Journal of Clinical InvestigationVol. 45, No. 7, 1966

Biochemical Findings in Normal and Osteoarthritic ArticularCartilage. II. Chondroitin Sulfate Concentration and

Chain Length, Water, and Ash Content*ALFREDJAY BOLLET t ANDJOANNEL. NANCE

(From the Departments of Preventive Medicine and Internal Medicine, University of VirginiaSchool of Medicine, Charlottesville, Va.)

Softening and erosion of cartilage, the charac-teristic early lesions of osteoarthritis, occur insites of decreased chondroitin sulfate concentra-tion. The role of aging and the specific biochemi-cal events that produce this alteration in glycos-aminoglycan composition have been under studyin several laboratories. Collins and McElligottfound increased radiosulfate fixation in these le-sions compared with normal articular cartilagefrom the same individuals, suggesting increasedsynthesis of polysaccharide (1). These observa-tions indicated an increased turnover of chon-droitin sulfate in osteoarthritic cartilage lesions,and attention has become focused on the mecha-nism responsible for breakdown of this glycos-aminoglycan.

As reported previously (2), when the cartilageproteinpolysaccharide complex was extracted fromhuman articular cartilage obtained at autopsy, adecrease in the concentration of chondroitin sulfatebut not protein was found in the areas of erosion.This observation pointed to a specific breakdownof the glycosaminoglycan, since proteolysis mightbe expected to cause removal of both componentssuch as occurs after papain injection into rabbits(3). An enzyme resembling testicular hyalu-ronidase could be responsible, but reports of thepresence of hyaluronidase in tissues other thantestis were unconvincing or unconfirmed. A re-evaluation of the tissue distribution of this enzymeresulted in the finding of an enzyme with most ofthe characteristics of testicular hyaluronidase in

* Submitted for publication August 3, 1965; acceptedMarch 24, 1966. This work was supported by U. S.Public Health Service grants AM03421 and AM21,934-5K3.

tAddress requests for reprints to Dr. Alfred Jay Bol-let, Dept. of Preventive Medicine, University of Vir-ginia School of Medicine, Charlottesville, Va.

urine, plasma, liver, spleen, kidney, synovial tissue,and synovial fluid, but no hyaluronidase activitywas detected in articular or costal cartilage (4).On the other hand, a proteolytic enzyme capableof degrading cartilage proteinpolysaccharide ispresent in chondrocytes (5-7). Whether proteaseor an enzyme resembling testicular hyaluronidaseis responsible for the increased turnover of chon-droitin sulfate in osteoarthritic cartilage matrixrequires more definitive analysis.

Cartilage proteinpolysaccharide probably con-sists of multiple chondroitin sulfate chains at-tached to a central protein core (8). One could,therefore, predict that proteolysis would releasepart of the protein and associated polysaccharidefrom the molecule, but the chondroitin sulfateremaining would be qualitatively unchanged. Onthe other hand, hyaluronidase would specificallyattack and degrade the chondroitin sulfate chains,leaving behind polysaccharide of lower averagechain length in the zones of decreased chondroitinsulfate concentration. In addition, keratan sulfateis probably attached to the same or a similar pro-tein core; hyaluronidase, which does not digestkeratan sulfate, would cause loss of only thechondroitin sulfate, whereas protease would causeboth glycosaminoglycans to be lost from the car-tilage matrix. Similar glycoproteins, which arealso present in cartilage matrix, are not affectedby hyaluronidase. This paper reports studies ofthe chain length of chondroitin sulfate and the rela-tive concentrations of chondroitin sulfate and neu-tral sugar in keratan sulfate and glycoprotein inhuman osteoarthritic and normal articular carti-lage; in addition, we studied the synthesis ofchondroitin sulfate in these lesions by cartilagesamples in vitro and determined their water andash content. We also analyzed the influence of

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BIOCHEMICAL FINDINGS IN NORMALAND OSTEOARTHRITICARTICULAR CARTILAGE 1171

age on these components in normal articularcartilage.

Methods

Human articular cartilage was obtained from the kneeat autopsy, using a scalpel blade to cut as deeply as pos-sible without removing underlying bone. Samples wereremoved from the posterior surface of the patella or fromthe condyles and patellar groove of the femur; abnormalareas were excised with care not to include surroundingnormal zones. Both normal and abnormal samples wereusually obtainable from the same joint. An attempt wasmade to grade the degree of abnormality from the grossfindings, as described previously (2). The cartilage wasstored frozen in sealed containers if not processed imme-diately; each specimen was diced, and weighed sampleswere used for analysis of the chondroitin sulfate concen-tration and at least one other determination. The termchondroitin sulfate as used in this study includes the totalof chondroitin-4-sulfate plus chondroitin-6-sulfate, sincethe methods used did not distinguish between these isomers.

Ethanol fractions were prepared from cartilage samplesweighing up to 250 mg after digestion in 5 ml of 0.05 Nphosphate buffer, pH 7.0, containing 2 mg per ml crystal-line trypsin 1 for 16 to 20 hours at 370 C and then ho-mogenization in a Kinematica 2 for 1 minute. Insolubledebris was removed by brief centrifugation; this residuewas found to have an average of 10.8%o (SEM ± 1.2%o)of the total hexosamine of the cartilage. Four ml of su-pernatant was brought to 40%o ethanol by the addition of4.0 ml of ethanol and 1.6 ml of aqueous 20% potassiumacetate. After standing at 40 C overnight, the precipitatewas removed by centrifugation and the supernatantbrought to 50%o ethanol concentration, allowed to standovernight at 40 C, and centrifuged. When needed, athird precipitate was collected in similar fashion bybringing the ethanol concentration in the supernatant to75%o.

Each precipitate was dissolved in 4 ml of water, andportions were used for determination of uronic acid withthe carbazole method of Dische (9) and hexosamine bythe method of Neuhaus and Letzring (10). Chondroitinsulfate chain length was determined by the procedure ofPartridge, Davis, and Adair (11), which gives a numberaverage molecular weight; reducing sugar determinationswere done by the method of Somogyi (12) after exposureof the glycosaminoglycan solution to 0.1 M sodium hy-droxide for 24 hours and neutralization. The concentra-tion of chondroitin sulfate used in the calculation wasbased on the uronic acid determination, multiplied by afactor of 2.67.

The anthrone method (13) was used to determine theneutral sugar in the ethanol precipitates obtained fromcartilage samples, using galactose as standard, after sub-traction of the color development attributable to the uronicacid present in each specimen. This value represents a

1 Pentex, Inc., Kankakee, Ill.2 Willems, Lucerne, Switzerland.

combination of the neutral sugar present in keratan sul-fate and glycoprotein.

The in vitro synthesis of polysaccharide was studiedusing cartilage samples placed in ice immediately afterremoval at autopsy; finely diced samples weighing a totalof 100 mg were incubated in Tyrode's solution containing'SO4 or acetate-'4C, 5 ,uc per ml, at 370 C for 2i hours,then washed five times with 0.15 M saline and incubatedovernight with trypsin as described above. Precipitateswere prepared at various ethanol concentrations, washed,dissolved in water, and analyzed for uronic acid and ra-dioactivity by liquid scintillation spectrometry. Before 'Swas counted, samples were hydrolyzed with 0.1 M sodiumsulfate and 6 N HCl for 4 hours at 1000 C. Specific ac-tivities were determined on the basis of the concentrationof uronic acid, which implies that the molar ratio of sul-fur and acetate to uronic acid was 1.0.

To determine the water content, diced cartilage sam-ples of approximately 50 mg were weighed and dried inacetone for 18 to 24 hours and heated at 600 C for an ad-ditional 18 to 24 hours; no further weight loss was ob-tained by additional heating. The dried samples wereweighed on a Kahn electrobalance to ± 0.01 mg andplaced in a muffle furnace at 1,0000 F for 48 hours inweighed pieces of aluminum foil in crucibles. Additionalheating at this temperature caused no further loss ofweight. After it had reached room temperature in adesiccator over calcium chloride, the ash was weighed onan electrobalance.

Concentrations are given on a wet weight basis exceptfor ash weights, which were determined as per cent ofdry weight. Figures in tables and text are means plusor minus standard errors; p values, calculated using thet test, are for differences from the normal samples exceptwhere indicated.

Results

Concentration of chondroitin sulfate. As inprevious studies (2), the concentration of chon-droitin sulfate was decreased in the samples con-sidered abnormal at autopsy (Tables I and II).A good correlation was observed between thegross estimate of the severity of the osteoarthritisand the decrease in chondroitin sulfate concentra-tion. In view of this, to obtain greater objectivityin dividing samples into minimal and advancedosteoarthritis for statistical calculations, we de-cided to use the chondroitin sulfate concentrationas the basis for division. Of the samples con-sidered abnormal at the time of autopsy, thosewith a concentration below 534 mg per 100 mlchondroitin sulfate (200 mg per 100 ml as uronicacid) were classified as advanced disease. A finaldecision whether or not early osteoarthritic changewas present was postponed at the time of autopsyin a few instances because of uncertainty concern-

ALFRED JAY BOLLET ANDJOANNEL. NANCE

TABLE I

Chondroitin sulfate in normal and osteoarthritic articular cartilage

40% ethanol fractionNo. of Hexosamine/obser- Chain length uronic acidvations Concentration Concentration (X 103) (molar ratio)

g/1oo ml g/100 mlNormal 26 1.84 4 0.13 1.57 4- 0.12 14.7 d 0.6 1.36 1 0.06

All osteoarthritic 21 0.871 d 0.093 0.706 41 0.09 9.1 =1 0.5 1.39 i 0.15samples p = <0.001 p = <0.001 p = <0.001

Minimalosteoarthritis 7 1.31 4d 0.17 1.08 i 0.18 11.1 i4: 0.7 1.45 i 0.15

p = <0.05 p = <0.05 p = <0.01

Advancedosteoarthritis* 14 0.651 i 0.045 0.519 i- 0.042 8.2 4± 0.5 1.37 i 0.09

p = <0.001 p = <0.001 p = <0.001

* p values for difference between minimal and advanced osteoarthritis are: total concentration of chondroitin sulfate(CS), <0.001; concentration of CS in 40%ethanol fraction, <0.001; chain length, <0.01.

ing the presence of softening. These sampleswere labeled doubtful, and those which had chon-droitin sulfate concentrations well in the normalrange (over 1.0 g per 100 ml) were classified as

normal; the others were considered minimal osteo-arthritis.

Chain length of chondroitin sulfate in the 40%ethanol fraction. The calculated values for chainlength and concentration of the chondroitin sulfatein the fraction obtained by precipitation at 40%ethanol concentration are given in Table I. Ahighly significant decrease in chain length was ob-served in the abnormal specimens, consistent withdepolymerization of the chondroitin sulfate. Agood correlation between the chondroitin sulfatechain length and concentration in this fraction(r = 0.88, p = < 0.001) was observed for the ab-normal specimens.

Evaluation of chain length method. The methodfor chain length, based on reducing sugar activityas an end group analysis, might be erroneous ifother reducing sugar was contaminating the speci-mens. When further attempts at purification were

made by using pronase or papain in addition toor in place of trypsin to degrade the proteinmoiety further, or by reprecipitation of the chon-droitin sulfate, the same or lower chain lengthvalues were found, rather than increased values,which might indicate separation from contaminat-ing reducing activity. No procedure was foundthat gave higher values for chain length. Keratansulfate was present in the trypsin digests, buthexosamine to uronic acid ratios and the anthronereaction for galactose showed that very little was

present in the fraction precipitated by 40%ethanol. More neutral sugar was present in the

TABLE II

Neutral sugar and chondroitin sulfate values found in normal and osteoarthritic articular cartilage

No. of Concentration ofobser- chondroitin sulfate/

vations Neutral sugar* Chondroitin sulfate neutral sugar

mg/100 ml g/100 mlNormal 32 595 ± 1.4 1.23 ±t 0.077 2.17 i 0.10

All osteoarthritic 30 395 i 2.6 0.462 i 0.044 1.23 ± 0.10samples (p = <0.01) (p = <0.001) (p = <0.001)

Minimal 11 480 ± 4.8 0.723 i 0.051 1.59 i 0.12osteoarthritis (p= <0.2) (p = <0.001) (p = <0.01)

Advanced 19 347 i 2.6 0.311 :1: 0.044 1.02 i 0.13osteoarthritist (p = <0.001) (p = <0.001) (p = <0.001)

* As galactose.t p values for difference between minimal and advanced osteoarthritis are: neutral sugar, <0.01; chondroitin sulfate,

<0.001; ratio of chondroitin sulfate to neutral sugar, <0.01.

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BIOCHEMICAL FINDINGS IN NORMALAND OSTEOARTHRITICARTICULAR CARTILAGE 1173

precipitate obtained at 50% ethanol; the 75%ethanol precipitate contained the most neutralsugar and was virtually devoid of uronic acid.On testing the 75% ethanol fraction, no reducingsugar activity was detected with or without priortreatment with sodium hydroxide while using neu-tral sugar (keratan sulfate plus glycoprotein)concentrations considerably higher than were pres-ent in the 40%o ethanol fractions used for chainlength determination. The molar ratio of hexosa-mine to uronic acid was essentially the same inthe 40% ethanol precipitates from the normaland abnormal specimens, indicating no greaterdegree of contamination in the abnormal. Nocorrelation was found between the value found forchain length and the ratio of hexosamine to uronicacid. It seems unlikely, therefore, that contamina-tion affected the reducing sugar values.

As a further check on the possible presence ofcontaminating reducing sugar, several sampleswere incubated with bovine testicular hyaluroni-dase,3 1 mg per ml in 0.5 M acetate buffer, forup to 48 hours at 370 C, and the chain lengthdetermination was repeated. The chain lengthsfound were never below the theoretical value fortetrasaccharide, ranging between 1,000 and 1,500;if contaminating reducing sugar was present, alower apparent molecular weight should have beenobtained. As a standard for comparison, a chainlength determination was performed on pure tetra-saccharide 4; a value of 946 was found, whichis close to the theoretical one.

When determinations of chain length were donewithout exposure to sodium hydroxide, highervalues were found, presumably due to incompleteseparation of the reducing ends of the polysac-charide from the peptide. A series of samples runin this fashion showed a mean 28.3 ± 2.67 ( x 103)for 24 normal samples and 12.3 + 1.24 ( x 103)for 22 osteoarthritic samples (p = < 0.001). Thesodium hydroxide treatment gave more consistentresults with lower values, particularly in the nor-mal samples, reflecting more complete exposureof reducing end groups. It is unlikely that sodiumhydroxide caused depolymerization of the chains,judging by the fact that serial observations madeat 24, 48, and 72 hours on the same samples gave

3 Sigma Chemical Co., St. Louis, Mo.4 Kindly supplied by Dr. Eugene A. Davidson, Durham,

N. C.

similar chain length values rather than evidencefor progressive degradation.

Neutral sugar. The concentration of neutralsugar, as galactose, in the ethanol-precipitatedfractions was decreased in the osteoarthritic carti-lage samples, although the samples showing mini-mal osteoarthritis were not significantly differentfrom normal (Table II). The decrease in keratansulfate plus glycoprotein concentration, therefore,was not so great as the fall in chondroitin sulfateconcentration. As a result, the ratio of chon-droitin sulfate to neutral sugar was considerablylower in the abnormal samples; as the severityof osteoarthritis increased, the ratio of chon-droitin sulfate to neutral sugar fell, indicatingproportionately greater loss of chondroitin sulfatethan of the components containing neutral sugar.

In vitro uptake of 35S04 and acetate-14C. Car-tilage samples obtained at autopsy were incubatedwith labeled precursors to study the nature of thecompounds synthesized. Previous reports of in-creased 35S uptake in osteoarthritic cartilage sam-ples compared with normal areas from the sameindividuals were based on radioautography orcounting digests of whole cartilage fragmentswashed after incubation (1). More convincingevidence that the label was actually incorporatedinto chondroitin sulfate was sought by separatingthe fractions obtained by ethanol precipitation.The incorporation of isotope into the chondroitinsulfate precipitated at 40%o ethanol was comparedin normal and abnormal sites from the same kneejoint. Presumably because of variations in timebetween death and autopsy, there were wide varia-tions in total radioactivity incorporated into thechondroitin sulfate between individuals; however,comparison of normal to abnormal sites in eachknee was possible. The specific activity of thechondroitin sulfate in the 40%v ethanol precipitatewas considerably higher in the osteoarthritic sam-ples compared with normal cartilage. On thebasis of the weight of the cartilage samples incu-bated, there was no consistent relationship be-tween the normal and abnormal sites. Only 20%of the abnormal samples showed higher incorpora-tion of radiosulfate than the corresponding normalsites; there was no relationship between severityof the osteoarthritis, concentration of chondroitinsulfate, or age of the individual and the 35S find-ings (Table III).

ALFREDJAY BOLLET ANDJOANNEL. NANCE

TABLE III

36SO4 and acetate-14C incorporation by normal and osteoarthritic cartilage samplesinto polysaccharide precipitated by 40% ethanol

A. Representative individual casesChondroitin

Isotopic sulfate con- Radioactivity incompound Patient Cartilage sample centration 40% fraction

% cpm/100 cpm/fjmolemg (103) (102)

35SO4 61-year-old Normal 1.12 7.3 12.9male Osteoarthritic 0.427 3.6 17.6

Osteoarthritic 0.475 5.7 25.1

62-year-old Normal 1.04 45.6 98.9male Osteoarthritic 0.155 15.6 215.0

Osteoarthritic 0.157 156.0 163.0Osteoarthritic 0.745 71.3 177.0

Acetate-'4C 80-year-old Normal 1.43 4.5 4.3female Normal 1.52 5.1 4.9

Osteoarthritic 0.921 2.3 11.2Osteoarthritic 0.334 2.3 3.6

B. Means of specific activityIsotopic No. of

compound Cartilage sample observations Mean (X 102)

35SO4 Normal 17 35.6Minimal osteoarthritis 10 81.6Advanced osteoarthritis 18 133.1

Acetate-'4C Normal 5 11.0Osteoarthritic 7 20.0

Water and ash content (Table IV). The watercontent of normal articular cartilage averaged67.9%, whereas in osteoarthritic cartilage sam-ples it averaged 74.8%o of the wet weight. Theash weights determined on the same cartilagesamples showed no significant difference betweennormal and abnormal sites.

Influence of age. The normal cartilage samplesfrom different individuals were compared on thebasis of age; almost all the samples were fromindividuals between the ages of 30 and 80 years.In that age range, no trend was noted with in-creasing age for the concentration of chondroitinsulfate or of compounds containing neutral sugar,the ratio of chondroitin sulfate to these com-

TABLE IV

Water content and ash weights of normal and osteoarthriticarticular cartilage

Concentra-No. of tion ofobser- Water chondroitin

vations content Ash content sulfate

%wet wt

Normal 26 67.9 40.84Osteoarthritic 24 74.8 1.33p <0.01

%dry wt

4.20 4-0.214.34 ±0.70

<0.7

g/loo ml1.20 40.0460.522 ±0.167

<0.001

pounds, the chain length of the chondroitin sul-fate, the water content, or the ash content of thesenormal cartilage samples (Figures 1 and 2).

Discussion

The decrease in average chain length of thechondroitin sulfate in osteoarthritic cartilage le-sions and the decreased ratio of chondroitin sulfateto compounds containing neutral sugar (glyco-protein plus keratan sulfate) suggest a role ofa depolymerase such as hyaluronidase in chon-droitin sulfate degradation in these lesions. Al-ternatively, the decreased chain length might resultfrom preferential synthesis of lower molecularweight chondroitin sulfate; an attempt to demon-strate this by separation of relatively low andrelatively high molecular weight fractions on thebasis of ethanol solubility showed a similar patternof radiosulfate incorporation in normal and osteo-arthritic cartilage samples. These results, how-ever, cannot be considered conclusive, and thealternative explanation, preferential synthesis oflower molecular weight chondroitin sulfate, re-mains a possibility. In addition, it should beclearly pointed out that these findings in no way

1174

BIOCHEMICAL FINDINGS IN NORMALAND OSTEOARTHRITICARTICULAR CARTILAGE 1175

3.00-

(gms.%)

2.75-

250-

2.25-

200-

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ISO-

L25-

.00-

220-

20D-

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12D-

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6D-

Il0 200 40 50 60 8 0so lo i 5'o40s 60 i go so

Age(Yeon) Age(Yrs)FIG. 1. THE CHONDROITIN SULFATE CONCENTRATION(A) AND CHAIN LENGTH (B) IN NORMALARTICULAR CARTILAGE

FROMTHE KNEEJOINT IN INDIVIDUALS OF VARIOUS AGES.

eliminate the possibility of a role of proteolyticenzyme or a combination of the two types of poly-saccharide removal. A combined effect seemslikely, since the decreased neutral sugar levelfound in the extracts of osteoarthritic cartilageindicates that the metabolic abnormality is notlimited to chondroitin sulfate. However, the de-crease in the concentration of compounds contain-ing neutral sugar is negligible in early lesions.Keratan sulfate and glycoprotein are not degradedby hyaluronidase; turnover of keratan sulfate inintervertebral disc tissue is exceedingly slow (14)and the mechanism of breakdown is not known.

7460-

720-

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In view of the possibility of a role of hyaluroni-dase in the loss of chondroitin sulfate from osteo-arthritic cartilage, the source of this enzyme comesunder scrutiny. Although an enzyme with char-acteristics of testicular hyaluronidase is widelydistributed in animal tissues and present in humanserum, synovial tissue, and synovial fluid (4), ithas not been found in articular or epiphyseal car-tilage in spite of numerous attempts by a varietyof methods in our laboratory. Hyaluronidase, anacid pH optimal hydrolytic enzyme, is associatedwith lysosomes in liver (15) ; several lysosomalenzymes are easily demonstrable in cartilage sam-

60-

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AVe (as) Age (Yesn)THE WATERCONTENT(A) ANDASH WEIGHT (B) OF NORMALARTICULAR CARTILAGE FROMTHE KNEE JOINT

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ALFRED JAY BOLLET AND JOANNEL. NANCE

ples, but hyaluronidase has not been identified.Further investigation of possible sources of theenzyme and of factors influencing its activity areneeded, as are studies of factors influencing therate of synthesis of chondroitin sulfate by normaland abnormal articular cartilage.

Although Collins and McElligott (1) reportedincreased radiosulfate uptake by osteoarthritic car-tilage lesions, our data failed to show consistentlyincreased incorporation into fractions containingchondroitin sulfate, on the basis of the weight ofcartilage samples incubated. Increase in the spe-cific activity of the radiosulfate or acetate-14Cwas virtually consistent, pointing to increasedturnover of the chondroitin sulfate in the osteo-arthritic lesions. These findings could have re-sulted from increased diffusion of isotope intoabnormal samples, but this seems unlikely sincethe cartilage was carefully diced into the smallestsize fragments obtainable to provide maximal sur-face area and to minimize this variable. Thegreater cellularity of osteoarthritic lesions (1, 2)could have been responsible for the increased 35Suptake; the data cannot be interpreted as showingincreased synthesis per cell but, nevertheless, theydo seem to indicate increased turnover in thelesions.

The finding of increased water content of osteo-arthritic cartilage was unexpected in view of thedecreased chondroitin sulfate concentration. Aproportionality between polysaccharide concentra-tion and water content is usually found in con-nective tissues, and the divergence in this instanceis interesting particularly since the lower chainlength of the chondroitin sulfate and the decreasedratio of chondroitin sulfate to protein (2) indicatethat the residual protein polysaccharide in theosteoarthritic lesions is qualitatively altered.

The failure to find a change in concentrationor qualitative nature of the polysaccharide withincreasing age in articular cartilage is consonantwith our earlier observations (2), and in contrastto the findings of Kaplan and Meyer on costalcartilage (16). The age ranges studied are lim-ited to older adults; no statements can be madecomparing children or young adults, but the agerange observed includes the period of most fre-quent development and progression of osteoarthri-tis. Other studies of articular cartilage have re-

vealed an absence of age dependent changes inthe concentration of hexosamine (17), total solids,percentage of extracellular and intracellular solids,total sulfate, and total potassium (18), althoughan increase in intracellular water with a decreasein extracellular water was found in older agegroups (18). The failure to find significant bio-chemical changes in aged but normal articularcartilage and the focal distribution of the bio-chemical as well as pathological changes in thisdisease point to the need for studies of focal fac-tors influencing metabolic phenomena in articularcartilage in analyzing the pathogenesis of osteo-arthritis.

Summary

Osteoarthritic cartilage lesions show a decreasein chondroitin sulfate concentration and chainlength and a decreased ratio of chondroitin sulfateto compounds containing neutral sugar, keratansulfate plus glycoprotein. This is evidence for arole of a depolymerase such as hyaluronidase inthe metabolic breakdown of chondroitin sulfate inthese lesions.

In vitro incubation with 35SO4 and acetate-'4Cresults in an increased specific activity of thechondroitin sulfate in osteoarthritic cartilage le-sions, indicating increased turnover of the poly-saccharide compared to normal cartilage sites.

The water content of osteoarthritic cartilage isincreased despite the decreased chondroitin sulfateconcentration. The ash content of these lesions isnot significantly altered.

Comparison of normal specimens from individ-uals ranging in age from 30 to 80 years revealedno influence of age on the concentration or chainlength of chondroitin sulfate, the concentration ofcompounds containing neutral sugar, the watercontent, or the ash content of articular cartilage.

References

1. Collins, D. H., and T. F. McElligott. Sulphate(35SQ4) uptake by chondrocytes in relation to his-tological changes in osteoarthritic human articu-lar cartilage. Ann. rheum. Dis. 1960, 19, 318.

2. Bollet, A. J., J. R. Handy, and B. C. Sturgill. Chon-droitin sulfate concentration and protein-poly-saccharide composition of articular cartilage inosteoarthritis. J. clin. Invest. 1963, 42, 853.

3. Greenawald, K. A., and T. T. Tsaltas. Papain in-duced chemical changes in adult rabbit cartilage

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BIOCHEMICAL FINDINGS IN NORMALAND OSTEOARTHRITICARTICULAR CARTILAGE

matrix. Proc. Soc. exp. Biol. (N. Y.) 1964, 117,885.

4. Bollet, A. J., W. M. Bonner, Jr., and J. L. Nance.The presence of hyaluronidase in various mam-malian tissues. J. biol. Chem. 1963, 238, 3522.

5. Chrisman, 0. D., and J. M. Fessel. Enzymatic degra-dation of chondromucoprotein by cell-free extractsof human cartilage. Surg. Forum 4962, 13, 444.

6. Tourtellotte, C. D., R. D. Campo, and D. D. Dzie-wiatkowski. Degradation of chondromucoproteinby an enzyme extracted from cartilage. Fed. Proc.1963, 22, 413.

7. Ali, S. Y. The degradation of cartilage matrix byan intracellular protease. Biochem. J. 1964, 93,611.

8. Partridge, S. M., and D. F. Elsden. The chemistryof connective tissue. 7. Dissociation of the chon-droitin sulfate-protein complex of cartilage withalkali. Biochem. J. 1961, 79, 26.

9. Dische, Z. A new specific color reaction of hexuronicacids. J. biol. Chem. 1947, 167, 189.

10. Neuhaus, 0. W., and M. Letzring. Determination ofhexosamines in conjunction with electrophoresison starch. Analyt. Chem. 1957, 29, 1230.

11. Partridge, S. M., H. F. Davis, and G. S. Adair. Thechemistry of connective tissues. 6. The constitu-

tion of the chondroitin sulfate-protein complex incartilage. Biochem. J. 1961, 79, 15.

12. Somogyi, M. A reagent for the copper-iodometric de-termination of very small amounts of sugar. J.biol. Chem. 1937, 117, 771.

13. Seifter, S., S. Dayton, B. Novic, and E. Muntwyler.The estimation of glycogen with the anthrone re-agent. Arch. Biochem. 1950, 25, 191.

14. Davidson, E. A., and W. Small. Metabolism in vivoof connective-tissue mucopolysaccharides. I. Chon-droitin sulfate C and keratosulfate of nucleus pul-posus. Biochim. biophys. Acta (Amst.) 1963, 69,445.

15. Aronson, N. N., Jr., and E. A. Davidson. Lysosomalhyaluronidase. J. biol. Chem. 1965, 240, 3222.

16. Kaplan, D., and K. Meyer. Ageing of human car-tilage. Nature (Lond.) 1959, 183, 1267.

17. Anderson, C. E., J. Ludoweig, H. A. Harper, andE. P. Engleman. The composition of the organiccomponent of human articular cartilage. Rela-tionship to age and degenerative joint disease. J.Bone Jt Surg. 1964, 46-A, 1176.

18. Miles, J. S., and L. Eichelberger. Biochemical stud-ies of human cartilage during the aging process.J. Amer. Geriat. Soc. 1964,12, 1.

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