Catabolism of Hyaluronan in the Knee Joint of the Rabbit

Matrix Vol. 12/1992, pp. 130-136 © 1992 by Gustav Fischer Verlag, Stuttgart

Catabolism of Hyaluronan in the Knee Joint of the Rabbit ULLA B. G. LAURENT 1 ,2, J. ROBERT E. FRASER 3 , ANNAENGSTROM-LAURENT1 ,4, ROLFK. REED 1 ,5, LAURITZB. DAHL 1 ,6 andTORVARDC. LAURENT1 1 2

3 4

5 6

Department of Medical and Physiological Chemistry and Department of Ophthalmology, University ofUppsala, Sweden; Department of Medicine, University of Melbourne, Australia; Department of Rheumatology, Central Hospital of Falun, Sweden; Department of Physiology, University of Bergen, Norway; and Department of Pediatrics, University of Troms0, Norway.

Abstract Catabolism of hyaluronan was studied by injecting hyaluronan labelled with [125 I]-tyramine cellobiose ([ 125 I]_TC) into knee joints of rabbits. After endocytosis [125 I]_TC remains intracellulady allowing localization of the site of catabolism. At 6 hours after injection 63 % could be recovered in and around the joint, while at 48 hours 32% remained locally. Chromatography showed that 12% of the injected tracer was degraded in joint tissues at 6 hours, increasing to 33 % at 24 hours. There was no apparent degradation within the joint fluid. No tracer was found in the regional lymph glands, but 16% of the injected tracer was detected in the liver at 24 hours. This investigation demonstrates that hyaluronan in the joint can be degraded both locally and in the liver. Key words: catabolism, hyaluronan, joint fluid, joint tissue.

Introduction Hyaluronan (hyaluronate, HYA) is the main glycosaminoglycan in normal synovial fluid (Meyer et aI., 1939) and responsible for the viscous and elastic properties of the fluid. The synovial fluid protects the cartilage and transports metabolites to and from the cartilage (Sundblad, 1965; Balazs, 1974). HYA is produced by the synovial lining cells (Baxter et aI., 1973) and can leave the joint via the lymphatic system either to be taken up in the local lymph nodes (Fraser et aI., 1988), or after entering the bloodstream, by the liver (Smedsrod et aI., 1990) where it is taken up and degraded. The concentration of HYA in normal synovial fluid in man is 2-3 mg/ml and in rabbit 3.9 mg/ml (Sundblad, 1965); the average molecular mass is several millions (Sundblad, 1965). The concentration and molecular mass

of HYA in inflamed joints are often reduced although the total content is increased due to the greater volume of fluid (Sundblad, 1965; Balazs et aI., 1967; Dahl et aI., 1985). Furthermore, an increased level of HYA has been observed in blood (Engstrom-Laurent and Hallgren, 1985), and urine (Laurent et aI., 1987) in inflammatory joint disease. Concentrated HYA was introduced in veterinary medicine by Rydell et a1. (1970). They injected 1% HYA and cortisone into arthritic joints of race horses and found an improvement of function that lasted longer than when cortisone was used alone. Since then HYA has been used in a number of studies on osteoarthritis in experimental animals and in man, as well as in tendon surgery to reduce adhesions (reviewed by Strachan et aI., 1990). As the HYA in the joint has proven to be of clinical interest we have turned our attention to its catabolic pathways. In a recent paper we studied its turnover rate in the

Hyaluronan Catabolism in Joint rabbit knee joint after injection of hyaluronan labelled with tritium in the acetyl groups (Brown et aI., 1991). We utilized the observation that this labelled HYA is almost quantitatively degraded to 3H2 0, the appearance of which can be followed in blood (Laurent et aI., 1988). HYA with a molecular mass of several millions, as in normal synovial fluid, had a half-life of 13 h while a HYA with lower molecular mass (M r = 0.09 X 106) exhibited a half-life of 10h. The present study was designed to find out the site of catabolism, especially if there is a local degradation of HYA within or around the joint cavity. HYA was labelled with 125 [ I]-tyramine cellobiose (Dahl et aI., 1988). This ligand will be retained in the lysosomes of the cells which have endocytosed and degraded the HYA (Pittman et aI., 1983; Dahl et aI., 1988). After extraction of labelled material and separation of high and low molecular mass fractions by gel chromatography one can estimate the amount of labelled HYA degraded in a tissue.

Materials and Methods Tracer preparation

Hydrazinolytically modified HYA substituted with tyramine-cellobiose (TC) was the same as described by Dahl et aI. (1988). The product contained one TC-ligand per 130 disaccharide units. The weight average molecular mass was 198,000 Da as determined by chromatography (Laurent and Granath, 1983; Lebel et aI., 1989). The product was iodinated as described previously (Laurent et aI., 1991) and transferred to phosphate-buffered saline. On the day of the animal experiments (which was one day after labelling) the [125 I]-TC-HYA was applied to a PD-lO column (Pharmacia, Uppsala). The first peak which contains high molecular weight material was collected. In this way radioactive ligand which had been spontaneously released from the polymer was removed from the solution to be injected. The released ligand is absorbed to the gel and eluted late in the chromatogram, after Vr and called "late peak" (Dahl et aI., 1988). Finally, the preparation was sterilized by ultrafiltration (MillexR-GS, Millipore SA, France). Animals

Eight male New Zealand White rabbits weighing 1.80 to 2.68 kg were used in the experiments. They had free access to food and water throughout the experimental period.

131

20 mg/ml, Bayer). An overdose of pentobarbital was used to sacrifice the animals, two at each time point, at 6, 12,24, 48 h after the injections. Intra-articular injections

Labelled hyaluronan (5.6 f..tg) was injected in a volume of 0.15 ml into the anterior compartment of the knee joint with a needle of 0.6 mm diameter inserted through the patellar ligament. The amount of radioactivity injected in each rabbit was 3.26 x 106 d. p. m. (54 x 103 Bq). The animals were returned to their cages after the injections. Recovery oflabelled HYA from the joint cavity

At the end of each study, immediately after the animal had been sacrificed, the injected joint was quickly and repeatedly irrigated with four separate volumes of isotonic saline to a total of approximately 5 mI. The precise amount of each volume injected and aspirated was determined by weight. For calculation of the total radioactivity remaining in the joint cavity at the end of the study interval, the unrecovered residue of irrigation fluid was assumed to contain the same level of [125 I]activity as the last volume recovered, and a corresponding amount was added to the sum of the activity measured in each volume aspirated. The joint fluid saved from two rabbits at each of the four time points was combined and sodium azide was added to give a final concentration of 0.02%. The material was then kept frozen until the day of chromatography, when it was thawed and centrifuged at 2000 r. p. m. Recovery ofsynovial lining and other tissue from joints

After removal of as much muscle as possible, each joint was removed intact by transection of the bones with margins > 1 cm beyond the upper and lower limits of the synovial cavity. The joint was then divided through the capsule and the cruciate ligaments. The broad anterosuperior expansion of the synovial lining and capsule was excised at its reflections on the femur and round the patella and set apart, since it was the most suitable portion for later extraction (later referred to as joint capsule). Cartilage pared from the femoral condyles was weighed and also kept for individual measurement. The remaining bulk of tissue was aggregated for determination of radioactivity, and then saved at - 20°C (see below). Recovery from other tissues

Anaesthesia

Before the intra-articular injections of tracer the animals were anaesthetized with ketamine 35 mg/kg (Ketalar R, 50 mg/ml, Parke-Davis) and xylazin 5 mg/kg (Rompun vet.

Regional lymph nodes in the popliteal and inguinal regions were collected as well as spleen and liver. The whole liver was weighed and the radioactivity determined on randomly selected samples. Urine was aspirated from the blad-

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der and kept frozen with 0.02% azide until chromatographed. Peripheral blood was also obtained. Radioactivity measurements

All specimens were weighed and radioactivity measured in a Packard 1566 Auto-Gamma counter.

and urine and on extracts of liver and joint capsule. Five weeks later the remaining part of the joints was taken out of the freezer, HYA was extracted, and chromatography performed. Three ml portions of the soluble extracts from each tissue CPM I ml 100.000

Extraction ofTC-HYA from the tissues

Extraction of [125 I]-TC-HYA was performed after determination of the radioactivity in samples from the joint capsule and the liver. The tissue samples from the two rabbits at each time point were homogenized by cutting the tissue into small pieces with a pair of scissors (except for the patella). The tissue was then submerged in 0.25 M phosphate buffer, 0.5 M NaCl with detergent (1 % Tween 20, Merck, Germany) and 0.02% sodium azide. The vials were rotated end-over-end at + 4 °C for 24-36 h. The samples were then kept frozen, (the patella was not frozen with the capsule) until the day of chromatography when they were thawed and centrifuged, first at 2000 r. p. m. and then by ultracentrifugation in a Beckman L8-M Ultracentrifuge for 45 min at 63,000 x g. To extract the labelled TC-HYA from the remaining part of the joints, containing bone, the tissues were cut with a sharp knife and then extracted and centrifuged twice. The mean recovery of label in the extractions was 82% (range 72-93%) from liver and 72% (range 70-76%) from joint tissues.

Joint fluid

vt

f

~

6 hr

50.000

~ 30.000

12 hr

15.000

10.000

\

50

100

150

200

250

24 hr

Chromatography

Chromatography on the combined material from two rabbits at each time point was carried out on synovial fluid

5.000

~

Table 1. Recovery of label (expressed as percentage of injected tracer). In joint"

In tissues collected b

Total recoveryC

6h

65.4 59.8

73.2 67.8

80.9-83.1 75.0-76.2

12h

51.3 47.3

74.1 60.7

82.3-88.9 68.3-75.3

Time

24h

49 47.4

65.6 67.7

84.2-105 78.9-91.5

48h

33.6 29.8

54.9 44.3

62.5-72.0 62.2-85.0

" Radioactivity in the joint cavity and surrounding tissues. b Sum of radioactivities in all collected tissues. CCalculation based on the assumption that the total extracellular space has the same concentration of label as blood and that the urinary samples are representative for all urine (daily urinary production 40-100 ml).

2.000

....-....

.... 48 hr

~ 1.000

50

100

~ 150

200

-

250

ml eluate

Fig. 1. Chromatograms of joint fluid at 6, 12, 24 and 48 h after injection of radioactively labelled hyaluronan into the knee joint. The peak eluted at void volume (Vo ) (from 50 to 120 ml) represents high molecular mass material and the peak at total volume (V,) (from 120 to 210 ml) low molecular mass material. The peak at 210-250ml represents the "late peak", a retarded peak which is discussed in the Methods. The ordinate has been adjusted so that the area under each chromatogram represents the total activity recovered.

Hyaluronan Catabolism in Joint were applied to a 1.6 x 84 cm Sephacryl S-300 column (Pharmacia, Uppsala, Sweden). The column was eluted with phosphate-buffered saline containing 1% Tween 20 and 0.02% sodium azide. The flow rate was maintained at 18 ml h -1, and 3 ml fractions were collected. Void volume (V 0) and total volume (Ve) were determined from the elution of the bacteria Serratia marcescens and 3H2 0, respectively. The radioactivity was essentially eluted in three peaks; one high molecular peak at V0> one low molecular peak at V e and one distinctly retarded peak ("late peak"). The definition of the Vo peak has somewhat arbitrarily been given to the material eluted between 50 and 120 ml and of the Ve peak to material eluted between 120 and 210 m!. The late Joint tissue

CPM/ml 100.000

6 hr

50.000

133

peak (210 - 25 0 ml) corresponds to spontaneously released ligand formed during the processing of the material and was supposed to have been released from both the high and the low molecular portions with equal rate.

Results Distribution Of 125 I_TC-HYA At 6 h the recovered synovial fluid contained 25% of the injected dose while at 48 h less than 1% of the injected dose was left. At the same time points altogether 63 % and 32%, respectively, could be recovered within or around the joint (TableI). In liver only 5 % was detected at 6 h. The amount increased to 14 -17% at the later time points. Less than 0.1 % and 0.05% was found in the spleen and regional lymph glands, respectively. The total radioactivity recovered from the tissues analyzed was 71 % at 6 hand 50% at 48 h. Assuming a urinary volume of 40-100 mllday and that radioactive material is evenly distributed between blood and extracellular space it is possible to account for about 80% of the injected dose (TableI).

Chromatograms 12 hr

80.000 -

40.000

Joint fluid. The material was of high molecular mass at the first time points, but some low molecular mass HYA could be detected at 24 and 48 h (Fig. 1). Joint tissue. Chromatograms originally run on extracts Table II. Comparison of results from chromatographies of extracts of the joint capsule and of the remaining part of the joint (stored for five weeks).

40.000

24 hr

20.000

Time

48 hr 20.000

6h 12h 24 h 48 h

100

150

200

250

ml eluate

Fig. 2. Chromatograms of extracts of the joint capsule at 6, 12, 24 and 48 h after injection of radioactively labelled hyaluronan into the knee joint. For definitions see Fig. 1. The ordinate is adjusted as in Fig. 1 to represent the total radioactivity recovered both in the joint capsule and the surrounding tissues (see also Methods and Table II).

9.5% 9% 11%

10%

0.70/0.30 0.62/0.38 0.26/0.74 0.23/0.77

Time

Joint tissue minus joint capsule ratio high/low mol weight "late peak"

6h 12 h 24h 48 h

28% 26% 33% 30%

10.000

50

Joint capsule ratio b high/low mol weight "late peak"·

0.67/0.33 0.56/0.44 0.29/0.71 0.21/0.79

• "late peak": material eluted between 210 and 250ml (see Methods, last paragraph, for definition. b ratio high/low mol weight: proportion eluted at Vo, 50-120 ml and V" 120-210ml, respectively.

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U. B. G. Laurent et al. Injection in joint (24 hr)

CPM Iml

Urine

2.000

1.000

Liver 10.000 5.000

50

100

150

200

250

experiments showed that the "late peak" had increased after five weeks storage, but that the proportion of high molecular mass to low molecular mass HYA was the same (Table II). Twelve percent of the injected tracer was recovered with low molecular mass at 6 h, increasing to 33 % of the injected dose at 24 h (Fig. 4). Urine. In all urine samples more than 95% of the radioactive material was of low molecular mass. Figure 3 shows a chromatogram of urine taken 24 h after injection of the label. Liver. All samples analysed contained more than 85% low molecular HYA (Fig. 3). The data on the distribution of labelled high and low molecular mass material in the various tissues has been collected in Figure 4.

ml eluate Fig. 3. Chromatograms of urine and of extract of the liver, both at 24 h after injection of radioactively labelled hyaluronan into the knee joint. The ordinate is adjusted as in Fig. 1.

• D

40 ~

VI

0 -0 -0

high MW lowMW

30

~

U ~

'c 0 20 0

0

10

6 12 24 48 Joint fluid

6 12 24 48 Joint tissue

6 122448 Hours Liver

Fig. 4. Percentage of injected dose recovered in the joint fluid, the tissue around the joint cavity and the liver at 6, 12, 24 and 48 h after injection of (125 I]-tyramine-cellobiose hyaluronan in the knee joint. High and low molecular mass fractions are shown in light and dark shade, respectively. Each value is the mean of two experimental animals.

of the joint capsule showed (Fig. 2) that low molecular mass material accounted for 32% at 6 hand 78% at 48 h. Five weeks later chromatograms were also run on extracts of the remaining part of the joint in order to check if the results obtained from extracts of the joint capsule were representative for all the tissue around the joint. These

Discussion Turnover of HYA in the rabbit knee joint has previously been studied by Denlinger (1982), who injected different volumes of concentrated (10 mg/ml) HYA and measured the amount left at different times after injection. From this study a half-life of 0.5 -1 day could be calculated. A similar rate of disappearance was found under more physiological conditions when tracer amounts of HYA were injected (Brown et aI., 1991). These kinetic studies give, however, no clue to the site of degradation. Antonas et a1. (1973) injected rt 4 C]-labelled HYA into the knee joint of rabbits and studied the uptake by autoradiography and also measured the radioactivity in tissue digests. In the synovium, activity was found at 2-48 h, and in the articular cartilage at 6 h - 7 days after injection. Activity was also found in the popliteal lymph node already at 15 min; also at 1 hand 6 h but not at 24 h. Radioactivity could be measured in plasma already at 1 h and the peak appeared at 3 h after the intraarticular injection. It was later shown in sheep that popliteal lymph contains at least ten times as much HYA as blood plasma (Laurent and Laurent, 1981) indicating that HYA is transported from the tissues via lymph to blood. It is then cleared from the circulation with a half-life of 2 - 5 min (Fraser et aI., 1981). The above observations made it probable that at least part of the HYA in the joints is removed via the lymphatics. Further support for this hypothesis was obtained when Engstrom-Laurent and Hallgren (1987) showed that physical exercise, especially in rheumatoid patients increases the HYA level of blood. The present investigation demonstrates that a considerable amount of the HYA in the joint is degraded locally. It is unlikely that any HYA is degraded in the synovial fluid itself; the small quantities of degradation products observed at the late time points (Figs. 1 and 4) presumably

Hyaluronan Catabolism in Joint are due to degradation in surrounding tissues. As up to 33 % of the total injected HYA could be recovered as low molecular mass material in the tissues surrounding the joint, this figure can be regarded as the minimum amount degraded locally. The observation is not completely unexpected as in a similar study on rabbit skin it was found that local degradation of [125 I]_TC_HYA amounted to about 25% in 24 h (Laurent et aI., 1991). There are two intrinsic uncertainties in this investigation which are due to the technique employed. During labelling the HYA becomes partly depolymerized and has a molecular weight at least ten times lower than native HYA. Secondly, substitution with TC introduces nonphysiological ligands on the polysaccharide although the groups are widely separated. We believe that these objections are contradicted by the facts that HYA of widely different molecular mass display similar catabolic rates (Brown et aI., 1991) and that the uptake of TC-HYA in liver endothelial cells is inhibited by native HYA (Dahl et aI., 1988). The latter observation indicates that the two polymers compete in binding to the same receptor. Another problem with the probe is a spontaneous release of a radioactive product that appears as a "late peak" in the chromatogram (Dahl et aI., 1988). The experiments described in Table II clearly demonstrate, however, that the release takes place to an equal extent from high and low molecular TC-HYA and that the ratio of high/low molecular material is constant even if the material has been stored for 5 weeks. A surprising finding, however, was that we could not detect any tracer in the popliteal nor in the inguinal lymph nodes, while after injecting TC-HYA in skin the local lymph nodes contained> 10% at 6-48 h (Laurent et aI., 1991). It has been shown previously that lymph nodes perfused with tritium-labelled high molecular HYA to a large extent extract the polymer (Fraser and Laurent, 1989). We have no explanation for the present observation except that high molecular HYA has a higher affinity to cellular receptors than low molecular mass polymer (Laurent et aI., 1986). From a functional and clinical point of view it is intriguing that hyaluronan in synovial fluid has such a rapid turnover and that, inspite of its large molecular size, it can be transported into the synovial tissue and lymph to be degraded or removed. The rapid turnover would not be necessary if the polysaccharide had an essentially mechanical function as a lubricant in the joint. We would therefore like to propose another possible function of hyaluronan, namely that of a scavenger substance. Particulate material, such as debris and inflammatory cells which are deleterious to the joint, could be immobilized in the viscous polymer network and transported away with the same rate as hyaluronan itself.

135

Acknowledgements This work was supported by grants from the Swedish Medical Research Council (Grant 03x-4), GustafV:s 80-Year Foundation, Magnus Bergvall's Foundation, Pharmacia AB and Ester AsbergLindberg's Foundation). References Antonas, K. N., Fraser, J. R. E. and Muirden, K. D.: Distribution of biologically labelled radioactive hyaluronic acid injected into joints. Ann. Rheum. Dis. 32: 103-111, 1973. Balazs, E.A.: The physical properties of synovial fluid and the special role of hyaluronic acid. In: Disorders ofthe Knee, ed. by Helfet, J., J. B. Lippincott, Philadelphia, 1974, pp. 63 - 75. Balazs, E.A., Watson, D., Duff, J.F. and Roseman, S.: Hyaluronic acid in synovial fluid. J. Molecular parameters of hyaluronic acid in normal and arthritic human fluids. Arthr. Rheum. 10: 357-376,1967. Baxter, E., Fraser, ]. R. E. and Harris, G. S.: Fractionation and recovery of secretions of synovial cells synthesized in culture with radioactive precursors. Ann. Rheum. Dis. 32: 35-40, 1973. Brown, T.]., Laurent, U. B. G. and Fraser, ]. R. E.: Turnover of hyaluronan in synovial joints: Elimination of labelled hyaluronan from the knee joint of the rabbit. Exp. Physiol. 76: 125 -134, 1991. Dahl, L.B., Dahl, J.M., Engstrom-Laurent, A. and Granath, K.: Concentration and molecular weight of sodium hyaluronate in synovial fluid from patients with rheumatoid arthritis and other arthropathies. Ann. Rheum. Dis. 44: 817-822, 1985. Dahl, L. B., Laurent, T. C. and Smedsmd, B.: Preparation of biologically intact radioiodinated hyaluronan of high specific radioactivity: coupling of 125I-tyramine-cellobiose to amino groups after partial N-deacetylation. Anal. Biochem. 175: 397-407,1988. Denlinger,].: Metabolism of sodium hyaluronate in articular and ocular tissues (Thesis for Docteur d'Universite en Sciences Naturelles), Lille, France, 1982. Engstrom-Laurent, A. and Hallgren, R.: Circulating hyaluronate in rheumatoid arthritis: relationship to inflammatory activity and the effect of corticosteroid therapy. Ann. Rheum. Dis. 44: 83-88,1985. Engstrom-Laurent, A. and Hallgren, R.: Circulating hyaluronic acid levels vary with physical activity in healthy subjects and in rheumatoid arthritis patients. Relationship to synovitis mass and morning stiffness. Arthr. Rheum. 30: 1333 -1338, 1987. Fraser,]. R. E., Kimpton, W. G., Laurent, T. c., Cahill, R. N. P. and Vakakis, N.: Uptake and degradation of hyaluronan in lymphatic tissue. Biochem.]. 256: 153-158,1988. Fraser, J. R. E. and Laurent, T. c.: Turnover and metabolism of hyaluronan. In: The Biology of Hyaluronan, Ciba Foundation Symposium 143, ed. by Evered, D. and Whelan,]., John Wiley and Sons, Chichester, 1989, pp. 41-54. Fraser,]. R. E., Laurent, T. c., Pertoft, H. and Baxter, E.: Plasma clearance, tissue distribution and metabolism of hyaluronic acid injected intravenously in the rabbit. Biochem.]. 200: 415 -424, 1981. Laurent, T. c., Fraser,]. R. E., Pertoft, H. and Smedsmd, B.: Binding of hyaluronate and chondroitin sulphate to liver endothelial cells. Biochem.]. 234: 653-658, 1986. Laurent, T. c., Lilja, K., Brunnberg, L., Engstrom-Laurent, A., Laurent, U. B. G., Lindqvist, U., Murata, K. and Ytterberg, D.:

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Urinary excretion of hyaluronan in man. Scand. J. Clin. Lab. Invest. 47: 793-799, 1987. Laurent, U. B. G., Dahl, L. B. and Reed, R. K.: Catabolism of hyaluronan in rabbit skin takes place locally, in lymph nodes and liver. Exp. Physiol. 76: 695 -703,1991. Laurent, U.B.G., Ftaser, J.R.E. and Laurent, T.e.: An experimental technique to study the turnover of concenttated hyaluronan in the anterior chamber of the rabbit. Exp. Eye Res. 46: 49-58, 1988. Laurent, UB.G. and Granath, K.A.: The moleculat weight of hyaluronate in the aqueous humour and vitreous body of rabbit and cattle eyes. Exp. Eye Res. 36: 481-492, 1983. Laurent, U. B. G. and Laurent, T. e.: On the origin of hyaluronate in blood. Biochem. Internat. 2: 195-200,1981. Lebel, L., Smith, L., Risberg, B., Lautent, T. and Gerdin, B.: Increased lymphatic elimination of interstitial hyaluronan during E. coli sepsis in sheep. Am. J. Physiol. 256: HI524-1531, 1989. Meyer, K., Smyth, E.M. and Dawson, M.H.: The isolation of a mucopolysaccharide from synovial fluid. J. BioI. Chem. 128: 319-327,1939.

Pittman, R.e., Carew, T.E., Glass, e.K., Green, S.R., Taylor, e. A. Jr. and Attie, A. D.: A radioiodinated, intracellularly trapped ligand for determining the sites of plasma protein degradation in vivo. Biochem. J. 212: 791-800, 1983. Rydell, N. W., Butler, J. and Balazs, E.A.: Hyaluronic acid in synovial fluid. VI Effect of intra-articular injection of hyaluronic acid on the clinical symptoms of arthritis in track horses. Acta Vet. Scand. 11: 139-155,1970. Smedsmd, B., Pertoft, H., Gustafsson, S. and Laurent, T. e.: Scavenger functions of the liver endothelial cell. Biochem. J. 266: 313-327, 1990. Strachan, R.K., Smith, P. and Gardner, D.L.: Hyaluronate in rheumatology and orthopaedics: Is there a role? Ann. Rheum. Dis. 49: 949-952, 1990. Sundblad, L.: Glycosaminoglycans and glycoproteins in synovial fluid. In: The Amino Sugars, VoI.IIA, ed. by Balazs, E.A. and Jeanloz, R. W., Academic Press, New York 1965, pp. 229- 250. Dr. Ulia Laurent, Department of Medical and Physiological Chemistry, Box 575, University of Uppsala S-75123, Uppsala, Sweden.