsin, to give Fractions 75 AI-BIII (Table IV). A second batch was ground in a colloid mill to a fine powder with 0 . 1 N-HC~ and dialysed in the cold against 0 .1 N-HCl and then digested with pepsin and trypsin, to give Fractions 83 A11 and RIII. There were no obvious gross differences between the products of the two procedures. The yields are only approximate. The total mucopolysaccharides isolated amounted to about 3 . 2 per cent. The major part of the muco- polysaccharide is chondroitin sulphate. Fraction 83 A111 contained both glucosamine and chondrosamine and mas

THE MUCOPOLPSACCHARIDES OF BONE 71

hydrolysed in part by pneumococcal hyaluronidase. It was for these reasons suspected t o contain hyaluronic acid mixed with sulphated compounds. On fractionation with ammonium sulphate and pyridine, two fractions were obtained, one obviously hyaluronic acid M VI lOA, the other a chondroitin sulphate. The total hyaluronate isolated from Fraction 83 AIII amounted to only N 280 mg. MTe do not know how many of the other fractions contain hyaluronate.

The high alcohol fractions not listed in Table IV gave a considerable anthrone reaction. Electrophoretically, these high alcohol fractions contained a component with a slightly lower mobility than the chondroitin sulphate and similar to keratosulphate. Attempts t o separate this component on ion-exchange resin were unsuccessful. It was, therefore, sub- jected t o hydrolysis with testicular enzyme to remove the chondroitin sulphate C which was obviously the main con- stituent. On fractionation of the resulting digest, Fraction D V 2GR was obtained which obviously is very similar to keratosulphate, that is, i t contains glucosamine, galactose and sulphate. The yield was only - 250 mg.

A major portion of the mucopolysaccharide fraction, (about one-half of the total), which presumably is a mixture, has not yet been fractionated. As far as we have determined thus far, this fraction contains no non-sulphated components and presumably is a mixture of chondroitin sulphates which are not completely sulphated.

Mass tissue cultures were grown by Drs. Grossfeld and Godman. The medium contained 10 per cent embryo extract in which about 4-5 mg. of mucopolysaccharide is present, largely hyaluronic acid. The i'eeding fluid, renewed every 4-5 days and pooled, contained both hyaluronate and chon- droitin sulphate in both cultures of long bone and of meni- branous bone. There seemed to be little difference between the quantities of polysaccharides in long bone and membranous bone. From 200 ml. of the tissue growth (containing some fluid), 114 mg. of polysaccharide were obtained; from 740 nd. of the culture fluid, 7 2 mg. of polysaccharide were obtained,

Yield mg.

I 26 I1 25

I11 7 . 2

[EID Hexos- Uronic so4 amine acid

-~

3 1 . 5 37.3 2 . 4 - 66 23.8 33.6 9 . 6 -25 - - - 30.1

THE R~UCOPOLYSACCHARIDES OF BONE 73

REFERENCES

DISTEFANO, V., NECMAN, W. F., and ROUSER, G. (1953). Arch. Biochem.

EASTOE, J. E., and EASTOE, B. (1954). Biochem. J . , 57, 453. MAURER, P., and HUDACK, S. S. (1952). Arch. Biochem. Biophys., 38, 49. ROGERS, H. J. (1951). Biochem. J. , 49, XII.

Biophys., 47, 218.

DISCUSSION

de Bernard: Do you think that ,!3-glucuronidase may be concerned with the synthesis of mucopolysaccharides?

Meyer: No, P-glucuronidase has nothing to do with the synthesis of sugar, nor has i t anything to do with the degradation of any of the polysaccharides.

de Bernard: Are these mucopolysaccharides protein-free with regard to electrophoretic mobility?

Meyer: Yes, they do not give a Biuret reaction; on analysis they appear to be protein-free, but of course this is within the limit of error of the method. I would not say that all these preparations were necessarily even analytically protein-free. In cartilage and in bone the binding of chondroitin sulphate to protein is certainly of a labile type, but they are not bound by covalent bonds.

BClanger: The keratosulphate that you are talking about is, of course, not related to keratin?

Meyer: No. We named this substance because it was first separated from cornea. The name does not in any way imply that it is derived from keratin. I n the cornea, keratosulphate is the main polysaccharide.

de Bernard: Are the different optical properties which you find in chondroitin sulphate a and c due to the degree of polymerization or to the molecular configuration?

Meyer: I do not yet know. We are investigating the structure of the chondroitin sulphates now.

hleuberger: Have you evidence of branching? Meyer: Yes, a t the present time this can be inferred only from enzy-

matic studies, no other method is useful. Neuberger: There is also a possibility that the sulphate group might

be attached a t different points, either 4 or 6. Meyer: Yes, but the hydrolysis curve for the sulphate is the same in

both, for instance if one is a primary sulphate the other would be a secondary, but no matter which one it is you would get differences in the hydrolysis.

Neuberger: How far, according to the results a t present obtained, is the particular mixture of mucopolysaccharides and sulphates character- istic of a particular type of connective tissue? In fact, if you were given a mixture could you guess from which type of connective tissue it is derived ?

Meyer: In skin and in cornea, yes; in bone, we believe yes. Of course this is mixed tissue; it might be quite different if we took only shaft

74 DISCUSSION

bone or only the plates in the epiphysis. In tendon, maybe. In heart valve, I believe yes. We have investigated only one species of heart valve, i.e. pig heart valve. In skin, chondroitin sulphate is the main polysaccharide but there is much more chondroitin sulphate seen in rodent skin than there is in pig skin. Pig skin, yes. We have not investi- gated human skin. This is the work of a group with which I am con- nected.

Kodicelc: I would like to put to you, in a slightly different form, the question which I asked Prof. Lacroix: what makes the bone calcify in comparison to the tendon? From your results, can you say that the difference in mucopolysaccharide set-up would justify our saying that the mucopolysaccharides are responsible for calcification ?

Meyer: I cannot say this. There is a large fraction and we do not know what i t is. This fraction is characterized by the fact that it is remarkably undersulphated; it does not contain chondroitin, a t least as far as we can tell from the ion-exchange experiments which distinguish very beautifully between sulphated and non-sulphated polysaccharides. I presume that this is the most interesting fraction, we suspect it is the fraction which picks up the sulphate which shows up as radioactive sulphate in various areas, as demonstrated by a number of people here. This could only be demonstrated in a cell-free isolated enzyme system, and I wish someone would do this.