Ann. rheum. Dis. (1972), 31, 513

Viscous interactions ofhyaluronic acid with some proteinsand neutral saccharides

J. R. E. FRASER, WIN KHEAN FOO, AND J. S. MARITZFrom the Departments ofMedicine and Statistics, University ofMelbourne, Australia

Synovial fluid greatly delays the centrifugal compres-sion of collagen gels, which can be interpreted as aviscous effect of hyaluronic acid (Fessler, 1960). Thiseffect is enhanced by adding serum, suggesting thatproteins in turn significantly influence the viscosityof hyaluronic acid (Baxter, Fraser, and Harris, 1971).Although the high viscosity of synovial fluid is

primarily due to hyaluronic acid, the effect of proteinson the viscosity of hyaluronic acid is nevertheless notentirely clear. The non-Newtonian (i.e. shear-dependent) element of synovial viscosity seems tostem largely from a complex of hyaluronic acid witha small specific group of proteins (Ogston andStanier, 1950, 1952; Silpananta, Dunstone, andOgston, 1968,1969), but any contribution to viscosityfrom the interaction of hyaluronic acid with the muchgreater residual fraction of synovial proteins is atpresent undefined.A commonly held view is that the effect of protein is

small and merely additive. Thus the viscosity ofsynovial fluid falls after treatment with hyaluronidaseto a low level close to that of an equal concentrationof serum proteins (-q,* 1X2; Sundblad, 1953), and ithas been found not to be significantly lowered bystep-wise separation of proteins, when assessed bymeasurement or derivation of intrinsic viscosity; thatis, in a state of limiting, or infinite, dilution (Balazsand Sundblad, 1959; Balazs, Watson, Duff, andRoseman, 1967). Brief observations in the course ofother studies have shown no change in viscosity onaddition of proteins to hyaluronic acid (Ropes,Robertson, Rossmeisl, Peabody, and Bauer, 1947;Ogston and Stanier, 1952; Johnston, 1955) andclinical references to the matter seem tacitly to agreewith Balazs and Sundblad (1959), who concluded thatin respect of viscosity the presence of proteins doesnot influence the behaviour of hyaluronic acid.

* ,7, = relative viscosity, or viscosity ratio.

However, inferences based on measurement ofintrinsic viscosity are not necessarily true of finiteconcentrations. Any material which alters thecharacter of the solvent can change the viscosity of asolution in either direction (Alfrey, Bartovics, andMark, 1942) and the viscosity of a mixture of homo-logous polymers can be assumed to be additive onlyat limiting dilution (Nichol, Bethune, Kegeles, andHess, 1964). Ogston (1962) calculated, on the basis ofhis previous osmotic studies and other physical data,that very dilute hyaluronic acid should be sensitiveto the presence ofalbumin although later observationsof viscosity did not confirm this prediction (Preston,Davies, and Ogston, 1965).The concentrations of proteins and hyaluronic acid

in synovial fluid vary from joint to joint and betweenspecies, and most of all in disease. In view of theapparent conflicts in the references cited, the effectsof mixing proteins and other viscous materials withhyaluronic have been studied in concentrationscloser to those that occur naturally, primarily interms of viscosity ratio since it is the derivative closestto absolute viscosity.

Materials

SYNOVIAL FLUIDPooled samples, taken from carpal joints of cattle imme-diately after slaughter, were chilled and centrifuged at25,000 G. for 30 to 60 min. Toluene was added as anti-septic, and aliquots were stored at -85°C. In the ninesamples used, the hyaluronic acid content was 15 ± 0-12mg./ml., and the protein content 159 ± 2-4 mg./ml.SERUMThis was prepared from blood of fasting normal humansubjects and stored as above.

BOVINE SERUM ALBUMIN, HUMAN GAMMAGLOBULINThese were obtained from the Commonwealth Serum

Accepted for publication April 24, 1972Correspondence: Dr. J. R. E. Fraser, University of Melbourne Department of Medicine, Royal Melbourne Hospital Post Office, Victoria, 3050,Australia

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514 Annals of the Rheumatic Diseases

Laboratories, Melbourne. Each showed a single com-ponent on immunoelectrophoresis with antiserum againstwhole serum. The globulin consisted of immunoglobulinG, 90 per cent. as monomer (Dr. S. Sutherland, personalcommunication).

FICOLLThis was the sucrose polymer of Pharmacia Ltd., with astated M, of 400,000.

CHEMICALSGlucose, sucrose, and polyethylene glycol 6000 were fromBritish Drug Houses Ltd.; glycerol from E. Merck.

BUFFERS0-05 M Sorensen's phosphate, pH 7-25 in 0 1 M NaCI wasused in preliminary work; the other was a phosphate-buffered saline with potassium, calcium, and magnesium,according to Dulbecco and Vogt (1954), pH 7 25.

Methods

PURIFICATION OF HYALURONIC ACIDThis was done by density gradient centrifugation ofsynovial fluid in CsCl at an initial density of 1-55. After90 hrs at 105,000 G, in a Beckman Spinco 50 Ti rotor, thebottom 3 ml. of the 10-11 ml. in each tube were pooled andthen exhaustively dialysed against 0-15 M NaCI, water, andfinally buffer. Protein content relative to that of hyaluronicacid (w/v) in this fraction was less than 2 per cent. Noattempt was made to separate the small amount ofchondromucoprotein which would be expected in theforegoing modification of the original method of Sil-pananta and others (1968).

Hyaluronic acid was measured against glucuronolactoneand hyaluronic acid standards by the method of Bitter andMuir (1962) with limited heating (Harris and Fraser, 1969).Protein was measured against crystalline bovine serumalbumin standards by the method of Lowry, Rosebrough,Farr, and Randall (1951).

SOLUTIONSSolutions of hyaluronic acid were thoroughly mixed bygentle rotation to avoid bubbling and denaturation ofadded proteins. Densities were determined by pycno-metry with triple-distilled water as a standard. To permituse of the relatively weakly buffered Dulbecco-Vogtsaline, the protein solutions, particularly albumin,required exhaustive dialysis to achieve a pH range of7-1-7-4 where viscosity of hyaluronic acid is not affectedby small differences in pH.

VISCOMETRYParticulate matter was removed from solutions by pre-liminary ultracentrifugation. All measurements weremade at 25°C.Four BS/U capillary viscometers were used. A type M2

with a flow rate of 150 sec. and a calculated mean shearrate of 800 sec.-1 for water at 25°C. was used for the initialstudies only. The remainder were done with type M4viscometers with corresponding flow times of 24 sec. andshear rates of 1300 sec.-'. A Hewlett Packard Auto-

viscometer was used for timing wherever possible. Allinstruments were calibrated by plotting .qIpt* againstlit2 with water at four temperatures. Kinetic energycorrections were zero, and the three M4 instrumentswere closely matched as shown by the intercepts of n/ptat l/t2 0 (Hewlett Packard Manual for Model 5901BAuto-viscometer).A cone-in-cone viscometer to the design of Dintenfass

(1963) was used for readings at controlled shear rates from7-3 sec.-1 upwards. Viscosity of a 25 per cent. solution ofglycerol in water at 25 5°C. was 1-77 ± 0-19 (n = 10) atshear rates between 7-3 and 183 sec-' compared with anexpected 1-80 (Sheely, 1932). Close correspondence wasalso obtained with hyaluronic acid solutions at similarshear rates in the capillary and cone-in-cone viscometers.Results from the capillary viscometers were corrected for

density, the maximum corrections being about 4 per cent.for concentrated sugar solutions. All results are expressedas viscosity ratios relative to buffer at 25°C., i.e. as multiplesof about 0 9 centipoises (9, = 1 0). The term -q will be usedto signify -q7 unless specified otherwise.

Results

SYNOVIAL FLUID

Stability of viscosity was first assessed with the M2viscometer in aliquots of a 1:1 (v/v) mixture ofsynovial fluid and serum. The initial -q, fell by 0 25 percent. after 3 days at room temperature, by 1 3 per cent.in 7 hrs at 25°C., and by 2 3 per cent. after freezingand thawing. On the whole, losses after freezing andthawing were infrequent and mainly related to smallprecipitates removed by the preliminary centrifu-gation.

Sixteen exploratory experiments were done inwhich synovial fluid was mixed with buffer or withwhole serum proteins, albumin, or other additives infinal concentrations selected to give viscosity ratiosabout 1 2 when measured alone in buffer. Thehyaluronic acid content of the synovial fluid dilutionsranged from 0-085 to 1-0 mg./ml., and the corres-ponding viscosity ratios from 17 37 to 1-34. Theseries of studies was primarily concerned withachieving pH control, but in every case, regardlessof efficiency of buffering, the viscosity of the synovialfluid was increased by the additive, and the order ofincrease in terms of specific increment (i.e. rysp = 7,-1,where buffer viscosity= 1) far exceeded the sum ofthe specific increments of each component as mea-sured in buffer alone. Examples are given in Tables Ito III, where pH was closely matched by previousdialysis against buffer.The preliminary results thus suggested that mixture

of any viscous solution with synovial fluid would leadto a multiplicative rather than an additive change inthe total viscosity of the mixture. Moreover, the orderof change seemed better related to viscosity of theadded solution rather than concentration of its solute.* X = viscosity, p = density, t = time of flow.

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Viscous interactions ofhyaluronic acid 515

Table I Effect ofadded serum protein on viscosity of Tablem Effect ofother additives on diluted synovialsynovialfluid in a range ofdilutions fluid

Viscosity Concentration

Serum Synovial Synovial Hyaluronic Synovial Serumalone fluid fluid acid protein protein

alone + serum (mg.lml.) (mg./ml.)

1-20 7T03 12-38 0-80 7 0 384 59 7-37 0 59 5-1 383-08 4 52 0 40 3-5 38182 2-54 0-19 1P7 381P34 1-87 0l10 0-8 38

pH of solutions in range 7-1 to 7-3.

ViscosityConcentration

Additive Synovial Synovial Ofalone fluid fluid additive

alone + additive (wlw)Glycerol 1-27 5-51 12-44 12-5%Glucose 1-22 5-51 11P09 8-5%Sucrose 1P20 5-51 11-84 8-3%Glycol 123 5-51 10-99 1P25%Ficoll 1-27 5-51 10-89 1-5%

pG of solutions in range 7 0 to 7-2.

Table II Effect of added glycerol on viscosity of synovial fluid in a range of dilutions

Viscosity Concentration

Glycerol Synovial Synovial Hyaluronic Synovial Glycerolalone fluid fluid acid protein

alone + glycerol (mg./ml.) (mg.fml.) (w/w)1-27 9-68 13-33 0-8 7-3 12-5%

3-60 4.97 0-4 3-6 12-5%2*17 2-84 0-2 1P8 12-5%

pH of solutions in range 7-2 to 7-25.

PURIFIED HYALURONIC A

Initially, sucrose, whole seglobulin, or albumin,* weretrations with a fixed conce* These solutes will be referred to as al

-o

0

+1

c

-o

_v_

1-

0

0U

l6

15 -

14

13

12

0

O 05 -dViscosity of additive

FIG. 1 Viscosity of solutionsvarious added solutes, comparisolutes alone. Capillary viscomscale is expanded. Concentr41-0 mg./ml. Single point on left:Curves, from above downwarsucrose, globulin, serum proteinCoefficients of variation for qper cent.

LCID acid (Fig. 1). The same magnifying effect on q, ofDrum proteins, gamma the mixtures was seen as with synovial fluid. Themixed in varied concen- order of change was distinctly different for eachmntration of hyaluronic solute. In terms of the viscosity of the additive alone,dditives. the effects of serum proteins and gamma globulin

were similar except for one outlying observation, butsignificantly less than that of sucrose and treater thanthat of albumin. These findings were next exploredin more detail to establish the relationships in termsof both viscosity and concentration.The concentrations of the various solutes were

varied by halving dilutions both in the pure solutionsand in the mixtures and all dilutions were done gravi-metrically for greater accuracy. The peak concentra-tion of hyaluronic acid ih each experiment was 0-8mg./ml., and each mixture was diluted with a stocksolution of additive, or with buffer alone, so that theconcentration of additive was kept constant orreduced in step with that of hyaluronic acid. Whenthe additive was constant, the viscosity ofthe mixtures

1.15 j.;2 1.25 1.3Q consistently exceeded a simple summative relation-ship from the highest to the lowest concentrations ofhyaluronic acid. When both solutes were diluted, the

of hyaluronic acid with resultant viscosity in each case approached that ofed with viscosity of those hyaluronic acid alone (Table IV, overleaf).ieter. Note that horizontal These data were submitted to a closer analysis ofation of hyaluronic acid the inter-relationships between the effects of thehyaluronic in buffer alone. e inte ships betin the ffects orrd: hyaluronic acid with several solutes. Preliminary plotting of the figures for

ts, or albumin respectively. hyaluronic acid alone suggested an empirical func-ranged from 0-03 to 0-46 tional relationship of the nature loge1H = aHCHnH

(H = , for hyaluronic acid, aH and nH are speific

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Table IV Viscosity ofpurified hyaluronic acid measured alone and with varying or constant concentrations ofsucrose or proteins

(A) Hyaluronic acid (HA) aloneConcentration (mg./ml.)ViscosityCoefficient of variation (%)

(B) With sucroseB1 Sucrose alone

Concentration (mg./ml.)ViscosityCoefficient of variation (0%)

B2 H.A. + sucrose (varying)Concentration (mg./ml.)ViscosityCoefficient of variation (0

B3 H.A. + sucrose (constant)Concentration (mg. /ml.)ViscosityCoefficient of variation (0

(C) With albuminCl Albumin alone

Concentration (mg. /ml.)ViscosityCoefficient of variation (0

C2 H.A. + albumin (varying)Concentration (mg./ml.)ViscosityCoefficient of variation (0

C3 H.A. + albumin (constant)Concentration (mg./ml.)ViscosityCoefficient of variation (0

(D) With serumDI Serum alone

Concentration (mg./ml.)ViscosityCoefficient of variation (0

D2 H.A. + serum (varying)Concentration (mg./ml.)ViscosityCoefficient of variation (0

D3 H.A. + serum (constant)Concentration (mg./ml.)ViscosityCoefficient of variation (0

(E) With gamma globulinEl Gamma globulin alone

Concentration (mg./ml.)Viscosity

Coefficient of variation (0

0-89-329(035)

821-182

(0-05)

8216-574

(0 27)

8216-954

(0*35)

531 245(043)

0-8 + 5314 117

(0-12)

0-8 + 5314-033(0-19)

31-51 219

(0-21)

0-8 + 31-514-199(0-42)

0-8 + 31-514-914

(0 06)

3251-250

(0-35)

E2 H.A. + gamma globulin (varying)Concentration (mg./ml.) 0-8 + 32 5

Viscosity 15-308Coefficient of variation (%) (0-31)

E3 H.A. + gamma globulin (constant)Concentration (mg./ml) 0-8 + 32 5Viscosity 15-343Coefficient of variation (%) (0 30)

043-526(0 04)

411-087(0-03)

414-711(0-15)

825-989(0 09)

2651-123

(0 36)

04+2654.459(0 09)

0-4 + 535-200(0-21)

15-751-102

(0 05)

04+ 15-754-461(0 28)

0-4+31-55-198(0-15)

16-251-116(035)

04+ 16254-649(0 27)

0 4 + 32 55608(030)

022-021(0-14)

2051-055

(0 26)

2052-404(0-15)

823.349(0-07)

13-31-058

(0 33)

0-2 + 13-32-323(0 07)

0-2 + 533-091(038)

7-881-056

(0 03)

0-2 + 7-882-355(0-13)

0.2 + 31-52 998(0-35)

81-054(0 09)

0-2 + 82-378(0-07)

0-2 + 32-53-262(0 36)

0-11-524

(0 50)

101-029

(0.21)

101-609

(0-14)

822355(0 16)

661-028

(0-11)

0.1 + 6-61-588(0 07)

0.1 + 532-271(0 20)

3.941-029(0 06)

01 + 3 91-583(0-18)

0-1 + 31-52219(0 05)

41-031(0-11)

0.1 + 41-603(0 06)

01 + 3252-371(0-31)

0051-256

(0 60)

N.D.

51-287(0 26)

821 904

(0 09)

3.31-019(025)

0*05 + 3-31-272(0-10)

005 + 531-913(030)

N.D.

0.05 + 21-268(0-12)

N.D.

N.D.

0.05 + 21-278

(0-13)

0-05 + 32-51-982

(0 40)

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Viscous interactions ofhyaluronic acid 517

constants, and CH = concentration of hyaluronicacid). Similar relationships were determined for theadditives, and the simplest multiplicative hypothesisfor predicting the viscosity of a mixture was testedaccording to the formula:

loge 'H+a = XHCHCH + OCaCana(Subscript a refers to the additive).

This was found to underestimate the observedresults. To obtain a better fit within a model of thistype, a correction factor with one further character-istic was introduced in the following form:

loge7]H+a = acHCH H + CaCaa + XHa CHHCaa

This model was fitted to all the data, using the methodof least squares to compute the various coefficients.These are shown in Table V.

Table V Coefficients for relationships betweenviscosity and concentration of solutes* (-q = e'cn, etc.See text)

Solutes aH 0a LHa nH na

Hyaluronic acid 2 604 0-7713(H)

+ sucrose (a) 2 604 0 0033 0 0052 0-7713 1-005+ albumin (a) 2-604 0-0061 00038 0-7713 0-980± serum 2-604 0 0086 0-0106 0-7713 0-965

proteins (a)+ gamma 2 604 0 0082 0 0067 0-7713 1-055

globulin (a)* mg./mi.

Expected values for viscosity were derived fromthese formulae, and agreed well with observed valuesas shown in Fig. 2.

NON-NEWTONIAN VISCOSITYHyaluronic acid, as it occurs in synovial fluid, showsa pseudoplastic type of non-Newtonian viscosity;that is, its viscosity increases as rate of shear falls.In capillary flow the shear rate, dv/dr, is inverselyrelated to viscosity,* and the increases in viscosityobserved abovemight therefore have been exaggeratedby a decrease in shear, though the lowest shear ratewould have been at least 75 sec.-1 (e.g. 1300/7, Expt. 1in Table I). Mixtures ofhyaluronic acid with a varietyofadditives were therefore studied in the cone-in-coneviscometer (Fig. 3). Small deflections of this instru-ment's torsion wire were difficult to read accurately.Multiple readings for buffer were therefore standard-ized by regression analysis constrained to zero rate ofshear, and the viscosity ratios were expressed as afactor of this regression. Estimates of the additivesgained from capillary studies were used for compari-sons in Fig. 3 (overleaf).

dv - P. where v = linear velocity, P pressure head, r = radius,and 1 = length of capillary.

18 -

6-

14 -

2 -

10-0

8-

4.-

2-

00.01 O*2 0-4

0 41Concentration of solutes (mg/ml)

0882

FIG. 2 Viscosity ofhyaluronic acidalone and with sucrose.Comparison of observed and predicted values as describedin text. Points indicate observed values, continuous linespredicted values.

Ordinate: viscosity ratio ('q,).Abscissa: concentrations. Upper, hyaluronic acid. Lower,

sucrose.(I) Hyaluronic acid + fixed concentration of sucrose

(82 mg./ml.).(II) Hyaluronic acid + varied concentration of sucrose(III) Hyaluronic acid alone.(IV) Sucrose alone.

Three conclusions can be drawn:(1) The additives increased the viscosity ofhyaluronicacid in a synergistic manner throughout the range ofshear rate examined;(2) Serum proteins caused a lesser increase in visco-sity than the non-ionic additives with a similar rangeof viscosity;(3) The factorial increase in viscosity tended toremain at least the same throughout the range of shearrate, and appeared to increase further at low shearrates with those additives that gave the greatestincrease in total viscosity. 7q, for most of the mixtures,except at the extremes of shear rate for serum M, felloutside 1 per cent. confidence limits for the regressionOf log197H against loge sec.-l.

DiscussionThe relationships between viscosity and concentra-tion were empirically derived. In the case ofhyaluronic acid the form is similar to that given byRopes and others (1947) and by Ragan and Meyer(1949). Different forms might be chosen for thesaccharides and proteins, but would not materiallyalter the interpretation of their interactions withhyaluronic acid.The total viscosity oftwo classes of solute behaving

ideally in a single solution might be expected to be

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i-

G)

0-j

30-

2-5 -

20

1 5 -

20 25 30 35 40 45 50

LO°e sec-t

FIG. 3 Effect ofadded solutes upon viscosity ofhyaluronicacid at controlled rates ofshear (sec.-').A: Hyaluronic acid alone:

Viscosities of additives alone were:B: Hyaluronic acid + serum M serum M, X 1 27,

loge = 0-2390C: Hyaluronic acid + serum G serum G; 7 1 23,

loge = 0-2070D: Hyaluronic acid + sucrose sucrose, r1 -20, lge

= 0-1823E: Hyaluronic acid + glucose glucose, 7 1-22, lge

= 0-1989F: Hyaluronic acid +glycerol glycerol, X 1-27, loge

= 0-2390Addition of the logarithms of -q, for each solute in the

mixed solutions to the figures for hyaluronic acid aloneshows that the resultant viscosities at all rates of shearexceeded the pr-oducts of the viscosities of the constituents,except in the case ofserum M.

derived from the product rather than the sum oftheir individual concentrations, or of their viscositiesmeasured separately, but the possible physical andchemical interactions between hyaluronic acid andproteins do not allow prediction of the direction ordegree of change in viscosity resulting from themixture.The serum proteins, albumin and gamma globulin,

which are not associated with hyaluronic acid inultrafilter residues of synovial fluid (Curtain, 1955),can clearly cause large increases in viscosity whenmixed with hyaluronic acid and do so in concentra-tions commonly found in joints. The relationshipsgiven here are consistent in form for two classes ofsolute, i.e. sucrose and protein, which differ greatlyin molecular weight and conformation, ionic struc-ture, and propensity for formation of different typesof physical and chemical bond. The general natureof the relationships would therefore seem a validbasis for analysing the viscous behaviour of hyalur-onic acid. Some variation in the constants shouldbe expected with different samples, although theformula for hyaluronic acid in Table IV gave avalue within 7 per cent. of the estimated value for

the batch of hyaluronic acid used in the previousexperiment.

Although the highest concentration of hyaluronicacid in this study was rather lower than that ofnormalhuman synovial fluid, it was well within the rangefound in synovial effusions, and the protein concen-trations covered the whole of the normal (13-20mg./ml.) and part of the abnormal range (up to 60mg./ml.) in synovial fluid. The increases in viscositybore a direct multiplicative relationship to theconcentrations of the hyaluronic acid and proteinor other added solute as expressed in the formulaegiven earlier. Furthermore, an additional magnifyingterm was required to gain a full estimate of viscosityin the presence of each added solute, whethersucrose or proteins. The magnitude of the firstcoefficient, xa, can be related to molecular weightof the solute, but there is a distinction in this respectbetween sucrose and the proteins in the secondcoefficient, CXHa-

Thus, the potential contribution of protein to theviscosity of synovial fluid can be grossly under-estimated if it is considered in terms of its viscosityalone. The relationships found in this study indicatethat intrinsic viscosity of hyaluronic acid can besafely derived from whole or fractionated synovialfluid if the protein is sufficiently diluted before finalextrapolation to zero concentration. However,intrinsic viscosity, by definition, cannot measure thecontribution of protein to the viscosity of synovialfluid in real concentrations although it is a validmolecular parameter of hyaluronic acid.

In seeking to explain the physical differencesbetween normal and rheumatoid synovial fluids,Ferguson, Boyle, and Nuki (1969) found that ureaand guanidine hydrochloride greatly reduced viscosityof osteoarthritic but not of rheumatoid synovialfluid, which suggests that a macromolecular complexexists in the former but not the latter. However, theseagents must affect the colligative properties of allconstituents of aqueous solutions and might havedifferent effects in the two classes of fluid simplybecause there are different ratios of protein tohyaluronic acid and different proportions of thesubclasses of protein in each type of fluid. The term'complex' implies a molecular association with orwithout obvious coacervation or phase separationand raises questions of selective bonds of physicalor chemical nature. We wish merely to draw attentionto the nature of the gross changes in viscosity withoutspeculating upon their mechanism at this stage.The techniques used in this study do not permit

any inferences about changes in elastic properties ofsynovial fluid (Ogston and Stanier, 1953; Balazs andGibbs, 1970) which require different methods.Nevertheless, increases in viscosity would indepen-dently impose increased work loads during a givenmovement or flow (Yang, 1961).

FEDcBA

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Viscous interactions ofhyaluronic acid 519

The viscous interaction of hyaluronic acid andproteins may be relevant to several aspects of synovialfunction. In synovial effusions, the concentrationof protein and the relative proportion of globulinsboth increase, which would tend to lessen the dropin viscosity due to concurrent dilution or to lesserpolymerization of the hyaluronic acid. The role ofviscosity in lubrication of joints is uncertain atpresent. Hyaluronic acid seems to be more importantin the lubrication of synovial membrane than ofcartilage (Radin, Paul, Swann, and Schottstaedt,1971). Increases in viscosity would, as noted above,increase the work-load of a joint especially in theperiarticular tissues where the rate of shear is likelyto be lower. The viscous reaction of protein withhyaluronic acid would thus contribute to 'morningstiffness' through inflammatory exudation andincrease in the concentration of proteins in thesurrounding connective tissue. This would be morepronounced if there is a temporary accumulation ofexuded protein as a result of reduced lymph flowwith overnight inactivity. Since diffusion is directlyrelated to viscosity, inflammatory exudation ofproteins into connective tissue ground substancewould also enhance the hindrance to diffusionpresented by hyaluronic acid.

Summary(1) Hyaluronic acid was freed from protein byultra-centrifugation of synovial fluid in caesiumchloride.

(2) The viscosity of solutions of this hyaluronic acidwas found to bear an exponential relation to its con-centration in the range 005 to 0-8 mg./ml.(3) The resultant viscosity of mixtures of hyaluronicacid with sucrose, total serum proteins, serumalbumin, or gamma globulin exceeded valuescalculated from the product of the viscosities ofeach component measured alone in equivalentconcentration, or the product of the exponentialfunctions relating the viscosity to the concentrationof each. This multiplicative effect of mixed soluteson total viscosity of hyaluronic acid is at least partlyindependent of rates of shear.(4) Similar results were consistently found in studieswith unfractionated synovial fluid and added viscoussolutes.(5) It is concluded that major proteins of synovialfluid, which are not known to form complexes withhyaluronic acid under physiological ionic conditions,can make a substantial contribution to the totalviscosity of the fluid. The possible significance of thisviscous interaction in inflamed joints and connectivetissue is discussed.

We are grateful to Prof. A. G. Ogston, F.R.S., for hisadvice and encouragement, to the Sunshine Foundationfor the generous gift of an autoviscometer, and to R. J.Gilbertson and Staff for facilities to collect synovial fluid.This work was supported by a grant from the NationalHealth and Medical Research Council of Australia.

References

ALFREY, T., BARTOVICS, A., AND MARK, H. (1942) J. Amer. chem. Soc., 64, 1557 (The effect of temperature andsolvent type on the intrinsic viscosity of high polymer solutions)

BALAZS, E. A., AND GIBBS, D. A. (1970) In 'Chemistry and Molecular Biology of the Intercellular Matrix',ed. E. A. Balazs. Academic Press, London and New York., AND SUNDBLAD, L. (1959) Acta Soc. med. upsal., 64, 137 (Viscosity of hyaluronic acid solutions containingproteins), WATSON, D., DUFF, I. F., AND ROSEMAN, S. (1967) Arthr. and Rheum., 10, 357 (Hyaluronic acid in synovialfluid. I. Molecular parameters of hyaluronic acid in normal and arthritis human fluids)

BAXTER, E., FRASER, J. R. E., AND HARRIS, G. S. (1971) Ann. rheum. Dis., 30, 419 (Interaction between hyaluronicacid and serum dispersed in collagen gels)

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