Annals ofthe Rheumatic Diseases 1992; 51: 214-219

Methylprednisolone acetate induced release ofcartilage proteoglycans: determination by highperformance liquid chromatography

Herkko Saari, Riitta-Mari Tulamo, Yrjo T Konttinen, Timo Sorsa

AbstractA high performance liquid chromatography(HPLC) procedure suitable for the simul-taneous determination of the molecular sizeand concentration of macromolecularhyaluronate and proteoglycans in synovialfluid has been developed. Irrigation of theequine tarsocrural joint with 20 ml physio-logical saline (PSS) caused a mild inflammationwith an increase of proteoglycans in thesynovial fluid over the baseline arthrocentesiscontrol sample. Proteoglycan and hyaluronatein the synovial fluid did not interact to formhyaluronate-proteoglycan aggregates, butseparated as distinct chromatographic peaks.This suggests that the cartilage derivedproteoglycans in synovial fluid in the inflamedjoint have been proteolytically cleaved fromthe non-covalent aggregates containing linkprotein and hyaluronate. Hyaluronidasedigestion completely abolished the hyaluronatepeak without affecting the proteoglycans.This seems to indicate that proteoglycan insynovial fluid is unable to interact withhyaluronate in synovial fluid to form cartilagetype aggregates.

Proteolytic degradation and the timedependent release into the synovial fluid ofsuch digested proteoglycan also resulted fromthe intra-articular injection of methylpred-nisolone acetate into normal tarsocruraljoints and joints irrigated with PSS. Theseproteoglycans were insensitive to hyalu-ronidase but may consist of a protein moietywith attached glycosaminoglycans, assuggested by their sensitivity to proteinaseand keratanase/chondroitinase digestion.These observations with cartilage treated withmethylprednisolone acetate and mildlystimulated articular cartilage are inconsistentwith earlier work on osteoarthritic andrheumatoid articular cartilage and haveintbresting implications for the pathogenesisand for the therapeutic action of intra-articular corticosteroids. A rapid HPLCprocedure applicable to unprocessed smallvolume samples of synovial fluid givesinformation simultaneously on hyaluronateand proteoglycan in synovial fluid which is notattainable with immunoradiometric or isotopetracer techniques. It therefore appears to beuseful for the analysis of cartilage turnoverand destruction in health and disease.

Intra-articular injections of glucocorticoids havebeen widely used in the treatment of arthritis.Although their beneficial effect of relieving theacute inflammatory response is well docu-

mented, reports of their influence on themetabolism of cartilage proteoglycans are stillcontradictory. Articular cartilage extracellularspace is composed of at least four differentproteoglycan populations, some of which areable to form aggregates with hyaluronate.' Inproteoglycan aggregates numerous proteoglycansubunits are non-covalently bound to a centralstrand of hyaluronate. The interaction is furtherstabilised by link proteins.2 The proteoglycansubunit consists of a central core protein towhich glycosaminoglycan side chains arecovalently bound. The core protein containsdifferent regions for the attachment of chond-roitin sulphate and keratan sulphate chains.The N terminal globular domain of the coreprotein possesses the ability to bind hyal-uronate.3 ' The unique viscoelastic propertiesof the articular cartilage are mainly due to thestrong water retention capacity of polyanionicproteoglycan aggregates. In addition, collagenfibres tend to oppose the considerable swellingpressure of hydrated proteoglycan aggregates.The proteoglycan content of cartilage has

been determined by various histochemicalstaining methods. Proteoglycan synthesis hasbeen studied by monitoring the incorporation of35SO4 into the cartilage proteoglycans and itsbreakdown by following the appearance oflabelled proteoglycans in the culture medium.5 6Immunological methods have been developedfor the determination of proteoglycans insynovial fluid, including several enzyme linkedimmunosorbent assays (ELISAs).7 8 Whetherthe effect of corticosteroids on the metabolismof proteoglycans in cartilage is anabolic orcatabolic seems to be dependent on the experi-mental model systems or proteoglycan assaymethod, and is still largely uncertain.The aim of this work was to develop a

reproducible method for the simultaneousdetermination of large proteoglycans andhyaluronate in synovial fluid samples withoutany preceding sample preparation. It wasintended to apply this method to determine thein vivo effects of intra-articular corticosteroidson cartilage glycosaminoglycan metabolism,using the equine tarsocrural joint as a modelsystem.

Materials and methodsEFFECT OF INTRA-ARTICULARMETHYLPREDNISOLONE ACETATEOne tarsocrural joint in five horses (Nos. 1, 2,3, 4 and 7) was injected at day 0 with 20 mlphysiological saline (PSS) to induce a slightinflammatory reaction. Twenty four hours afterirrigation (day 1), the same joint was injected

Fourth Departmentof MedicineHelsinki UniversityCentral Hospital,Helsinki School ofVeterinary Medicine,Department ofPeriodontology,University of Helsinki,Helsinki, FinlandH SaariR-M TulamoY T KonttinenT SorsaCorrespondence to:Dr H Saari,Fourth Departmentof Medicine,Helsinki UniversityCentral Hospital,Unioninkatu 38,SF-00170 Helsinki,Finland.Accepted for publication18 March 1991

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with 100 mg (2 8 ml) methylprednisolone acetate(MPA). Each injection was performed aftertaking a sample by the aseptic arthrocentesistechnique. In addition, samples of synovialfluid were collected 2, 3, 4, 7, and 11 days afterirrigation with PSS (table 1).

CONTROLSIn each of these horses, the contralateral jointwas irrigated at day 0 and, instead of MPA, wasinjected on day 1 with 2-8 ml PSS as a control.Three horses (Nos. 5, 6, and 8) had onetarsocrural joint subjected to repeated samplingonly as an arthrocentesis control. In two of these(Nos. 6 and 8) the contralateral joint wasirrigated with 20 ml PSS on day 0 and injectedwith 2-8 ml PSS on day 1 for comparison withsimilarly treated joints in horses injected withMPA. Three horses (Nos. 9, 10 and 11) were

injected in one tarsocrural joint with 100 mg ofMPA without a preceding PSS injection todifferentiate between the effects ofPSS irrigationand injection.

CLINICAL SIGNSThe horses were observed for signs of lameness;exercise was restricted and they were kept atrest in their stalls. Clinically identifiable grosschanges in the joints were evaluated at eachsynovial sampling by visual and manualexamination.

COLLECTION OF SYNOVIAL FLUID

Synovial fluid samples were obtained using an

aseptic technique before the injections (day 0),on day 1 (24 hours after irrigation) and on days2, 4, 7, and 11 after injection. Each sample was

immediately transferred into a sterile test tubeand centrifuged at 1500 g for 10 minutes topellet the cells. The synovial fluid samples were

stored without preservatives at -20°C forHPLC analysis.The synovial fluid volume was evaluated by

forced aspiration into the syringe at eachsampling time; 5 ml was taken for furtherstudies and the rest was carefully injected backinto the joint. Total protein was determined bythe method of Lowry et al9 and the leucocytecount was performed on a 1:20 dilution using a

standard haemocytometer (HaemocytometerCounting Chamber, American Optical Cor-poration).

HIGH PERFORMANCE LIQUID CHROMATOGRAPHYThe HPLC determination was performed usingan LKB 2150 chromatographic pump (with asample loop of 50 ,ul) coupled with an LKB2151 online UV detector.'1'2 Hyaluronateand proteoglycan in synovial fluid were detectedat a wavelength of 206 nm and the chromato-grams were recorded with an LKB 2220recording integrator. The elution buffer was 50mM sodium phosphate, pH 6 50, and theconstant flow rate was 1-0 ml/min. TSK 5000PW (30 cmx7T5 mm) and TSK 6000 PW (60cmx7 5 mm) size exclusion columns wereobtained from Toyo Soda.

Synovial fluid samples were diluted to 1:20 to1:40 with elution buffer, and 50 id was im-mediately injected into the HPLC apparatus.

CALIBRATION OF THE COLUMNSFor determination of the concentration ofhyaluronate, a calibration graph was obtainedusing Healon (Pharmacia Diagnostica, S-75182)with the appropriate dilutions. The molecularweight calibration was performed as describedby Saari.'3 Proteoglycan concentrations insynovial fluid were calculated using Arteparon,a commercial proteoglycan mixture obtainedfrom Luitpold-Werk.The detection limits for hyaluronate and

proteoglycans in synovial fluid were less than 51ig/ml.

CHEMICALSHyaluronidase from Streptomyces hyalurolyticus(type IV), chondroitinase ABC from Proteusvulgaris and keratanase from Pseudomonasspecies (EC 3.2.1.103) were purchased fromSigma. MPA (Depo-Medrol 40 mg/ml) wasfrom Upjohn. Sodium chloride, 9 mg/ml, wasobtained from Orion Oy. All other chemicalswere of the highesr commercial purity available.

ResultsDETERMINATION OF HYALURONATE ANDPROTEOGLYCAN IN SYNOVIAL FLUID WITH HPLCA single injection of MPA (100 mg) into theequine tarsocrural joint induced a progressiverelease of proteoglycan into the synovial fluid(fig 1, day 0-11). At the same time thehyaluronate content of the synovial fluidincreased, but not to the same degree as theproteoglycan content. The peaks of hyaluronateand proteoglycan were easily separated from

Table I Experimental protocol for 105 synovial fluid samples

Day Horse (leg)

1 2 3 4 5 6 7 8 9, 10, 11

Left Right Left Right Left Right Left Right Left Right Left Right Left Right Left Right Left Right

0 PSS PSS PSS PSS PSS PSS PSS PSS S - PSS S PSS PSS S PSS - -20 20 20 20 20 20 20 20 20 20 20 20

1 MPA PSS PSS MPA MPA PSS PSS MPA S - PSS S MPA PSS S PSS MPA -100 2-8 2-8 100 100 2-8 2-8 100 2-8 100 2 8 2-8 100

2,4, 7, 11S S S S S S S S S - S S S S S S S -

Abbreviations: PSS 20=20 ml of physiological saline solution; MPA 100=100 mg of methylprednisolone acetate; PSS 2-8=2-8 ml of physiological saline solution;S=sample, arthrocentesis only.

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each other and the other components of synovialfluid.

Hyaluronidase treatment of the synovial fluidsample (obtained at day 11) resulted in thecomplete disappearance of the hyaluronatecontaining peak, whereas the proteoglycan peak(retention time 15-52 minutes) was not affectedby hyaluronidase treatment. If digestion wascontinued with protease or with a combinationof chondroitinase ABC and keratanase, theproteoglycan peak was also degraded (lowercurve in fig 2A).The proteoglycan fraction was collected after

hyaluronidase treatment from the terminal endof the HPLC capillary tube and further chroma-tographed on a TSK 5000 PW column. Figure2B shows the chromatogram obtained for thenative, large molecular size proteoglycan(retention time 8-34 minutes) and the degrada-tion products after subjection to chondroitinaseABC and keratanase (retention time 12-14minutes). Thelower curve shows chromatogramsof pure chondroitinase ABC and keratanaseenzymes; the results indicate that no markedinterference with the determination of proteo-glycans occurred.

EFFECT OF ARTHOCENTESIS, PSS IRRIGATION ANDMPA ON THE CONCENTRATIONS OF HYALURONATEAND PROTEOGLYCAN IN SYNOVIAL FLUIDThe injection of PSS into the joint induces aslight inflammatory reaction 4 with concomitantleucocyte infiltration and an increase in theprotein content in synovial fluid (table 2). Inthis model there was a slight increase ofproteoglycans in the synovial fluid over thebaseline concentration and arthrocentesiscontrols. Figure 3A shows that this returned tothe baseline concentration in seven days. Theintra-articular injection of 100 mg MPA on day

Figure I High perfornance liquid chromatography ofequine tarsocruraljoint synovialfluid. A mass of100 mg ofmethylprednisolone acetate (MPA) was injected intra-articularly at day 0. A time course study ofthe effects ofMPA on proteoglycan (PG) and hyaluronate (HA) insynovialfluid was performed by drawing samples at days 0,1, 2, 4, 7, and 11. MPA induced a time dependent release oflarge molecular size proteoglycan into equine synovialfluid.Hyaluronate and proteoglycan are eluted in differentfractions: hyaluronate at the retention times 1246-12S56 andproteoglycan 1550-15S52 minutes.

Figure 2 (A)HPLC chromatogram ofequine synovialfluid after treatment with hyaluronidase;hyaluronidase totally degrades the hyaluronate peakconfirming its identity. The proteoglycan peak is not affectedby hyaluronidase treatment (upper curve in A). However, ifthe incubation ofthe synovialfluid is continued afterhyaluronidase treatment with a combination ofchondroitinase ABC and keratanase, the proteoglycan peakdisappears (lower curve in A). (B) The proteoglycan peakwith a retention time of15 62 min on the TSK 6000PWcolumn was collectedfrom theHPLC outlet. Theproteoglycan fraction wasfurther chromatographed on aTSK 5000PW column due to its more advantageous range oflinear resolutionfor proteoglycan andproteoglycandegradation products. The proteoglycan peak elutes with aretention time of8-34 minutes and its degradation productsafter digestion with chondroitinase ABC and keratanase elutewith a retention time 12 14 minutes. (C) ChondriotinaseABC and keratanase were chromatographed alone withoutsynovialfluid; this control shows that they do not interferewith theHPLC resultsfor the proteoglycan fractions.

Figure 3 (A) Equine proteoglycan (PG) in synovialfluidand (B) hyaluronate (HA) in synovialfluid concentrations(mglml) in various experimental groups. Samples werecollected on the days indicated. (a) Arthocentesis alone,(n=3); (b) irrigation with physiological saline (PSS)(n= 7); PSSfollowed by (c) 100 mgMPA intra-articularlyone day afterPSS irrigation (n=5) and (d)MPA alone(n=3).

AA= 206 nm

A

1 5.52

0

w

z12.14

C

0 10 20

Retention time (min)

I

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Table 2 Summary of results. Values given are mean (range) of sample of synovial fluid

Day Arthrocentesis (n=3)* PSS irrigation (n=7)

WBC Prot Vol PG HA WBC Prot Vol PG HA(x 1091) (mg/mi) (ml) (mg/ml) (mg/ml) (x 1091l) (mg/mi) (ml) (mg/ml) (mg/ml)

0 0-05 11-6 18-0 0-02 0-36 0-17 10-4 12-0 0-02 0-400-02-0-08 2-2-19-0 8-0-28-0 0-007 030-0 50 005-0 38 3-2-21-2 2-5-19-0 0-0 14 0 16-0-82

1 0-15 10-3 16-5 0-05 0-42 2-02 16-8 18-0 0-05 0-450-02-0-33 6-3-15-2 8 0-25-0 0-0 09 0 28-0 50 0-10-6 36 6-1-34-0 3-0-25 0 0-0-14 0-24-0-78

2 0-35 11-1 17-0 0-09 0-38 2-50 15-4 19-5 0-22 0-590-16-0-67 3-6-15-2 6-0-28-0 0-04-0-14 0-22-0 52 0-31-9-59 46-43-2 3-0-34 0 0-0-59 0-28-0-%

4 0-30 15-0 17-5 0-07 0-53 0-40 14-9 16-3 0-20 0-590-04-0-72 5-8-31-1 4-0-31-0 0-0-18 0-40-0-72 0-02-1-33 7-3-24-6 3-5-23-0 0-0-56 0-26-0-84

7 0-34 15-4 18-0 0-06 0-44 0-13 12-1 14-5 0-07 0-530-15-0-68 7-3-27-0 4-0-32-0 0-0-17 0-38-0-52 0-05-0-27 5-1-18-2 5-0-25-0 0-0 15 0-22-0-98

11 0-16 12-3 25 0-05 0-37 0-15 10-1 13-7 0-03 0-430-04-0-23 6-2-20-3 13-0-38-0 0-0-16 0-24-0-50 0-03-0-38 5-6-16-0 4-0-27-0 0-0-18 0-22-0-84

Day PSS imrgation + MPA (n=S)* MPA (n=3)*

WBC Prot Vol PG HA Vol PG HA(x 1091l) (mg/ml) (ml) (mg/mi) (mg/ml) (ml) (mg/ml) (mg/ml)

0 0-09 9-8 9-6 0-07 0-63 - - -0-06-0-13 5-6-19-2 4-0-20-0 0-0-19 0-44-0-%

1 4-36 20-6 15-1 0-13 0-66 12-5 - 0-360-05-8-58 10-0-38-2 4-5-35-0 0-0-50 0-38-1-12 10-0-15-0 0-32-0-48

2 4-46 20-6 8-3 0-47 0-86 10-0 0-43 0-722-80-7-15 8-2-37-0 2-0-13-0 0-1-17 0-38-1-54 8-0-12-0 0-28-0-75 0-42-0-96

4 0-63 14-0 10-0 0-39 0-69 6-5 1-48 0-940-03-1-02 10-1-16-2 4-0-20-0 0-0-84 0-40-0-96 5-5-8-0 0-66-1-62 0-70-1-26

7 0-20 13-4 6-0 0-77 0-85 5-5 1-65 1-270-10-0 30 8-2-18-6 1-5-14-0 0-19-1-17 0-76-0-94 3-5-7-5 1-22-1-98 0-98-1-44

11 0-24 11-6 4-3 0-90 0-94 7-5 1-10 0-640-07-0-42 5-6-19-2 1-0-10-0 0-28-1-33 0-62-1-20 4-0-9-5 0-82-1-32 0-36-0-86

*Abbreviations: (WBC) white blood cell count; (Prot) protein concentration; (Vol) volume of synovial fluid; (PG) proteoglycanconcentration; (HA) hyaluronate concentration; (PSS) physiological saline solution; (MPA) methylprednisolone acetate.

1 did not reverse the effect of PSS irrigation,but caused an increased concentration ofproteoglycan in the synovial fluid, this increasebeing at a maximum as late as 11 days after PSSinjection. Interestingly, the injection of MPAwithout preceding irrigation with PSS producedan even higher and immediate appearance oflarge molecular size proteoglycans in the synovialfluid. The effect of MPA was at a maximum onday 7, after which it began to diminish. Figure3B shows that in all experiments the effect ofMPA on the hyaluronate concentration insynovial fluid was fairly similar to the effect onproteoglycan, although the extent of theresponse was less (Fig 3B). These results showthat MPA induces a degradative action oncartilage aggregates of proteoglycans.

Corticosteroids may cause rapid dehydrationof the swollen joint. We therefore calculated thetotal concentrations of proteoglycan andhyaluronate in synovial fluid by taking intoaccount the volume of synovial fluid.

Figure 4 shows the total concentrations ofproteoglycan and hyaluronate in synovial fluidwhen the volume corrections were made. ThePSS and PSS plus MPA induce an initial releaseof proteoglycans at nearly the same magnitude.When MPA was present, the proteoglycancontent of synovial fluid remained at theconstant high value throughout the observationperiod. MPA alone drastically increased thetotal proteoglycan concentration in synovialfluid, which reached its maximum value in fourdays, with a slight decrease towards the end ofthe follow up period.The corresponding total determinations of

hyaluronate in synovial fluid gave very differentresults. PSS and PSS plus MPA caused a

Figure 4 (A) Total equine proteoglycan (PG) and (B)hyaluronate (HA) in synovialfluid (mg) in the experimentalgroups offig 3. Cakulations were based on the correspondingvolumes ofsynovialfluid. The effect of(a) arthrocentesisalone, (b) PSS irrigation, (c) PSS plus MPA and(d) MPA are shozvn.

moderate increase in hyaluronate in synovialfluid, but the hyaluronate concentration beganto decrease at day 1 (MPA) and day 2 (PSS plusMPA), reaching the original value during thesample collection period. MPA alone caused anincrease in the total hyaluronate concentration,which remained constant until day 7, where-after it fell and reached the original level on day11. The increase of total hyaluronate in synovialfluid with PSS irrigation seems mainly to be dueto the increased volume of synovial fluid (from amean of 12-0 ml to a maximum value of 19-5 mlat day 2) and the simultaneous slight increase inhyaluronate concentration (0-4 mg/ml at day 0and 0-59 mg/ml at days 2 and 3). At the sametime the protein content and leucocyte countincreased in synovial fluid samples and decreasedin parallel with the total hyaluronate concen-tration (table 2). Thus, it is reasonable tosuggest that the slight inflammatory condition

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obtained with the injection of PSS causes firstthe stimulation of hyaluronate synthesis insynovial fluid, which then causes the retentionof water in the joint resulting in an increasedvolume of synovial fluid. This was then followedby the reduction of hyaluronate synthesis to itsbasal level with a parallel volume depletion anddecrease of total hyaluronate in synovial fluid tonear the original value. In the experimentswhere MPA was injected after PSS the volumereduction occurred faster. Table 2 summarisesthe results obtained with this experimentalmethod.

DiscussionSubunits of proteoglycan are released fromarticular cartilage into the synovial fluid fromlarge hyaluronate-proteoglycan aggregates bythe action of enzymes which digest the hyalu-ronate binding globular region from theproteoglycan core proteins. Two distinctmetalloproteases, able to degrade proteo-glycans,'5 have been isolated from humanarticular cartilage. These matrix metallo-proteases are secreted in the extracellular spacein the zymogen form. They can be activated byorganomercury compounds or by trypsin andtheir production is stimulated by interleukin1.16 They are zinc enzymes which requirecalcium for full activity. Neutral proteoglycandegrading enzymes from human articularcartilage have been identified as stromelysin.17These cartilage matrix metalloproteinases maybe involved in the MPA induced release ofproteoglycan into the synovial fluid fromnormal or mildly irritated cartilage. This con-clusion is indirect and is based on the inabilityofproteoglycan in synovial fluid spontaneously toaggregate with hyaluronate in synovial fluid andis supported by evidence provided by theinsensitivity of the proteoglycan HPLC peak tohyaluronidase action.

In all joints treated withMPA the proteoglycanconcentration in synovial fluid stayed consider-ably high during the sample collection period,indicating that the continuous degradation ofcartilage matrix proteoglycan aggregates occursduring this time. The effects of MPA on thecartilage may occur over a long time, asmethylprednisolone, the main metabolite ofMPA, could be detected in synovial fluid evenseveral weeks after a single intra-articularinjection of MPA.'8To calculate the total amounts of hyaluronate

and proteoglycan in synovial fluid, it wasnecessary to consider their concentrations withrespect to the volume of synovial fluid ondifferent days of sample aspiration. We usedthe forced aspiration of synovial fluid withsimultaneous measurement of the synovial fluidvolume. When measured with an isotopic tech-nique, the mean equine tarsocrural joint fluidvolume is 40 ml."9 Forced aspiration is not themost reliable method with which to measuresynovial fluid volumes, because about 40% ofthe synovial fluid still remains in the jointcavity. 0 However, if care is taken to aspirate atthe same time of day, careful estimations of thevolume of synovial fluid can be obtained.

Various histochemical staining methods havebeen used to assess the degenerative changes inthe articular cartilage. For example, tissueshave been stained using haematoxylin, eosin,toluidine blue and safranine O.2"`23 As a result,the formation of ulcers, loss of metachromasia,and cyst formation were observed as signs ofproteoglycan degradation.2' The appearance ofcartilage derived proteoglycans in the synovialfluid has been studied using several immuno-logical techniques, including the use of anti-bodies against different molecular structures ofproteoglycans. ELISA methods have beendeveloped for the determination of proteoglycancore protein hyaluronate binding region24 andfor extracted articular cartilage proteoglycans.8Monoclonal antibodies have been used todetermine keratan sulphate epitopes fromsynovial fluid.25 Many factors may influence theimmunological methods, and these can affectthe specificity and hamper the reliable deter-mination of proteoglycans released in synovialfluid. Also, they do not give any information onthe molecular size of the determined proteo-glycan. We describe here a rapid HPLCprocedure for the simultaneous assessment ofthe concentration and molecular size and formof hyaluronate and proteoglycan in synovialfluid. As this assay can be performed onunprocessed samples and less than 5 ,ul of nativesynovial fluid is required, it is useful for thestudy of cartilage metabolism and destruction.The HPLC chromatogram (fig 1) shows that

the hyaluronate and proteoglycan fractions inthe synovial fluid do not interact stably, as seenin cartilage, as their chromatographic peaks areeluted separately. This is probably due to thedigestion of the hyaluronate binding region ofthe core protein by MPA induced matrixmetalloproteases, which have been shown to becapable of degrading proteoglycan aggregates.The insensitivity of the proteoglycan peak tohyaluronidase treatment supports this sug-gestion. The proteoglycan peak may consist ofproteoglycan subunits lacking the hyaluronatebinding core protein domain. It is suggestedthat MPA in vivo causes the healthy equinearticular cartilage to express proteolytic enzymeactivity, which cleaves the hyaluronate bindingregion from proteoglycan aggregates andsubsequently causes the release of proteo-glycan subunits into the synovial fluid. Thisalso suggests that normal cartilage turnover maybe a proteinase mediated process.

In various tissue culture experiments cortico-steroids have been shown to depress thedegradation of cartilage derived from patientswith osteoarthritis and rheumatoid arthritis byinhibiting the production of various inflam-matory mediators and decreasing the activity ofproteolytic enzymes.5 2629 Corticosteroids havealso been shown to decrease the concentrationof proteoglycans in synovial fluid in the treat-ment of inflammatory joint diseases, beingindicative for the decreased breakdown ofcartilage proteoglycan aggregates.8 30

Using HPLC we were able to see the rapidrelease of large molecular size proteoglycansinto synovial fluid during PSS induced irri-gation. MPA alone or given intra-articularly at

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day 1 after PSS irrigation surprisingly increasedthe cartilage degradation as measured in termsof proteoglycan and hyaluronate concentrationsin synovial fluid. We used the tarsocrural jointirrigated with PSS as an experimental modeland there might be species differences in theaction of MPA, but this seems unlikely.Considerable ethical reasons do not allowsimilar experiments to be performed on humans.The discrepancies between cartilage in normalsubjects and patients with osteoarthritis orrheumatoid arthritis implies different modes ofaction of MPA. In patients with osteoarthritisand rheumatoid arthritis, MPA may inducelipomodulin gene activation3' and suppress theexpression of, for example, the interleukin 1gene in the well described conventional way.32This might decrease the intrinsic and extrinsicdegradation of cartilage extraceilular matrix. Incontrast, according to our findings, MPAincreases the release of proteoglycan andhyaluronate into synovial fluid from normal ormildly stimulated cartilage. Whether this is dueto some hitherto unrecognised alternative signaltransduction pathway or is based on physico-chemical properties of the MPA suspension is atpresent unknown.

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4 Paulsson M, Morgelin M, Wiedemann H, et al. Extended andglobular protein domains in cartilage proteoglycans.BiochemJ3 1987; 245: 763-72.

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6 Sledge C B, Steinberg J J. Co-cultivation models of jointdestruction. In: Dingle J T, Bordon J L, eds. Cellularinteractions. Amsterdam: Elsevier/North-Holland,Biomedical Press, 1981: 263-80.

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10 Saari H T, Konttinen Y T, Santavirta S. Synovial fluidhyaluronate: a study using high performance liquidchromatography with size exclusion column. MedicalScience Research 1989; 17: 99-101.

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performance liquid chromatography. Ann Rheum Dis 1989;48: 565-70.

12 Saari H T, Konttinen Y T, Tulamo R M, Antti-Poika I,Honkanen V. Concentration and degree of polymerizationof hyaluronate in equine synovial fluid. AmJI Vet Res 1989;50: 2060-3.

13 Saari H T. Oxygen derived free radicals and synovial fluidhyaluronate. Ann Rheun Dis 1991; 50: 389-92.

14 Wagner A E, McIlwraith C W, Martin G S. Effect of intra-articular injection of orgotein and sakine solution on equinesynovia. Am J Vet Res 1982; 43: 594-7.

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