NONDIALYZABLEHEXOSEOF HUMANSYNOVIAL FLUID * t

By JOHNSANDSONt AND DAVID HAMERMAN§ WITH THE TECHNICAL ASSISTANCE OF

HILDA SCHUSTER

(From the Department of Medicine, Albert Einstein College of Medicine, and the BronxMunicipal Hospital Center, New York, N. Y.)

(Submitted for publication November 24, 1959; accepted January 29, 1960)

Studies that show differences in the componentsof synovial fluids in osteoarthritis and rheumatoidarthritis may be helpful diagnostically. Synovialfluids from joints involved by rheumatoid arthritiscontain a high concentration of protein, and elec-trophoretic studies reveal increased a2-globulins(1, 2). Since this protein fraction contains thehighest concentration of hexose among the, elec-trophoretically separated proteins of serum (3),determination of nondialyzable hexose concentra-tion in synovial fluid was undertaken. In synovialfluid from osteoarthritic joints the nondialyzablehexose concentration, determined with the an-throne reagent, was similar to or lower than thenormal level, while in synovial fluid from jointsinvolved by rheumatoid arthritis it was elevated.

The first step in this study was to identify thecarbohydrate components of dialyzed synovialfluid that contributed to the anthrone reaction.Synovial fluid was dialyzed and then lyophilized.The lyophilized solids were hydrolyzed in sul-furic acid, and the carbohydrates in the hydroly-sate were separated by a combination of zone elec-trophoresis in borate buffer and paper chromatog-raphy. Galactose, mannose, small amounts offucose and glucose, and hyalobiuronic acid werefound.

MATERIALS AND METHODS

I. Identification of carbohydrate components

Synovial fluid from normal subjects. Synovial fluidwas aspirated from apparently normal knee joints ofdeceased subjects. Fluids from 8 to 10 normal kneeswere pooled to provide about 10 ml. Three different

* Part of this work was presented before the SecondPan-American Congress on Rheumatic Diseases, June1959.

t Supported by Graduate Training Grant 2A-5082 fromthe National Institute of Arthritis and Metabolic Dis-eases, and by a grant from the N. Y. State Chapter,Arthritis and Rheumatism Foundation.

t Fellow of the Arthritis and Rheumatism Foundation.§ Markle Scholar in Medical Science.

pools of normal synovial fluid were collected. Thep-oled fluids were diluted with an equal volume of abuffer (0.03 M NaHCO3 and 0.15 M NaCl, pH 8.1),dialyzed against 500 ml of distilled water (changedtwice daily) for 48 hours at 20 C, and lyophilized.

Synovial fluid from patients with primary osteoarthri-tis or rheumatoid arthritis.' About 10 ml of synovialfluid was obtained from the involved knee joint of 1 pa-tient with primary osteoarthritis and from 2 patients withrheumatoid arthritis. These fluids were treated in thesame manner as the pooled normal synovial fluids.

Normal serum. The total protein-bound hexose ofpooled normal serum was precipitated with ethanol ac-cording to the method of Winzler (4), and dried in avacuum over P205.

Hydrolysis. Lyophilized solids (80 mg) of poolednormal synovial fluid were hydrolyzed in 1 ml of 2 NH2SO4 in a sealed tube for 2 hours at 1000 C. The hy-drolysate was adjusted to pH 5.0 with Ba(OH)2, andthe insoluble BaSO4 removed by centrifugation. Ly-ophilized solids of osteoarthritic synovial fluid (100 mg),rheumatoid synovial fluid (100 mg), and the dried pre-cipitate of normal serum (100 mg) were similarly hydro-lyzed and neutralized.

Zone electrophoresis. The technique employed wasmodified from that of Miiller-Eberhard and Kunkel (5).Blocks (4 X 13 X 1/4 inches) of polyvinyl chloride (GeonResin no. 435, obtained from B. F. Goodrich) were used.The hydrolysate of normal synovial fluid was applied tothe block in a slit (1'2 X 1/4 X1/4 inches) 3 inches from thecathodal end of the block. A mixture of authentic glu-cose, galactose and mannose was applied to a slit in theblock 2 inches lateral to the hydrolysate. Electropho-resis was carried out for 9 hours in borate buffer (0.05M Na2B4O7 10 H2O and 0.15 M HSBO:, pH 8.5) at avoltage gradient of 12.5 volts per cm at 2° C. Theblock was blotted dry and divided longitudinally intotwo equal parts. Each part was cut into 1/4 inch seg-ments starting at the anodal end. Each segment wasplaced in a 15 ml sintered glass funnel (medium porosity),and elution was performed with 3 additions (2.5 ml each)of distilled water by applying air pressure to the funnel.The final volume of each eluate was made up to 8 ml.After analysis for hexose, the eluates of some segments

1 For simplicity, synovial fluids obtained from involvedjoints of patients with osteoarthritis or rheumatoid ar-thritis will be termed osteoarthritic synovial fluid andrheumatoid synovial fluid, respectively.

782

NONDIALYZABLEHEXOSEOF HUMANSYNOVIAL FLUID

were pooled to obtain a larger quantity of the isolatedcarbohydrates.

Electrophoresis of the hydrolysates of osteoarthriticsynovial fluid, rheumatoid synovial fluid, and normal se-rum was similarly performed.

Removal of sodium borate. It was necessary to re-move sodium borate from the eluates prior to chromatog-raphy. The eluates containing hexoses were passedthrough a column (5 X 0.9 cm) of Amberlite 1R-120(H' form). The effluent was evaporated to dryness ina vacuum, methanol (3 additions of 5 ml each) added,and the methyl borate removed by vacuum distillation at30° C (6). The eluates containing hyalobiuronic acidwere passed through a column (3 X 0.9 cm) of equalparts (by weight) of carbon and celite (7). The so-dium borate was washed from the column with distilledwater (3 additions of 10 ml each). The hyalobiuronicacid was then eluted from the column with 10 ml of30 per cent (vol/vol) aqueous ethanol.

Chromatography. Ascending paper chromatographywas performed using Whatman no. 1 paper at roomtemperature for 16 hours. Hexoses were separated inisopropyl alcohol-water (8: 2) (8), and the chromato-grams were stained with aniline hydrogen phthalate.Eluates containing hyalobiuronic acid were chromato-graphed in butanol-acetic acid-water (6: 2: 2), and thechromatograms were stained with either aniline hydro-gen phthalate or p-dimethylaminobenzaldehyde in HCI(8).

Analytical methods. All the eluates were analyzed forhexoses by an anthrone method (9). Anthrone reagentwas prepared by adding 100 ml of C.P. H.SO4 to 0.2 gof anthrone. Five ml of anthrone reagent was added toPyrex test tubes (25 X 150 mm) in water at 10° C. Analiquot (2.5 ml) of each eluate was layered on top of theanthrone reagent. A reagent blank (2.5 ml of the Na-HCO3-NaCl buffer) and the hexose standard (1 ml con-taining 50 ,ug per ml of galactose and 50 Ag per ml of man-nose plus 1.5 ml of buffer) were similarly prepared. Thetubes were covered with glass marbles, shaken, removedfrom the water, and allowed to stand at room tempera-ture for 5 minutes. The tubes were immersed in waterat 1000 C for exactly 6 minutes, and then cooled in waterat 100 C for 5 minutes. Optical density was determinedin a Beckman DU spectrophotometer at 625 m/u.

Some of the eluates were also analyzed for hexosa-mine (10) and hexuronic acid (11). Lyophilized non-dialyzable solids of synovial fluids were analyzed forhexose, hexosamine and nitrogen (12).

11. Determination of nondialyzable hexose concentration

Synovial fluid was obtained from two groups of sub-jects with apparently normal knee joints. The firstgroup consisted of 22 deceased subjects of varying ageswho had no evidence of acute illness. The second group,was composed of living subjects in coma from an acutecerebral vascular accident. The methods of aspirationand preservation of the fluid are described elsewhere(10). Synovial fluid was also obtained from involved

knee joints of patients with active rheumatoid arthritisor primary osteoarthritis. The diagnosis of definite rheu-matoid arthritis fulfilled the criteria of the AmericanRheumatism Association (13). The diagnosis of pri-mary osteoarthritis was established by history, physicalexamination, X-ray changes, normal erythrocyte sedi-mentation rate, and negative latex fixation test. Serumwas obtained from the living subjects at the time syno-vial fluid was aspirated.

Dialysis. To a weighed aliquot (0.2 g) of normal orosteoarthritic synovial fluid was added 2.5 ml of a buffer(0.03 M NaHCO3, 0.15 M NaCl, pH 8.1). To rheu-matoid synovial fluid (0.15 g) 3 ml of buffer was added.Each diluted fluid was mixed, added to cellulose casing(Visking, 8%2 inch) and dialyzed against 500 ml of buf-fer changed twice daily for two days at 20 C. Toweighed aliquots (0.25 g) of serum from normal sub-jects or patients with osteoarthritis was added 3 ml ofbuffer. A smaller aliquot (0.15 g) of serum from pa-tients with rheumatoid arthritis was similarly dilutedwith buffer. The diluted sera were then dialyzed againstbuffer.

Alcohol precipitation. In a few synovial fluids andsera, the values for nondialyzable hexose were comparedwith protein-bound hexose precipitated by alcohol.Weighed aliquots similar to those described above weretaken. Normal synovial fluid was diluted 1: 5 with tapwater; rheumatoid synovial fluid was diluted 1: 1; se-rum was not diluted. To the diluted fluids and sera 10ml of cold absolute ethanol was added with constantstirring. After standing for 30 minutes at 20 C, thetubes were centrifuged, the precipitate washed once inethanol, and then dissolved in 2 or 3 ml of buffer.

Hexose analysis. To 5 ml of anthrone reagent inPyrex test tubes immersed in water (100 C), 0.5 mlbuffer and 2 ml 2 of diluted dialyzed synovial fluid werecarefully added down the side of the tube. For analysisof serum, 1.5 ml of buffer and 1 ml of diluted, dlialyzedserum were used. A hexose standard (1 ml containing50 ,ug per ml of galactose and 50 ,ig per ml of mannoseplus 1.5 ml of buffer) and a reagent blank (2.5 ml ofbuffer) were similarly prepared. The determination wascarried out as described above.

Protein determination. A biuret method was used(14). When the total volume of synovial fluid was lessthan 0.3 g a modified Folin method (15) was employedto permit analysis of protein in 0.05 g of synovial fluid.Normal serum, whose nitrogen was determined by amicro-Kjeldahl method (12), was used as a standard.Values for protein obtained by the biuret and Folinmethod on the same synovial fluids agreed within 5 percent.

Anthrone reaction of isolated hyaluronidase-digestedhyaluronate. Synovial fluid was digested with hyaluron-idase, and the hyaluronate fragments isolated by zoneelectrophoresis in a phosphate buffer (0.05 M, pH 6.5)on blocks of polyvinyl chloride (16). The molar ratio

2 The density of diluted synovial fluid is 1, and gramsand milliliters may be used interchangeably.

783

JOHN SANDSONAND DAVID HAMERMAN

TABLE I

Analyses of nondialyzable solids of human synovial fluid

Weight of solids(mg/g) of

synovial fluid Hexose Hexosamine Nitrogen

Normal 19.9 1.8 5.1 12.2Normal 17.7 1.8 5.5Normal 17.0 2.0Osteoarthritis 39.8 1.6 3.1 12.1Rheumatoid arthritis 64.4 1.3 2.0 10.2Rheumatoid arthritis 43.8 1.5 2.8

of hexuronic acid to hexosamine of the isolated hyalu-ronate fragments was 1. No protein was detected by theFolin method (15) and chromatography after acid hy-drolysis revealed hexosamine but no amino acids. Theanthrone reaction of an aliquot of this material contain-ing 400 ,ug of hexuronic acid per ml was determined.

RESULTS

I. Identification of carbohydrate components

Analytical data. The hexose, hexosamine andnitrogen content of the nondialyzable solids of

HEXOSEOPTICALDENSITY

.25

.20

.15

.10

.0 5

AUTHENTIC GLUCOSESUGARS

GALACTC

normal, osteoarthritic and rheumatoid synovialfluid are shown in Table I.

Hydrolysate of normal synovial fluid. An-throne-reacting carbohydrates in the acid hydroly-sate of nondialyzable solids of normal fluid wereseparated into five zones by electrophoresis (Fig-ure 1). Three of these five zones (Zones I, IIand IV) had mobilities nearly identical with thoseof authentic glucose, galactose and mannose. Thecarbohydrates in the pooled eluates of these zoneswere studied by chromatography, and glucose,

)SE MANNOSE

0

HYDROLYSATEOFNORMAL

SYNOVIAL FLUID

HExuRONIC. w ACID .,0 - 6@0.

HEXOSAMINE OR-GINo0 ANODE--- 1----- - r --- -- l--I

14 16 18 20 22 24 26 28 30 32SEGMENTNUMBER

ZONE I - FZONFH5 >i~

34 36 38 40 44

Izot; 1.- ZONEx

FIG. 1. ZONE ELECTROPHORESIS OF AN ACID HYDROLYSATE OF NONDIALYZABLESOLIDS OF NORMALHUMANSYNOVIAL FLUID, AND OF AUTHENTIC HEXOSESIN BORATEBUFFER, PH 8.5. Eluates were analyzed for carbohydrate with the anthrone reagentand the optical density was plotted for each segment. Eluates within each of fivezones were then pooled for chromatographic studies.

784

NONDIALYZABLEHEXOSEOF HUMANSYNOVIAL FLUID

f a

N.. /

I i

Fucose Mannose Glucose Galactose Zone I Zone I ZoneN ZoneFIG. 2. ASCENDINGPAPER CHROMATOGRAMOF HEXOSESIN ZONES I THROUGHIV OBTAINED

AFTER ZONE ELECTROPHORESIS, AND OF AUTHENTIC HEXOSES. Isopropyl alcohol-water solvent(8: 2); aniline hydrogen phthalate stain. Zone I, glucose; Zone II, galactose; Zone III, tracesof fucose and mannose; Zone IV, mannose.

I.1I ¶

weV wet _.

JIBA Zonm_V N-mAct2BA

FIG. 3. ASCENDING PAPER CHROMATOGRAMOF HYALURONATEFRAGMENTS IN ZONE V.

Butanol-acetic acid-water solvent (6 : 2 : 2); p-dimethyl aminobenzaldehyde in HCl stain.HBA, hyalobiuronic acid (red); N-AcHBA, N-acetyl hyalobiuronic acid (purple). In ZoneV the faint purple stain of one component (dotted line) faded; the other component stainedred.

785

i ...

I

4.

JOHN SANDSONAND DAVID HAMERMAN

galactose and mannose were identified in theirrespective zones (Figure 2). Fucose was foundin Zone III between galactose and mannose.

The fifth zone moved more slowly than man-nose, and contained hexosamine and hexuronicacid. After sodium borate was removed, the mo-lar ratio of hexuronic acid to hexosamine in thepooled eluates of this zone was 1. Chromatog-raphy revealed two carbohydrates (Figure 3).That present in highest concentration migrateda distance identical to authentic hyalobiuronicacid,3 and stained red with p-dimethylamino-benzaldehyde in HCL. The other sugar, presentin trace amounts, migrated a distance identicalto authentic N-acetyl hyalobiuronic acid,3 andstained purple with p-dimethylaminobenzaldehydein HC1.

3 Kindly supplied by Dr. Karl Meyer, College ofPhysicians and Surgeons.

Hydrolysates of osteoarthritic and rheumatoidsynovial fluids. Glucose, galactose, fucose, man-nose, hyalobiuronic acid and trace amounts ofN-acetyl hyalobiuronic acid were identified in theacid hydrolysates of nondialyzable solids of thesesynovial fluids.

Hydrolysate of normal serum. Glucose, galac-tose, fucose and mannose but no hyalobiuronicacid or N-acetyl hyalobiuronic acid were foundin the acid hydrolysate of the ethanol precipitateof serum.

II. Concentration of nondialyzable hexose insynovial fluid

The mean value for the nondialyzable hexoseconcentration in synovial fluids from 22 deceasedsubjects with normal joints was 0.54 mg per g(Figure 4 and Table II). Synovial fluid and

TABLE II

Levels of nondialyzable hexose in synovial fluids and sera

Normal

Cerebral vascularaccidents

Osteoarthritis

Rheumatoid arthritis

5462456470

MonGreCuoMcNTio

RagPap

TimCouAckMur

DeG

KocCas

MacBoy

SweNieScoCam

Lt.Rt.Rt.Lt.

Rt.Lt.t

Rt.Lt.t

Rt.Lt.

* Standard deviation.t Analysis of serum from 10 living normal subject>.$ Uninvolved knee.

Nondialyzable hexose

Synovial fluid Serum

mg/g

0.54 i 0.11*

0.610.430.750.660.69

0.350.360.450.530.510.470.56

0.780.741.061.061.250.880.820.910.780.440.971.100.641.071.090.700.860.88

mg/g

1.10 i 0.09*t1.901.401.561.671.68

1.091.201.151.181.041.010.93

1.881.47

1.47

1.48

1.481.751.241.58

Synovial fluidprotein

mg/g25.3 4.6*

18.325.629.828.423.5

23.723.224.626.728.430.426.8

53.762.550.5

50.540.7

34.8

53.563.0

46.443.052.544.044.7

786

NOND:T\LYZABJ.E HEXOSEOF HIUMAN SYNOVITAL FLUID

OSTEOARTHRITICSYNOVIAL FLUID

40 60 80 100AGE IN YEARS

RHEUMATOIDSYNOVIAL

FLUID

*D

20 40 60 80

FIG. 4. NONDIALYZABLE HEXOSE CONCENTRATIONOF HUMANSYNOVIAL

FLUIDS. The shaded area represents one standard deviation from the mean

value (0.54 mg per g) of normal synovial fluid.

serum were also obtained from living, comatosesubjects with normal knee joints but wvith elevatedacute phase reactants due to an acute cerebralvascular accident. Although the nondialyzablehexose concentrations in these sera were elevated,hexose concentrations in the dialvzed svnovialfluids were normal in four of the five cases.

The nondialyzable hexose concentration was

not elevated in seven osteoarthritic fluids (Figure4 and Table II). The volume of each of thesefluids was less than 3 ml except for Tio (50 ml)and Cuo (12 ml). The nondialyzable hexoseconcentration in the sera of the patients with pri-mary osteoarthritis ws as normal. The synovialfluid protein concentration was normal in all theosteoarthritic fluids.

The nondialyzable hexose concentration was

above 0.70 mg per g in all the rheumatoid fluidsfrom involved knees (Figure 4 and Table II).The nondialyzable hexose concentration in thesera was usually increased. The protein concen-

tration was increased in all the rheumatoid fluids.In two patients wvith rheumatoid arthritis, synovialfluid was simultaneously obtained from an ap-

parently uninvolved knee [normal physical find-ings, negative X-ray, volume of synovial fluid less

than 2 mil, and normal hyaluronate hexosamineconcentration (17)] and from an involved knee.\Nondialyzable hexose concentration was normalin the normal knee, and elevated in the involvedknee (Table II).

Precipitation of protein-boutnd hexose by al-cohol. Addition of alcohol to synovial fluids or

serum precipitated the total protein-bound hexose.Values obtained by this method were similar to

TABLE III

Hexose levels of synovial fluid and serum: comparison ofvalues obtained after dialysis or alcohol precipitation

Ilexose

Nondialyzable Alcohol ppt.

mg/g mg, g

Svnovial fluid:Normal 0.79 0.77

0.43 0.390.60 0.560.46 0.45

Rheumatoid arthritis 1.12 1.19

SeruLm:Normal 1.20 1.19

Rheumatoid arthritis 1.60 1.551.48 1.50

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JOHN SANDSONAND DAVID HAMERMAN

hexose levels obtained after dialysis of synovialfluid or serum (Table III).

Contribution of hyaluronidase-digested hyalu-ronate to the anthrone reaction. A solution ofprotein-free hyaluronate fragments containing400 ug hexuronic acid per ml (1,000 jug hyalu-ronate per ml) gave an anthrone reaction equiva-lent to 25 Mtg per ml of hexose.

DISCUSSION

The carbohydrates in an acid hydrolysate of thenondialyzable solids of synovial fluid were sepa-rated by zone electrophoresis on blocks of poly-vinyl chloride in borate buffer at pH 8.5. Neutralmonosaccharides in the hydrolysates formed nega-tively charged complexes with borate and mi-grated in the electric field (18). Good separationof glucose, galactose and mannose was accom-plished.4 Fucose was not completely separated byelectrophoresis but was identified by paper chro-matography. The use of polyvinyl chloride in-stead of starch as the supporting medium per-mitted elution without contamination with car-bohydrate.

Galactose, mannose and small amounts of glu-cose and fucose were identified in the acid hy-drolysates of dialyzed, lyophilized synovial fluidsand in the ethanol precipitate of serum. Thefinding of glucose in these hydrolysates is of in-terest since in a recent discussion glucose wasthought not to be one of the hexoses bound tohuman serum proteins (19). Hyaluronate frag-ments isolated from the hydrolysates of nondialy-zable solids of normal and rheumatoid synovialfluids were identified as hyalobiuronic acid.

The nondialyzable hexose concentration in hu-man synovial fluids was determined with the an-throne reagent using a solution containing equalamounts of galactose and mannose as a standard.The small volumes of viscous fluid taken foranalysis were weighed rather than pipetted. Theanthrone reaction of isolated protein-free hyalu-ronate was small (2.5 per cent of the hyaluronateconcentration) and, therefore, no correction wasmade in the hexose levels for the hyaluronate ofsynovial fluid.

The mean value for the nondialyzable hexose4 Free hexosamine in the acid hydrolysates was swept

off the cathodal end of the block and was not recoveredin these studies.

concentration in synovial fluid obtained from sub-jects with normal joints was 0.54 mg per g. Non-dialyzable hexose levels in synovial fluids obtainedfrom osteoarthritic knee joints were normal ordecreased. Similar results were found by Decker,McKenzie, McGuckin and Slocumb (2). Thenondialyzable hexose concentrations in synovialfluids from joints involved by rheumatoid arthri-tis were increased. Determination of the non-dialyzable hexose concentration in synovial fluidmay be a means for differentiating osteoarthriticfrom rheumatoid synovial fluid.

Studies reported here support the view (20)that the normal synovial membrane selectivelyadds components of serum to synovial fluid.High serum levels of protein-bound hexose werenot accompanied by an increase in the concentra-tion of nondialyzable hexose in synovial fluid ob-tained from normal joints of patients with acutecerebrovascular accidents and from the uninvolvedknees of patients with active rheumatoid arthritis.

There is evidence that nondialyzable hexosesare bound to proteins in synovial fluid (21).Since the protein concentration was elevated inall the rheumatoid fluids, increased hexose levelswere expected. However, hexose levels were notalways proportional to the increased protein con-centration, and it is likely that the very high hex-ose level in some fluids was due chiefly to an in-crease in proteins rich in hexose that entered theinflamed joint.

SUMMARY

1. Pooled normal human synovial fluids, andsynovial fluids from joints involved by primaryosteoarthritis or rheumatoid arthritis were dia-lyzed and lyophilized. These lyophilized solidswere hydrolyzed in acid, and the carbohydratecomponents were separated by a combination ofzone electrophoresis in borate buffer at pH 8.5,and ascending paper chromatography. Glucose,galactose, fucose, mannose, hyalobiuronic acid andtraces of N-acetyl hyalobiuronic acid were found inall the hydrolysates.

2. Nondialyzable hexose concentration in nor-mal synovial fluid and fluid from joints involvedby primary osteoarthritis or rheumatoid arthritiswas determined with the anthrone reagent. Ahexose standard containing equal amounts ofgalactose and mannose was used.

788

NONDIALYZABLEHEXOSEOF HUMANSYNOVIAL FLUID

3. The mean value for the nondialyzable hexoseconcentration in normal synovial fluid was 0.54mg per g. Osteoarthritic synovial fluids hadnormal or lower levels, while rheumatoid synovialfluids had values above 0.7 mg per g. Determina-tion of the concentration of nondialyzable hexosemay be useful in differentiating osteoarthritic andrheumatoid synovial fluids.

REFERENCES

1. Perlmann, G. E., Ropes, M. W., Kaufman, D., andBauer, W. The electrophoretic patterns of pro-teins in synovial fluid and serum in rheumatoidarthritis. J. clin. Invest. 1954, 33, 319.

2. Decker, B., McKenzie, B. F., McGuckin, W. F., andSlocumb, C. H. Comparative distribution of theproteins and glycoproteins of serum and synovialfluid. Arth. Rheum. 1959, 2, 162.

3. Winzler, R. J. Glycoproteins of plasma in CibaFound. Symp. on the Chemistry and Biology ofMucopolysaccharides, G. E. W. Wolstenholme andM. O'Connor, Eds. Boston, Little, Brown andCo., 1958, p. 245.

4. Winzler, R. J. Determinations of serum glycopro-teins in Methods of Biochemical Analysis, D.Glick, Ed. New York, Interscience Publishers,1955, vol. II, p. 279.

5. Miuller-Eberhard, H. J., and Kunkel, H. G. Thecarbohydrate of y-globulin and myeloma proteins.J. exp. Med. 1956, 104, 253.

6. Zill, L. P., Khym, J. X., and Cheniae, G. M. Fur-ther studies on the separation of the borate com-plexes of sugars and related compounds by ion-exchange chromatography. J. Amer. chem. Soc.1953, 75, 1339.

7. Whistler, R. L., and Durso, D. F. Chromatographicseparation of sugars on charcoal. J. Amer. chem.Soc. 1950, 72, 677.

8. Smith, I. Chromatographic Techniques. London,Heinemann, 1958.

9. Scott, T. A., Jr., and Melvin, E. H. Determinationof dextran with anthrone. Analyt. Chem. 1953,25, 1656.

10. Hamerman, D., and Schuster, H. Hyaluronate innormal human synovial fluid. J. clin. Invest. 1958,37, 57.

11. Dische, Z. A new specific color reaction of hexu-ronic acids. J. biol. Chem. 1947, 167, 189.

12. Kabat, E. A., and Mayer, M. M. Experimental Im-munochemistry. Springfield, Charles C Thomas,1948.

13. Committee of the American Rheumatism Association.1958 Revision of diagnostic criteria f6r rheuma-toid arthritis. Arth. Rheum. 1959, 2, 16.

14. Weichselbaum, T. E. An accurate and rapid methodfor the determination of proteins in small amountsof blood serum and plasma. Amer. J. clin. Path.,Technical section. 1946, 16, 40.

15. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., andRandall, R. J. Protein measurement with theFolin phenol reagent. J. biol. Chem. 1951, 193,265.

16. Hamerman, D., and Sandson, J. Zone electrophoresisof human synovial fluid. Fed. Proc. 1959, 18, 241.

17. Hamerman, D., and Schuster, H. Synovial fluidhyaluronate in rheumatoid arthritis. Arth. Rheum.1958, 1, 523.

18. Foster, A. B. Zone electrophoresis of carbohydrates.Advanc. Carbohyd. Chem. 1957, 12, 81.

19. Winzler, R. J. Discussion in Ciba Found. Symp. onThe Chemistry and Biology of Mucopolysac-charides, G. E. W. Wolstenholme, and M.O'Connor, Eds. Boston, Little, Brown and Co.,1958, p. 137.

20. Ropes, M. W., and Bauer, W. Synovial FluidChanges in Joint Disease. Cambridge, HarvardUniv. Press, 1953.

21. Schmid, K., and MacNair, M. B. Characterizationof the proteins of human synovial fluid in certaindisease states. J. clin. Invest. 1956, 35, 814.

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