Synovial Fluid Hyaluronate in Rheumatoid Arthritis By DAVIDHAMERMAN AND HILDASCHUSTEFI By several analytic methods, the hyaluronate hexosamine concentration of synovial fluid was found low in the joint effusions of rheumatoid arthritis in comparison to normal synovial fluid. .4n increase in nonhyaluronate hexosamine was observed in rheumatoid synovial fluid of increased volume. Relative viscosity measurements after equilibrating hexosamine Concentration by dilution revealed few differences between rheumatoid and normal fluid. Plure methodos analytic monstrava basse concentrationes de hexosamina hyaluronate in le effusiones de liquido synovial de patientes con arthritis rheumatoide, comparate con normal liquido synovial. Esseva observate un augment0 de hexosamina non-hyaluronate in rheumatoide liquido synovial de volumine supranormal. Mesurationes de viscositate post equilibration del concentration de hexosamina per dilution non revelava multe differentias inter liquido rheumatoide e normal. T HE CONCENTRATION of hyaluronate in synovial fluid from patients with rheumatoid arthritis* and in normal synovial fluid has been determined by a number of workers. Using different methods, some workers have reported a similar concentration,lP2 and others a decreased c ~ n c e n t r a t i o n ~ ~ ~ of hyaluronate in rheumatoid synovial fluid compared with normal fluid. In this repor:, the hyaluronate concentration of rheumatoid synovial fluid was determined by several methods described in a previous study of normal synovial fluid.6 Hyaluronate was precipitated in mucin clots from rheumatoid synovial fluid by adding acetic acid or a cobalt salt to the fluid, and was measured indirectly by determining hexosamine in these clots. A part of the total hexosamine of synovial fluid is not a component of hyaluronate, and it was shown in the method adopted that no hexosamine other than hyaluronate hexosamine was precipitated in mucin clots and that no hyaluronate hexosamine remained in the supernatant after a mucin clot was formed. Another method used to determine hyaluronate in rheumatoid synovial fluid was to measure the fall in hexosamine after digestion of the fluid with testicular hyaluronidase and dialysis of the digestion products. Figures for hyaluronate hexosamine in rheumatoid synovial fluid obtained by a mucin clot method and by the hyaluronidase digestion method agreed. Normal and rheumatoid synovial fluids were diluted by adding a buffer to the fluids until the hyaluronate hexosamine concentration of each fluid, measured by the hyaluronidase digestion method, was the same. The relative From the Department of Medicine, Albert Einstein College of Medicine, and the Bronx Munictpal Hospital Center, New York, N.Y. This study was supported by Graduate Training Grant 2A-5082 (a) (C), National Institute of Arthritis and Metabolic Diseases, Nattcnal Institutes of Health, and by a grant from the New York State Chapter, Arthritis and Rheumatha Foundation. One of the authors (D.H.) is Markk Scholor in Medtcal Science. 'Synovial fluid obtained from patients with rheumatoid arthritis is more briefly noted hereafter RS rheumatoid synovial fluid. 523 524 DAVID HAMERMAN AND HILDA SCHUSTER viscosity of these normal and rheumatoid fluids of equivalent hyaluronate hexosamine concentration was then compared. METHODS AND PROCEDURES .itetkods Patients with definite rheumatoid arthritis: with active disease at some time in the course of these studies, were used. All patients were female. Synovial fluid was obtained from the knee joints. Normal synovial fluid was obtained from the knees of eight deceased and two living subjects without evidence of joint disease and within the age range of most of the patients (50 to 66 years). Samples of synovial fluid for analysis were weighed rather than pipeted, and results are expressed in terms of grams rather than milliliters of synovial fluid. The density of rheumatoid synovial fluid was found not to differ from water within the limits of the precision of the method. Grams and milliliters may therefore be used interchangeably. 1. Hexosamine determinations on synooial fluid and mucin clots.-The method of measuring total hexosamine in synovial fluid, the technic of forming mucin clots by adding acetic acid or a complex cobalt salt to synovial fluid, and the method of analyzing for hexosaniine in mucin clots have been reported in detail." One additional method to precipitate hyaluronate hexosaniine in mucin clots from rheumatoid synovial fluid was studied. Synovial fluid (0.3Gm. to 0.4 Gm. ) was weighed in Pyrex test tubes ( 13 by 100 mm. ) and diluted 1:l with tap water. A mucin clot was formed in the cold by adding a volume of glacial acetic acid equal to 1 per cent of the total volume of diluted synovial fluid. 2. Hyaluronidase digestion of synoviol fluid and mucins.-This was carried out exactly as previously described." Hexosamine of a dialyzed control less hexosamine of a hyaluronidase-treated dialyzed sample was a measiire of hyaluronate hexosamine. Procedures 1. Herosomine of mucin clots.-By contrast with the findings in nornial synovial fluid, all the methods used to precipitate hyaluronate hexosamine in mucin clots from rheumatoid synovial fluids did not give the same results (table 1). There was always less hexosamine in mucins precipitated with glacial acetic acid from synovial fluids diluted 1:5, and more hexosaniine in mucins precipitated with the cobalt salt. These differences were due to incomplete precipitation of hyaluronate hexosamine by the former method, and to precipitation of proteins containing nonhyaluronate hexosamine by the latter method. The failure o f glacial acetic acid to precipitate all the hyaluronate hexosamine from fluids diluted 1:5 was shown by the considerable loss of hexosamine following hyaluronidase digestion and didysis of these mucin supernatants (table 1). Precipitation by the cobalt salt of protcins containing nonhyaluronate hexosamine was shown by the high nitrogen to hexosainine ratios of these mucins and by the failure of streptococcal hyaluronidase to digest and render dialyzable all the hexosamine in these mucins. Almost all the hexosamine in mucins precipitated by glacial acetic acid from fluids diluted 1:1 was digested by hyaluronidase and rendered dialyzable. No hexosamine was lost after hyaluronidase digestion and dialysis of these mucin supernatants. This method was therefore used to determine hyaluronate hexosamine in rheumatoid synovial fluids by the mucin clot technic. 2. Hyaluronidase digestion of synovial fluid.-Incubation of synovial fluid at pH 4.8 with testicular hyaluronidase and subsequent dialysis removed that part of the total hexosamine that was hyaluronate hexosaniine. Table 1 shows that in normal synovial fluid a figure for hyaluronate hexosamine obtained by this method agreed closely with hexosamine of any mucin. In rheumatoid synovial fluids hyaluronate hexosamine obtained by the method of hyaluronidase digestion agreed only with the hexosamine of rnucins precipitated by glacial acetic acid from fluids diluted 1:l with water. Rheumatoid synovial fluids were examined for the presence of hyaluronidase inhibitors,' and none were found. Table 2 shows that testicular hyaluronidase completely digested and rendered dialyzable the hexosamhe of chondroitin sulfate or a mucin solution (as a source of hyaluronate ) in rheumatoid synovial fluid. 525 SYNOVIAL FLUID HYALURONATE I N RHEUMATOID ARTHRmS TABLE1.-Studies on the Specificitu of Methods to Determine Hyaluimate Hexosamine in Rheumatoid Synooial Fluids’ Results of hyaluronidase digestion and dialysis HyalurMucin Control Total hx. onate h r . molar ratio hx. mg./Gm. ma./Gm. N./hx. mg./Grn. Sample After 76TRU T. Hyd. hx. ma./Gm. After Hx. 75TRU FIX. rendered S. Hyd. rendered dialyzable hx. dialyzable % mg./Gm. % ~ Synovial fluid GL mucin Z 10% A. mucin f co. mucin GI. sup?. 10% A. sup’t. co. sup’t. 1.60 Synovial fluid GI. mucin p G1’. m u c h S 10% A. mucin co. mucin ? G1. sup’t. Gl’. sup’t. 10% A. sup’t. co. 3upY. .84 Synovial fluid 1 2 a f 16.1 266 240 .067 .027 20.4 .228 16.8 253 .024 .021 .120 .124 .27 -20 -28 .a2 .43 1.37 89 80 .030 .028 R8 92 8 1 .031 88 .177 .122 .166 .117 4 A24 .421 32 88 .313 .227 .069 .066 26.1 .299 .964 294 204 220 .222 .om 78 72 74 .211 42 76 68 62 69.4 10% A. mucin 19.2 24.6 co. mucin .64 61.6 .606 .240 .241 274 .la4 .189 .210 .210 21 7 6 6 384 37 .014 82 76 .058 GB .lo3 G1’. sup’t. 236 .05Y .OQ2 206 co. sup%. .236 365 .l54 10% A. sup?. 74 18.1 19.4 -51 .49 .b4 g GI’.mucin S 1.18 1.22 1.26 1.23 219 .076 18 7 7 *Abbreviations : hx.. hexosamine; N.. nitrogen; GI.. g!aeial acetic acid added to synovial fluid diluted 1:5; GI,., glacial acetic acid added to synovial fluid dilutpd 1:l: 10% A., 10% acetic acid; Co., cobalt salt; sup’t.. supernatant; T. Hyd., teaticular hyaluronidase; S. Hyd.. streptococcal hyaluronidase; TRU. turbidity reducing units. TABLE?.-Digestion b y Testiculur Hyuluroniduse of Substrutes Added to Rheumatoid Synotrial Fluid Hx. of substrate digested by T. Hyd. and rendered dia!yzable Substrate (%) Chondroitin sulfate Chondroitin sulfate rheumatoid S.F. Normal mucin Noniial mucin + rheumatoid S.F. + 97 95 89 81 kXJLTS 1. Herosumine in rheumatoid synoviul fluid.-The total hexosamine and lryaluronate hexosamine in normal fluids and in rheumatoid synovial fluids are compared in table 3. Hyaluronate hexosamine in rheumatoid fluids was determined by hexosamine analyses of mucin clots formed with acetic acid. In some rheumatoid fluids the technic of hyaluronidase digestion was also used to measure hyaluronate hexosamine. In all rheumitoid effusions whose volume” was slightly or considerably greater than normal, the hyaluronate ‘Figures for the volume of synovial fluid obtained from the knees of patients with rheumatoid arthritis should be regarded as approximate. A slight inaccuracy in the estimation of the volume can arise from failure to withdraw completely all the fluid from a joint. 528 DAVID HAMERMAN AND HILDA SCHUSTER TABLE3.-Synoviol Fluid Hynhrronate Herosumfne Levels in Patients with Rhcumutoid Arthritis Hyaluronate hx. mg./Gm. S.F. Total Age Normal hx. Knee mg./Gm. :0-66 Mucin GL 1.41 -c.20+ 2 44 16 12 .14 1.07 6 <1 13 1.77 <1 14 66 Rt. 1.22 10%A. 1.26 GI. 61 Rt. Lt. Rt. Rt. 1.17 lOO/,A. 32 3/67 Rt. 1.36 GI. .18 11/66 Rt. 1.00 lOO/,A. .29 Lt. 1.37 CP. .49 .61 Rt. 1.31 Gl’. .06 Lt. 1.43 GI. 56 9/67 Lt. 1.44 GI.. 4/67 Rt. 1.40 1.08 GI’. G1’. 1.14 G1’. G1’. 1.26 1.23 1.26 1.65 GI’ .42 .43 4/66 8/66 11/67t 12/67# Bre. 1.69 *.la* Duration of RheumVolume arthritis. atoid T.Hyd. ml. years Nodules Factor Rei. 9/66 Nor. Swe. WaL 16 27 6 + 10 + + + 0 0 0 0 30 68 44 + -I- 0 63 4/67 3/67 GL GL o -I- 0/6 40 0/11 I 4 10 12 o + .64 20 2 + + .72 .64 10 2.5 + .66 .40 .K6 10 6 1.5 0 2 o + 0/6 0 0 f ~ 3/61 Gor. 69 Lt. Cin. 36 9/67 6/67 .30 4- Rt. Lt. <1 <1 Pol. 4 6/67 Bau. 64 Rt. 1.89 GI’. 15 f 8/67 Ali. 26 Lt. 1.69 Gl’. 1.04 <1 0/6 0 9/67 Pan. 67 Rt. G1’. 1.34 <I 14 + + 10/67 Sus. 28 Lt. G1’. 1.37 <I 7 0 0 GI. 1.32 <1 0/7 0 0 11167 Cil. Lt. 1.64 Rt. .84 2 .37 ~ 26 ~~ 64 Gl’. .28 Summaw 11 No. of fluids Volume S.F. mL Rheumatoid Arthritis H x . mg./Gm. S.F. Normal Hx. mg./Gm. S.F. .27 >5 0 30 3 8 26 <1 Total 1.29 1.22 1.66 Hyate .43 .27 Total 1.69 1.41 Hrate 1.30 *These 5gures are the mean value of 8 normal cases & the standard deviation. 1Th-e h r e s are the mean value of 10 normal cases f the standard deviation. #Thhpatient was on oral cortisone at the time of these studies. hexosamine concentration was reduced by a third to a fifth of normal. The total hexosamine concentration of these rheumatoid fluids was usually only slightly less than normal. The total hexosamine and hyaluronate hexosamine concentration were normal in fluids of a volume of 1ml. or less obtained from the knees of some patients with rheumatoid arthritis. Fluids of greater than normal volume and low hyaluronate hexosamine concentration were obtained from patients who complained of stiff and painful knees. X-rays of these knees showed increased soft-tissue density in the 527 SYNOVIAL FLUID HYALURONATE IN RHEUMATOID ARTHRITIS suprapatellar recess." Synovial fluids of normal volume and normal hyaluronate hexosamine concentration were obtained from patients who had minor or no complaints referable to the knees but whose disease was nonetheless tictive. In one patient (Cin., table O), however, normal fluid was obtained at a time when the disease had spontaneously remitted. Several months prior to this, when the disease was active and there were complaints of pain and stiffness in the knees, fluid of slightly increased volume and very low hyaluronate hexosamine concentration was obtained. 2. Relative viscosity of normal and rheumatoid synouial fltiids.-The relative viscosity of eight fluids of greater than normal volume and of low hyaluronate hexosamine concentration from patients with rheumatoid arthritis was compared with the viscosity of eight fluids from normal subjects. Prior to these studies, the hyaluronate hexosamine concentration of each fluid was reduced to about 0.25 mg./Gm. by adding a buffer (0.1 M NaCl and 0.1 M NaHC03. pH 8.1) to the fluids, and the fluids were then dialyzed in this buffer. The hyaluronate hexosamine concentration of each fluid was determined by the method of hyaluronidase digestion. Relative viscosity of the fluids was measured in an Ostwald viscosimeter in a constant temperature bath at 37°C. Table 4 shows that at an equivalent hyaluronate hexosamine concentration some rheumatoid fluids had a relative viscosity equal to that of the normal fluids, but slightly lower values were more frequently noted. The relative viscosity of mucins precipitated from rheumatoid synovial fluids and dissolved in buffer was lower than the viscosity of normal mucin solutions of equivalent hvaluronate hexosamine concentration (table 4). It was recognized that normal synovial fluids required a greater dilution TABLE4.--Relrrtiue Viscositu of Normal and Rheumatoid Synouial Fluids of Similar Hyaltwonate Hexosamine Concentration Dissolved mucins Diluted, dialyzed wnovial fluid Hy. hx. - ma./Gm. vrel.8 .23 4.9 4.9 1a Normal .28 .28 .24 .94 26 .26 3/67 K.2 Much Clot GI. lOO/,A. co. Hy. hx. mg./Gm. prel.. .27 28 .28 3.4 25 .26 .27 1.6 4.8 6.7 K.6 5.6 6.1 4.9 6.1 Lt. 27 6.4 Gor. Lt. .26 6.6 3/67 Nor. Rt. .21 4.8 4/67 swc. Lt. 26 4.6 9/67 GI'. 1OO/,A. co. 4/67 Cin. Rt. Lt. .26 .20 .21 4.7 6.0 6.2 Rt. 25 4.8 Lt. 11/57 Cil. C1'. co. -31 .31 1.6 3.0 1.4 3.3 *greI. ir the rehtive viscosity, or ratio of the flow time in seconds, of synovial fluid or mucin to the flow time of buffer (0.1 Y NaCI, 0.1 M NaHCOa) in an Oatwald viscosimeter at 87' C. 528 DAVID HAMERMAN AND HILDA SCHUSTER TABLES.-Effects of Zntra-Articular Znjection of Hydrocortisone or Saline on Rheumtoid Sunoijial Fluid Hyaluronate hx. mg./Gm. Volume Total hx. Total N. Knee ml. mg./Gm. much T. Hyd.mg./Gm. 11/56 Swe. Rt. Rt. Lt. Lt. 30 8 60 60 1.00 Lt. 40 12 1.37 1.68 GL’. 20 1.43 1.40 GI’. .96 1.17 1.31 lO%A. 10%A. IO%A. 1OPJoA. .29 .47 .43 -41 Diluted. dialyzed S.P. Hy. hx. mg./Gm. qrel. Intra-articular injection none 6.9 4.6 6.6 7.3 7 days after steroid none 7 days after NaCl 4/57 Swe. Lt. 3/61 Gor. 7 G1’. Rt. Rt. Lt. Lt. 10 1 6 7 .61 1.00 7.7 6.1 .24 .27 4.6, 6.1? none 5 days after steroicl .66 -11 .64 -67 7.7 6.7 .27 .26 6.4 6.7 7 days after steroid GI’. ~ ~~~ 4/61 Cin. .49 1.08 ~ 1.40 1.46 1.08 .99 GI’. GL’. GI’. G1’. ~ .65 .01 .40 .34 .66 .89 .30 -26 6.8 6.1 6.8 1.4 none ~~ .26 .24 .20 -21 none 4.7 4.3 6.0 6.2 7 days after steroid none 7 days after NaCl ~ +The relative viscosity of this synovial fluid before dilution and dialysis was 11. !The relative viscosity of this synovial fluid before dilution and diiilysis was 603. with buffer than did rheumatoid fluids to bring the hyaluronate hexosamine concentration to 0.25 mg./Gm., and as a result the final protein concentration of the normal fluids was only a sixth of that of the rheumatoid fluids. The possibility that a low protein concentration might decrease the relative viscosity of the normal fluids apart from changes in the hyaluronate was tested. Serum was added to diluted and dialyzed normal synovial fluids, but no increase in the viscosity of these fluids was observed. 3. Effects of intra-articular injection of hydrocortisone.-Synovial fluid was obtained from the knees of some patients with rheumatoid arthritis prior to and one week after the intra-articular injection of 37.5 mg. (one and one-half ml.) of hydrocortisone. In two cases synovial fluid was simultaneously withdrawn from the opposite knee before and after intra-articular injection of saline. Intra-articular injection of hydrocortisone led to these results (table 5 ) : ( 1) reduction in the volume of synovial fluid of 70 to 90 per cent; ( 2 ) a rise in the concentration of hyaluronate hexosamine in the fluid of 20 to 50 per cent; and ( 3 ) a slight reduction of total nitrogen of the order of 20 per cent, or no change. * These changes in volume, hyaluronate hexosamine concentration and total nitrogen were not observed in synovial fluid obtained after *Despite this small change in total nitrogen of synovial fluids, intra-articular hydrocortiqone injection led to a marked decrease in the nitrogen to hevosamine ratio of mucins formed with the cobalt salt. High ratios were consequences of the fact that proteins (chiefly alpha-, globulins) containing nonhyaluronate hexosamine precipitated in mucins fornied in rheumatoid fluid with the cobalt salt. Steroid injection led to changes in synovial fluid which resulted in decreased precipitation of nonhyaluronate hewosamine by the cobalt salt, and hence to lower nitrogen to hexosamine ratios of these mucins. These changes in the fluid were: an increase in hyaluronate hexosamine concentration; a slight decrease in the concentration of alpha-, glohulins and a slight increase in the albumin concentration? SYNOVIAL FLUID HYALURONATE IN RHEUMATOID ARTHRITIS 529 intra-articular injection of saline. Hydrocortisone did not bring about <any change in the relative viscosity of synovial fluid as shown by similar viscosities of all fluids diluted to an equivalent hyaluronate hexosamine concentration and dialyzed (table 5 ) . DISCUSSION A hyaluronate hexosamine in rheumatoid synovial fluid was determined in several ways. Hyaluronate hexosamine was completely precipitated in mucin clots by adding glacial acetic acid to synovial fluid. Hyaliironate hexosamine in rheumatoid synovial fluid was digested by testicular hyaluronidase and rendered dialyzable. The results obtained by these methods generally agreed. The average concentration of hyaluronate hexosamine in rheumatoid fluid of greater than normal volume was 0.4 mg./Gm. or less compared with 1.4 mg./Gm. in fluids from normal subjects. The volume of most of these rheumatoid fluids was much greater than normal and thus the total amonnt of hyaluronate hexosamine in the fluids exceeded normal, as has been previously noted.'J Hyaluronate hexosamine constituted only about 30 per cent of the total hexosamine of these rheumatoid fluids instead of 85 per cent as was the case in the normal fluids. This large amount of nonhyaluronate hexosamine in these rheumatoid fluids is presumably caused by an increased concentration of plasma proteins containing hexosamine. Some criteria can be offered for abnormal and normal synovial fluids from patients with rheumatoid arthritis. .4bnormal fluids are those of increased volume and low hyaluronate hexosamine concentration. These changes in the fluid presumably result from inflammation of the synovialis of the knee. By contrast, normal fluids from patients with rheumatoid arthritis are of normal volume and normal hyaluronate hexosamine concentration, and inflammation of the synovialis in these knees is presumed to be slight or absent. However, many of the patients from whom normal synovial fluid was obtained showed signs of disease activity, such as inflammation of other joints, subcutaneous nodules and an elevated erythrocyte sedimentation rate. The finding of normal synovial fluid in patients with disease activity supports the view of Egelius, Jonsson and SundbladlO that connective tissues are affected only at some sites and not involved diffusely by the inflammatory process of rheumatoid arthritis. Studies reported in this paper have compared the relative viscosity of synovial fluids from normal subjects with the viscosity of fluids of increased volume and low hyaluronate concentration from patients with rheumatoid arthritis. The approach used was first to bring the hyaluronate hexosamine concentration of all the fluids to the same level (0.25 mg./Gm.) by diluting them with a buffer. The method of hyaluronidase digestion was used to determine accurately the hyaluronate hexosamine concentration. Comparison of the relative viscosities of normal and rheumatoid fluids of equivalent hyaluronate hexosamine concentration revealed similar values, or a slight reduction in the viscosity of some rheumatoid fluids. These results suggest that the state of polymerization of hyaluronate is similar in normal and in rheumatoid fluids or that it is only slightly lower in some rheumatoid fluids. These 530 DAVID HAMERMAN AND HILDA SCHUSTER findings, which differ from those of other workers1Js4 who have reported that hyaluronate is incompletely polymerized in rheumatoid synovial fluid, must be considered in relation to the conditions employed in this study. First, the methods of measuring hyaluronate are not the same as those of other workers, and thus, the finding of figures for hyaluronate hexosamine in normal synovial fluid which are higher than those reported by others necessitated a considerably greater dilution of these fluids than of the rheumatoid fluids to reduce the hyaluronate hexosamine concentration to 0.25 mg./Gm. Second, the fact that dilution of all the fluids to this concentration of hyaluronate hexosamine minimized the effects of interaction of hyaluronate molecules which occurs at higher concentrations. It must be emphasized that measurements of relative viscosity of synovial fluids may not reveal information about the shape of hyaluronate molecules or about a state of the hyaluronate which depends on its conibination with part of the protein of synovial fluid." Changes in synovial fluid which come about as a result of changes in shape or in this state of hyaluronate can be detected by viscosity determinations at varying velocity gradients. When normal and rheumatoid fluids have been compared in this way, changes in the physical properties of hyaluronate in rheumatoid synovial fluids have been noted."1".13 These changes in hyaluronate have been thought to impair the effectiveness of rheumatoid fluids as a lubricant between joint ~ u r f a c e s . ~The ~ J ~low concentration of hyaluronate in rheumatoid effusions may also contribute to an inadequate lubricating action of these fluids. The particular suitability of synovial fluid as a lubricant in the joint has been related14 to the properties of hyaluronate when it is present in synovial fluids at a concentration of 0.2 Gm./100 ml. and above." At this concentration, hyaluronate molecules overlap, chains interpenetrate, and fluids possess special viscous and elastic properties not shared by dilute fluids. The concentration of hyaluronate is almost always greater than 0.2 Gm./lOO ml. in synovial fluid from normal joints, but is considerably less than this in fluids from inflamed joints of patients with rheumatoid arthritis. SUMMARY 1. Methods were studied to determine accurately hyaluronate concentration of synovial fluid from patients with rheumatoid arthritis. In one method, hyaluronate was precipitated in mucin clots by adding glacial acetic acid to weighed aliquots of rheumatoid fluid and was measured indirectly by determining hexosamine in these clots. 2. These clots contained all the hyaluronate hexosamine of synovial fluid, and the hexosamine of these clots was almost completely digested and rendered dialyzable by streptococcal hyaluronidase. Nonhyaluronate hexosamine remained in the supernatant and was not digested by hyaluronidase. 3. In a second method, hyaluronate in rheumatoid fluids was measured by the fall in hexosamine following hyaluronidase digestion and dialysis. Figures *This concentration of hyaluronate corresponds to :I hyaluronate hexosamine concentration of 0.08 Gm./100 ml., assuming 40 per cent hexosamine in hyaluronate. SYNOVIAL FLUID HYALURONATE IN RHEUMATOID ARTHRITIS 531 for hyaluronate hexosamine obtained by this method generally agreed with those of the mucin clot technic. 4. The average hyaluronate hexosamine concentration of rheumatoid synovial fluid whose volume exceeded normal was 0.4 mg./Gm. The average hyaluronate hexosamine concentration of synovial fluid from normal subjects was 1.4 mg./Gm. Hyaluronate hexosamine constituted only 30 per cent of the total hexosamine of rheumatoid synovial fluid. By contrast, hyaluronate hexosamine constituted 85 per cent of the total hexosamine of normal synovial fluids. 5. The hyaluronate hexosamine concentration of some of the rheumatoid and normal fluids was brought to 0.35 mg./Gm. by diluting the fluids with a buffer. Comparison of the relative viscosities of these fluids then revealed similar values, or a slight reduction in viscosity of some rheumatoid fluids. It is suggested that the state of polymerization of hyaluronate in normal and in rheumatoid fluids is similar, or only slightly lower in some rheumatoid fluids. REFERENCES 9. Sandson, J. and Hamerman, D.: Paper electrophoresis of synovial fluid in rheumatoid arthritis. Data to be published. 10. Egelius, N., Jonsson, R. and Sundblad, L.: Studies of hyaluronic acid in acid in synovial fluid. Acta SOC. med. rheumatoid arthritis. Ann. Rheumat. upsal. 58:113, 1953. 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