Clhemical characterization of mucopolysaccharides in myxoma

J. clin. Path. (1968), 21, 582-589

Clhemical and histochemical characterizationof mucopolysaccharides in a jaw myxoma

J. J. HODSON AND R. E. S. PROUT

From the Departments of Oral Pathology and Biochemistry, University of Sheffield

SYNOPSIS Chemical and histochemical analyses, including testicular and staphylococcal hyaluroni-dase digestion, have been made of a jaw myxoma and the results show the presence of two acidmucopolysaccharides. Of the total mucopolysaccharide present 80% was hyaluronic acid and 20%chondroitin sulphate. The high content of non-sulphated mucopolysaccharide would seem toexplain the paucity of fibres characteristic of the myxoma. It is suggested that myxomas generallyprobably have a similar high hyaluronic acid content. It is considered that the cell concerned is a

mesenchymal cell elaborating non-sulphated mucopolysaccharide and may be called a 'myxoblast';it is metabolically different from the sulphated-mucopolysaccharide-collagen-producing fibroblast.The high hyaluronic acid content is much greater than that found in embryonic connective tissueand may be a significant factor in the neoplastic behaviour of the myxomatous tissue. The aggressivebehaviour of the myxoma is against a simple reversion to embryonic mesenchyme. It is concludedthat the myxoblast is an active mucopolysaccharide-secreting cell and that mucin in the myxomais not a sign of cell degeneration of preexisting fibroblasts or collagen.

Myxomas are connective tissue tumours composedof stellate fibroblast-like cells dispersed in a meta-chromatic substance and resembling the myxoma-tous tissue of the umbilical cord. In pure form, ie,sparse in fibre production, they are rare. Theliterature shows that by far the largest number occurin the heart. Myxomatous tissue may be present inneoplasms in conjunction with other tissues, as, forinstance, in liposarcomas, ameloblastomas, andneurofibromas.The nature ofthemucopolysaccharidein the myxomas has not been elucidated, but thatfound in some other tumours, eg, some mesotheliomasof pleura, has been shown to consist of onlyhyaluronic acid (Wagner, Munday, and Harington,1962). Stout (1948) suggested that the soft tissuemyxoma might contain hyaluronic acid, andPearce, Weller, and Steinberg (1952) thought thatthe fluid in a recurrent myxomatous cyst of theskin was probably hyaluronic acid. Sedano andGorlin (1965) submitted a jaw myxoma to limitedhistochemical tests and suggested that it mightcontain either hyaluronic acid or chrondroitinsulphate.The myxoma of bone is of particular importance

in that, with few possible exceptions (Bauer andHarell, 1954; Perou, Kolis, Zaeske, and Borja 1967),it is only found in the maxilla and mandible. InReceived for publication 29 January 1968.

behaviour the myxoma is locally infiltrative andmay cause extensive bone destruction. It is not knownto metastasize. Histologically it is similar to the softtissue myxomas except that in some cases odonto-genic epithelial residues may sometimes be includedwithin its substance.

In a study of myxomas in the jaws and oral softtissues those in bone appear to arise from mesen-chyme of the marrow and also, possibly but lesscommonly, from myxomatous tissue arising in thefollicle of the unerupted tooth crown.The purpose of the present study was to elucidate

the nature of the intercellular material secreted bythe cells of the jaw myxoma by chemical analysis,and to correlate the findings with those obtainedhistochemically. The results are discussed from ahistogenic viewpoint. No myxoma appears to havebeen previously chemically analysed.

MATERIALS AND METHODS

A girl of 14 presented with a large hard swelling of thebody of the right mandible associated with an uneruptedpremolar. The tumour had reached and partially resorbedthe inferior cortical bone of the mandible. At operationit shelled out fairly easily. Three years after the operationthe tumour recurred and a hemimandibulectomy wasnecessary. Grossly, the mass (5 0 cm x 40 cm) washeavy and slimy and the bisected surface was grey and

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Chemical and histochemical characterization of mucopolysaccharides in a jaw myxoma

FIG. 1. Cut surface of myxoma. The opaque white ovalarea in the centre consists of collagen and at the lowerborder are spicules of bone regenerating from the peri-osteum.

translucent with small areas of white fibrous tissue (Fig. 1).The tissue was in formalin for a few hours during transitby post. Half of the tumour was stored at - 15°C andused for the chemical analysis and part of the other half,after fixation in buffered formol-saline, was used formicroscopical and histochemical studies.

CHEMICAL ANALYSES

The tumour tissue was weighed and homogenized inice-cold acetone in a Waring blender. The tissue was thenextracted with 7% (w/v) sodium acetate at 0°C for oneweek. The extract was acidified to pH 4 with acetic acid,treated with 1-5 volumes of ethyl alcohol and allowed tostand at - 15°C for 48 hr after which the precipitate wassedimented. A portion of the precipitate in aqueoussolution was analysed by electrophoresis using Oxoidcellulose acetate paper and 0-04M veronal bufferpH 8-3.The samples were run at 20 v/cm 2 m amp for 1-5 hours.The electrophoresis papers were stained for mucopoly-saccharide using alcian blue (Heremans and Vaerman,1958; Nanto, 1963).

Separation of the mucopolysaccharide material wascarried out using a column ofDEAE Sephadex A25 in theacid state, using 40 ml portions of sodium chloridesolution of progressively increasing molarity in 0-01Mhydrochloric acid as eluant (Schmidt, 1962). The eluatefractions were dialysed against distilled water, evaporatedto dryness in vacuo and the residue was redissolved indistilled water. Qualitative analysis was carried out byelectrophoresis. Quantitative analysis of the uronic acidcontent of the samples was carried out using the Bitterand Muir (1962) modification of the Dische (1947)carbazole method.The tissue residue remaining after the initial extraction

with sodium acetate was digested with pepsin at pH 2and 35°C for five days. Zinc acetate (10% w/v) wasadded in equal volume to precipitate any protein present

and the solution was then neutralized to pH 7-2, filteredand dialysed against water; the dialysate was acidified topH 4 and mucopolysaccharides were precipitated bythe addition of 1-5 volumes of ethyl alcohol.

HISTOCHEMISTRY

Parts of the tumour from various areas were blocked inparaffin wax and sections cut at 6, 10, and 151u thick.Control sections of umbilical cord and mucous salivarygland were used. The chemical analyses by Meyer,Davidson, Linker, and Hoffman (1956) and Balazs andJeanloz (1965) showed that in addition to hyaluronicacid, the umbilical cord also contains chondroitinsulphate as a minor component. Our histochemical testswith bovine and bacterial hyaluronidase also indicatedthe presence of the two mucopolysaccharides.

In addition to various routine stains, includingLaidlaw's reticulin stain, the following methods wereused: (1) azure A, 0-01 % in 30% ethanol, with andwithout alcohol dehydration of the section (Kramer andWindrum, 1954); (2) alcian blue, 001 % in 3% aceticacid (Steedman, 1950), with neutral red and chlorantin-fast red counterstains; (3) toluidine blue, 0-5%; (4) Hale'sdialysed iron method for acid mucopolysaccharides(Thompson, 1966); (5) Gomori's aldehyde fuchsin (Scottand Clayton, 1953), and the aldehyde fuchsin-alcian bluesequence described by Spicer and Mayer (1960); (6) peri-odic-acid-Schiff reaction (PAS).

ENZYMIC DIGESTION

A number of sections from various areas were stainedwith azure A, toluidine blue, alcian blue, and Hale'smethod before and after extraction with bovine testicularhyaluronidase (Pearse, 1960), and staphylococcal hyalur-onidase1. This latter, like streptococcal hyaluronidase,digests only non-sulphated acid mucopolysaccharides(Meyer, 1947; Linker, Hoffman, Meyer, Sampson, andKorn, 1960). The experimental sections were incubatedfor 3 hr at 37°C in bovine and bacterial hyaluronidaseprepared at a concentration of 1 mg/ml in normal saline.The controls were stained after incubation in normalsaline only. Comparing the staining after incubation insaline with untreated stained sections showed a reductionin intensity suggesting that incubation in normal salineremoved some of the polysaccharide. This observationneeds further investigation.

RESULTS OF CHEMICAL ANALYSES

The qualitative analysis of the tumour tissue byextraction with sodium acetate showed that bothhyaluronic acid and chondroitin sulphate werepresent. Analysis of the pepsin digest showed that afurther portion ofhyaluronic acid had been extracted.These results are summarized in Table I. Thequantitative results showing the amounts of muco-polysaccharide extracted from the tumour tissue aregiven in Table II.'Organon Laboratories Ltd.

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J. J. Hodson and R. E. S. Prout

TABLE ICHEMICAL ANALYSIS OF TUMOUR TISSUE

Electrophoresis ofSodium Acetate Extract againstKnown Standards' Pepsin Digest

Hyaluronic acid and chondroitin Hyaluronic acid only presentsulphate

'Hyaluronic acid, Koch Light Co. Chondroitin sulphate, KochLight Co.

TABLE IIANALYSIS OF Ml

4-3 g TUMOUR TISS

Hyaluronic acidNaOAc extractPepsin digestChondroitin sulphateNaOAc extractPepsin digestTotal

HI'

ROUTINE STAINS TI

scopical structure o

collagen, fine collaglwith large areas ofthrough which ranmucin and small sca

were present. The tissThe greater the quewas the collagen-retilogy of the bipolar -

bore only a superiembryonic myxomaoccasional mast cell i

New bone formaticexposed periosteum

AZURE A AND TOLl

generally highly mevaried in intensity.

HALE'S METHOD AN

produced strongly pmetachromatic reactcontrast between the

PERIODIC ACID-SCHIFthe collagen fibres a

fibres had the reddis

GOMORI S ALDEHYDE FUCHSIN The staining of themucin was only moderate with areas of weakercoloration. This stain was also used in conjunctionwith alcian blue according to the method of Spicerand Meyer (1960) and although the aldehydefuchsin-alcian blue (AF.AB) sequence suggested ademonstration of both sulphated and non-sulphatedmucopolysaccharides, the technique requires furtherinvestigation.

ENZYME DIGESTION

UCOPOLYSACCHARIDES FROMUE USING A SEPHADEX COLUMN TESTICULAR HYALURONIDASE Metachromasia, alcianFraction Eluted in M NaCI Weight of blue, and Hale staining of the mucin were abolishedin O'OIM HCI Mucopoly- after incubation with the enzyme (Figs. 5 and 6).I II IIl IV saccharide This reaction, together with the metachromatic

reactions and positive results with Hale's method and050 1-25 1-50 2-00 alcian blue, indicated the presence of acid muco-

polysaccharide. Slight metachromasia of the fine+ - - - 2 50 fibres was noted, particularly in the thicker sections+ - - - 2.00 treated.-

+ + - 1-10-- - - 000 STAPHYLOCOCCAL HYALURONIDASE The results

5s60 showed a reduction in the amount of metachromatic-mucin and also in the intensity of staining with

STOCHEMISTRY Hale's ferrocyanide solution and alcian blue. Thiswas also confirmed in four other oral myxomas,

iese showed a general micro- three of them from the jaw bones. Figures 8 and 9of interlacing narrow bundles of are an attempt to illustrate this reduction in stainingen and probably reticulin fibres intensity. The observation made earlier that someintercellular mucinous material mucopolysaccharide appeared to be lost in thefine fibres (Figs. 2-4). Pools of saline-incubated section does not affect the reductiontttered foci of compact collagen illustrated here since the sections treated with.ue was only moderatelyvascular. bacterial hyaluronidase are compared with theantity of mucin the less dense control saline-treated sections; the reduction wasiculin component. The morpho- somewhat greater compared with the normallyand stellate cells of the tumour prepared sections. The reduction in staining afterficial resemblance to those of treatment with the bacterial enzyme was apparentlytous tissue (Fig. 6). Only an due to digestion of the hyaluronic acid component.was present in a low-power field.rn was taking place from the DISCUSSION(Fig. 7).

The chemical analysis showed that the myxoma

UIDINE BLUE The mucin was contained hyaluronic acid and chondroitin sulphate.tachromatic but in some areas The quantitative analysis indicated that of the two

acid mucopolysaccharides present, hyaluronic acidformed the larger component amounting to 80% ofthe total mucopolysaccharide; 44*5% of the total

ID ALCIAN BLUE These stais mucopolysaccharide was protein-bound hyaluronic

ositive results and paralleled the acid. The chemical analysis confirmed the results

Lions, but gave a more striking suggested by the histochemical tests. The various

mucin and the fibre structure. histochemical methods used indicated the presenceof acid mucopolysaccharide which was entirely

F The mucin did not stain but digested by testicular hyaluronidase. Digestion withLppeared pale pink and the finer bacterial hyaluronidase, however, did not remove allh colour of reticulin. the metachromatic, alcian blue, and Hale-positive

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collagen fibres with interveningmyxomatous areas leading intomyxomatous tissue of the bulkof the tumour. Haematoxylinand Masson's light green x 42.

FIG. 3. Higher magnificationof narrow collagen bundles andirregularly orientated fibresbecoming sparser below, the lattercomprising the bulk of thetumour. A vessel is shown in the

r top half. Laidlaw's reticulin,x 225.

FIG. 4. Sparse collagen andreticulin fibres irregularlyorientated within and roundmucopolysaccharide-groundsubstance. Laidlaw'sreticulin, x 225.

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FIG. 5. Higher power of areanear spicule of regeneratingbone (lower right), showingmetachromasia of highlymucinous tissue. Controlsection for Fig. 7 incubatedin saline, undecalcified;toluidine blue, x 225.

FIG. 6. Loss of mucin inadjacent section after digestionwith testicular hyaluronidase.Note widely spacedmucopoly-saccharide-producing cells,and bone as in Figure 5.Undecalcified; toluidineblue, x 225.

FIG. 7. Part of area wheremyxoma resorbed corticalbone. Bony spicules havedeveloped from the periosteumseen at the lower border;compare Figure 1. Interveningtissue pure myxoma.Haematoxylin and eosin, x 42.

FIG. 6.

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Chemical and histochemical characterization of mucopolysaccharides in a jaw inyxoma

material. As staphylococcal, like streptococcal,hyaluronidase only digests hyaluronic acid, thereduction in staining suggested that hyaluronic acidhad been removed.The results support Stout's (1948) original idea

that the myxoma might contain hyaluronic acidalthough he cited this as the only component. Thefindings do not support Grishman (1952), Tighe andMeachim (1962), and Tighe (1963) who considerthat the mucopolysaccharide present is chondroitinsulphate. Their investigations, however, were con-fined to studies of soft tissue myxomas. Tighe andMeachim (1962) and Tighe (1963) used histochemicalmethods together with testicular hyaluronidase, andin addition the former authors used radioactivesulphate fixation. Grishman (1952) used bothtesticular and bacterial hyaluronidase but was

unsuccessful with the latter.In the sections examined, no specific foci of mucin

entirely digested by the bacterial enzyme could bedetected and it would appear that both sulphatedand non-sulphated acid mucopolysaccharides were

probably mixed together. The different areas stainedby aldehyde fuchsin and alcian blue, using thetechnique of Spicer and Meyer (1960) to differentiateacid mucopolysaccharides, suggest, however, thepossibility that some of the stellate cells may belimited to the production of either sulphated or

non-sulphated mucopolysaccharides, ie, may not be

FIG. 8. Area nearperiosteum. Controlsection for Fig. 9incubated in saline.Hale's dialysed ironand metanilyellow, x 225.

FIG. 9. Adjacentsection after digestionwith staphylococcalhyaluronidase showingreduction in stainingdue to loss ofhyaluronic acid. Hale'sdialysed iron andmetanilyellow, x 225.

FIG. 9.

products of the same cell. Further techniques needto be developed to localize the origin of the differentpolysaccharides. The similarity of the microscopicstructure between the jaw myxomas and those inother sites suggests the possibility that all myxomasprobably contain at least two mucopolysaccharides.Meyer et al (1956) from their chemical analyticalstudies suggest that tumours of mesodermal originproduce only one type of mucopolysaccharide, thesulphated group as in the chondrosarcoma or

hyaluronic acid as in the liposarcoma. In our case,however, we found two types present with hyaluronicacid as the larger component. It is interesting thatother tumours with mucinous areas have been foundto contain hyaluronic acid, as for instance theliposarcoma (Meyer et al, 1956; Tighe, 1963). Ourhistochemical and enzyme studies of a case ofliposarcoma showed the presence of both hyaluronicacid and chondroitin sulphate. In 13 of the 16mesiotheliomas analysed by Wagner et al (1962),streptococcal hyaluronidase removed all the meta-chromasia in the sections, indicating the presence ofhyaluronic acid only. Chemical analysis of thepleural fluids from five cases showed the presence ofhyaluronic acid.Although the metachromasia of hyaluronic acid

is said to be weaker than the sulphated muco-

polysaccharides, Tighe (1963) thought that itsconcentration in tissues was probably never high

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enough to contribute to metachromasia; however,umbilical cord shows a positive metachromasiapresumably largely due to hyaluronic acid. Meyer(1947) found a concentration of about 1% showedtypical metachromasia in smears. In the presentstudy it would appear that the quantity of hyaluronicacid present was sufficient to affect the intensity ofthe metachromasia after bacterial digestion; thiswas also supported by the observable reduction inthe intensity of the staining using Hale's colloidaliron method and alcian blue.The controversial question of fixation of tissue for

demonstrating acid mucopolysaccharides is worthyof comment here. Tighe (1963), quoting Grishman(1948), states that as hyaluronic acid is soluble inwater, formol saline fixation is not recommended.Pearce et al (1952) considered that mucin stains onformalin-fixed tissue yielded inconclusive results.In our histochemical controls, umbilical cord fixedin both buffered formol-saline and Camoy'sfixative showed little difference in the staining of themucin. Using the bacterial enzyme we found astriking loss of mucopolysaccharide, apparentlyhyaluronic acid and an undigested amount mostlyround the central vessels of chondroitin sulphate.In a full discussion on the question of fixation andmucopolysaccharides, Curran (1961) found thatformalin is an excellent fixative but draws attentionto the 'unpredictability of fixation in general andfixation of acid mucopolysaccharide in particular'.

There is strong evidence that the 'fibroblast cell'is responsible for the production of both hyaluronicacid and the sulphated acid mucopolysaccharides(Gersh and Catchpole, 1949; Curran and Kennedy,1955; Grossfeld, Meyer, and Godman, 1955; Meyeret al, 1956; Green and Hamerman, 1964; Curran,Lovell, and Clark, 1966). Asboe-Hansen (1950)suggests that mast cells are the main source ofhyaluronic acid. The few mast cells present in thejaw myxoma does not suggest that they are the sourceof the hyaluronic acid. Kennedy (1960) states that ontopographical grounds the fibroblast is probablyresponsible. As previously suggested it wouldappear that in the present tumour the stellate cellsproduced both sulphated and non-sulphated muco-polysaccharides, although it is possible that eachsubstance may be the product of a different stellatecell.

Tighe and Meachim (1962), following up Meyer's(1951) suggestion that fibroblasts secrete acid muco-polysaccharides together with a soluble protein,'pre-collagen', consider that sulphated mucopoly-saccharides are involved in collagen formation andput forward the idea that the stellate cells of myxo-matous tissue produce sulphated mucopolysacchar-ides but fail to form precollagen, thus accounting

for the accumulation of mucin and a paucity offibres, or in other words, 'the retention of mucinswithin the growth and the relative failure of fibreproduction'. As the quantity of hyaluronic acid inour case is not normal for connective tissue itself itcannot be considered a 'retention' substance. On thebasis of our results, the high hyaluronic acid and theminor sulphated mucopolysaccharide content couldexplain the paucity of collagen fibres characteristicof the tumour. However, chemical analyses of tissuecultures of one line of mouse fibroblasts havesuggested that during collagen synthesis the poly-saccharide produced by the same cells probablyconsists almost exclusively of hyaluronate. It is ofparticular interest that another fibroblast lineappeared to lose its ability to synthesize collagenbut continued to synthesize hyaluronate at the samerate as the previous line (Green and Hamerman,1964). It is suggested that the more mucinous thetumour the more likely it is to be aggressive. Curranet al (1956) have further elucidated the role ofmucopolysaccharides in fibrogenesis by light- andelectron-microscope techniques. The question ariseswhether the secretion of a larger quantity thannormal ofhyaluronic acid is a sign ofsevere metabolicdisturbance of the stellate cell and whether perhapsthis plays a role in its neoplastic behaviour. Thepostulation of merely a reversion to embryonicconnective tissue is not consistent with the invasiveand destructive behaviour of the myxoma. Meyeret al (1956) noted with surprise the differences inmucopolysaccharides 'in view of the relative histo-logical similarity of the tissues and especially of thecells which are believed to produce the acid poly-saccharides'.

Since little fibre production is taking place in thepurer forms of myxoma we believe that the stellatecell is in fact a mesenchymal cell which has failed todifferentiate sufficiently to play its role in collagenformation, ie, it is not strictly a collagen-producing,mucopolysaccharide-secreting fibroblast, but amesenchymal cell (or metabolically poorly differenti-ated or immature fibroblast) secreting mucopoly-saccharides in excessive and abnormal proportions.

This ability to produce mucin appears to be aproperty of an active viable cell and it is almostcertainly wrong to postulate a degenerative process,as most writers do when describing the myxoma orfibromyxoma as a fibroma undergoing myxomatousdegeneration. The old term 'myxoblast' would be asuitable name for the mucopolysaccharide-secretingmesenchymal cell and would serve to distinguish itfrom the collagen-mucopolysaccharide-producingfibroblast. The myxoma would then be considered atumour of neoplastic myxoblasts. The importanthistogenetic point here is that 'myxomatous de-

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generation' of a fibroma postulates a prior formationof fibrous tissue, whereas we suggest an initialformation of mucopolysaccharide by a cell producinglittle or no precollagen and hence little or no fibreformation and which has become neoplastic.Non-neoplastic mucopolysaccharide-producing cellsare common enough in the granulation tissue ofinflammatory lesions, the stroma of neoplastictissue and indeed in the fibroma itself.The collagen found in varying amounts in all

myxomas indicates that there are present mesenchymalcells which have differentiated into collagen-producing fibroblasts. Stout (1948) suggests that thecollagen present is akin to the fibrosis seen in manytumours but the morphology is not suggestive of this.The term 'fibromyxoma' is used where there is anappreciable fibrous content. It would be interestingto know whether in the largely fibrous tumours(fibromas) the intercellular ground-substance ischondroitin sulphate and whether the highlymucinous myxoma tumours are characterized by ahigh hyaluronic acid component as in the presentcase.We are indebted to Professor W. Bartley for his help andadvice with the chemical analysis, to Mr M. Rudland,FIMLT for technical assistance, to Mr S. Ireland, FDS,RCS, for the specimen, and to Organon Laboratories Ltdfor the gift of the staphylococcal hyaluronidase.

REFERENCES

Asboe-Hansen, G. (1950). Ann. rheum. Dis., 9, 149.

Balazs, E. A., and Jeanloz, R. W. (1965). The Amino Sugars, p. 219.Academic Press, New York.

Bauer, W. H., and Harell, A. (1954). J. Bone JISurg., 36-A, 263.Bitter, T., and Muir, H. M. (1962). Anal. Biochem., 4, 330.Curran, R. C. (1961). Biochemical Society Symposia, 20, 24.-, and Kennedy, J. S. (1955). J. Path. Bact., 70, 449.-, Lovell, D., and Clark, A. E. (1966). Ibid., 91, 429.Dische, Z. (1947). J. Biol. Chem., 167, 189.Gersh, I., and Catchpole, H. R. (1949). Amer. J. Anat., 85, 457.Green, H., and Hamerman, D. (1964). Nature (Lond.), 201, 710.Grishman, E. (1948). Bull int. Ass. med. Mus., 28, 104.

(1952). Cancer (Philad.), 5, 700.Grossfeld, H., Meyer, K., and Godman, G. (1955). Proc. Soc. exp.

Biol. (N. Y.), 88, 31.Heremans, J., and Vaerman, J. P. (1958). Clin. chim. Acta, 3, 430.Kennedy, J. S. (1960). J. Path. Bact., 80, 359.Kramer, H., and Windrum, G. M. (1955). J. Histochem., 3, 227.Linker, A., Hoffman, P., Meyer, K., Sampson, P., and Korn, E. D.

(1960). J. biol. Chem., 235, 3061.Meyer, K. (1947). Physiol. Rev., 27, 335.

(1951). Connect. Tissues, Trans. Confs. Josiah Macy Jr Fdn, 1,32.Davidson, E., Linker, A., and Hoffman, P. (1956). Biochim.biophys. Acta (Amst.), 21, 506.

Nanto, V. (1963). Acta chem. scand., 17, 857.Pearce, A. E., Weller, R. W., and Steinberg, N. (1952). Arch. Surg.,

64, 835.Pearse, A. G. E. (1960). Histochemistry, 2nd ed. Churchill, London.Perou, M. L., Kolis, J. A., Zaeske, E. V., and Borja, S. R. (1967).

Cancer (Philad.), 20, 1030.Sedano, H. O., and Gorlin, R. J. (1965). Arch. oral Biol., 10, 727.Schmidt, M. (1962). Biochim. biophys. Acta (Amst.), 63, 346.Scott, H. R., and Clayton, B. P. (1953). J. Histochem., 1, 336.Spicer, S. S., and Meyer, D. B. (1960). Amer. J. clin. Path., 33, 453.Steedman, H. F. (1950). Quart. J. micr. Sci., 91, 477.Stout, A. P. (1948). Ann. Surg., 127, 706.Thompson, S. W. (1966). Selected Histochemical and Histopatho-

logical Methods. Thomas, Springfield, Ill.Tighe, J. R. (1963). J. Path. Bact., 86, 141.

and Meachim, G. (1962). Ibid., 83, 195.Wagner, J. C., Munday, D. E., and Harington, J. S. (1962). Ibid., 84,

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Clhemical characterization of mucopolysaccharides in myxoma