For the purpose to reveal the characteris6cs of the synovial fluid of the chronic rheumatoid arthritis the proteins of the synovial fluid and blood serum have been analysed by employing the methods of electrophoresis, gel filtration on Sephadex G-200 column and ultracentrifugation. Waaler-Rose test and latex fixation test have also been made on each protein fraction, and the following results were obtained. 1) The total protein level of synovial fluid, which is 3/5 of that of the serum, is slightly higher than that of control. 2) Fractionation of the synovial proteins by electrophoresis revealed nearly the same protein contents in each fraction in percentage as that of comparable fraction of the serum protein, with a slight increase in &#947;-globulin fraction. 3) The fractionation by Sephadex column G-200 give three peaks both in serum and synovial fluid, 19 S, 7Sand 4S. 4) 19S fraction of the synovial fluid, which is mainly of &#947;-globulin, showed a higher level than that of the synovial fluid from the controls. 5) Rheumatoid tests gave positive reaction in the 1st peak containing
19S &#947;-globulin from the synovial fluid and blood serum.</p

Seno, Takashi

English

Okayama University Scientific Achievement Repository

Acta Medica OkayamaVolume 22, Issue 1 1968 Article 4FEBRUARY 1968Protein concentration of synovial fluid inchronic rheumatoid arthritis. Estimation ofprotein in the synovial fluid of chronicrheumatoid arthritis by gel filtration and paperelectrophoresisTakashi Seno∗∗Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Protein concentration of synovial fluid inchronic rheumatoid arthritis. Estimation ofprotein in the synovial fluid of chronicrheumatoid arthritis by gel filtration and paperelectrophoresis∗Takashi SenoAbstractFor the purpose to reveal the characteris6cs of the synovial fluid of the chronic rheumatoidarthritis the proteins of the synovial fluid and blood serum have been analysed by employingthe methods of electrophoresis, gel filtration on Sephadex G-200 column and ultracentrifugation.Waaler-Rose test and latex fixation test have also been made on each protein fraction, and thefollowing results were obtained. 1) The total protein level of synovial fluid, which is 3/5 of thatof the serum, is slightly higher than that of control. 2) Fractionation of the synovial proteins byelectrophoresis revealed nearly the same protein contents in each fraction in percentage as thatof comparable fraction of the serum protein, with a slight increase in γ-globulin fraction. 3) Thefractionation by Sephadex column G-200 give three peaks both in serum and synovial fluid, 19 S,7Sand 4S. 4) 19S fraction of the synovial fluid, which is mainly of γ-globulin, showed a higherlevel than that of the synovial fluid from the controls. 5) Rheumatoid tests gave positive reactionin the 1st peak containing 19S γ-globulin from the synovial fluid and blood serum.∗PMID: 4238237 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOLActa Med. Okayama 22, 51-58 (1968)PROTEIN CONCENTRATION OF SYNOVIAL FLUID INCHRONIC RHEUMATOID ARTHRITISESTIMATION OF PROTEIN IN THE SYNOVIAL FLUIDOF CHRONIC RHEUMATOID ARTHRITIS BY GELFILTRATION AND PAPER ELECTROPHORESISTakashi SENODepartment of Orthopedic Surgery, Okayama University Medical School, Okayama, Japan(Director: Profs. M. Ogata* and T. Kodama)Received for publication, October 22, 1967There are many reports dealing with the assay of serum proteins inchronic rheumatoid arthritis by means of electrophoresis (1,2), columnchromatography (2) and ultracentrifugation (1,2,3) revealing the specificchange to the disease but only a few reports on the analysis of the proteinsof synovial fluid by the assays of paperchromatography and discelectro-phoresis, that is to show most clearly the changes specific to chronic rheu-matoid arthritis (4).Since the introduction of the gel filtration method for protein assayby PORATH (1959) the analysis and the purification (5) of the serum proteinsin chronic rheumatoid arthritis have made a great advance. Therefore,it would be worthwhile to analyze the synovial fluid proteins by applyingthe same method, though some papers have already appeared. In viewof this, the author attempted to study change of proteins in the synovialfluid of chronic rheumatoid arthritic joints by the gel filtration on Sepha-dex G.200 column, and found that the synovial proteins can be separatedinto three fractions just as in the case of serum proteins and the precipitinconstant of the fractions are identical with that of the serum protein. Itwas also found that the percentage of the second peak increases along withthe increase of total protein in synovial fluid. The present comunicationdescribes briefly the results obtained by using gel filtration technique andpaper. electrophoresis.MATERIALS AND METHODSBlood serum and synovial fluid from the patients :of chronic rheumatoid* Department of Public Health, Okayama University Medical SchoolMain part of this work was presented at the Tenth Annual Meeting of Japan Rheu-matism Association in April, 1966.511Seno: Protein concentration of synovial fluid in chronic rheumatoidProduced by The Berkeley Electronic Press, 196852 T. SENOrathritis served as materials.The serum was obtained from the blood drawn from the cubital vein ofsix patients whose RA test of serum proved positive. Synovial fluid was obtain-ed from 16 patients with chronic rheumatoid arthritis, and four controls, twopatients with osteoarthritis and two of traumatic arthritis. Of all these samplestotal protein contents were measured by Hitachi proteinometer.Paper-electrophoresis (6, 7) was carried out with 0.01 ml each of serum andsynovial fluid employing Toyo filter paper No. 50 soaked with buffer, and0.5 rnA of the current per 1 cm width of the filter paper at 5°C with Verona1buffer, pH 8.5 (lM=O.II). After running the paper was dried, stained withBPB stain for 30 minutes, treated with acetic acid, dried, and each fractionwas eluted in 10 ml of 2M NaHC03 solution. The optical density of eacheluate was measured by the Hitachi electrospectrometer, GPO type, at theoptical density of 570 m,u, and the protein contents of each eluate was com·puted.Gel filtration on Sephadex column, 3.0 x 43.0 cm: The elution was con-ducted with 3 ml of synovial fluid and 0.9% NaCl solution. The synovial fluidwas pretreated with hyaluronidase in order to lower the viscosity (8) of thesynovial fluid to make its separation easier, i. e. 3 ml of synovial fluid wereadded with 200 units of Harotase (hyaluronidase, a product of Takeda Pharma.ceutical Co.). After mixing well, the mixture was centrifuged ft 2,500 r. p. m.for 10 minutes, 'and 2 ml of the supernatant were used for the gel filtration,one drop per ten seconds.The protein contents of the collected fractions were measured with thespectrophotmeter (Hitachi type) at the optical density of 280 m,u. The proteinratio of eluted fractions was calculated from the ratio of the areas givingby the protein concentraction curves of individual fractions. Waaler-Rosetest (9) (S. S. C. A. - test) and latex fixation test (10) (LFT) were made in eachprotein fraction to determine rheumatoid factol.Sedimention constants were estimated in the eluates obtained from twofractions giving the individual peaks which were put into vins kin tube andcondensed to 1 % in Sephadex G.25 and subjected to the ultracentrifugation(Hitachi DCA-! type analytical ultracentrifugation apparatus) as 52,300 r. p.m. for 10 minutes.Double diffusion test: The precipitin reaction in agar was conducted withanti-human 19 S and anti-human 7S of globulin goat serum (the products ofMann Research Lab.) against the eluate from each peak as antigen.RESULTSTotal protein contents of the synovial fluid of the rheumatoid arthri·tis were 4.50 gldl in mean and somewhat higher than that from thesynovial fluid of arthritis other than rheumatoid arthritis 4.45 in mean.Total serum protein of the patients was 7.8 gldl in mean and relativelylow comparing to the value of normal human serum, 8.13 gldl (Tables2Acta Medica Okayama, Vol. 22 [1968], Iss. 1, Art. 4http://escholarship.lib.okayama-u.ac.jp/amo/vol22/iss1/4Synovial Protein in Rheumatoid Arthritis 531, 2).The analysis of the synovial fluid protein by electropaperchromato-graphy gave the similar levels in every constitution, albumin and globu-lins, as those in serum, with a relatively large value in r-globulin (Table-1).Table 1 Proteinometric and Electropaperchromatographic Analysesof Chronic Rheumatoid ArthritisTotalIAlbuminIGlobulinProtein (%) I 1 I(g/dl) (l:1 (%) (l:2 (%) fJ (%) '1 (%)Synovial Fluid * 4.34 36.66 7.95 9.01 13.57 32.28±0.67 ±0.08 ±0.08 ±0.08 ±0.75Serum ** 8.05 37.96 7.39 10.31 14.16 30.04±0.87 ±0.87 ±0.15 ±0.23 ±0.63* mean value of 11 cases ** mean value of 12 casesTable 2 Proteinometric and Gel filtration analyses of Blood Serumand Synovial Fluid on Sephadex G-200 ColumnData on 16 patients of Rheumatoid Arthritis, 4 patientsof Arthritis other than Rheumatic Disease and 3 NormalIndividualsMaterial I Diseases Ic= Total I P,otein F"ction by S,pbadoa G-200 'olumnprotein (g/dl) Peak 1 (%) I Peak 2 (%) I Peak 3 (%)(l9S) (7S) (4S or albumin)Rheumatoid 16 4.76 13.03 45.67 41.17arthritis ± 1.17 *3.97 ±10.20 ±14.60SynovialFluid Arthritis 4.45 8.4 42.90 48.5other than 4Irheumatism ±0.97 ±2.5 ±12.00 ±13.3Rheumatoid 6 7.65 21.24 40.85 37.85arthritis ±0.70 ±2.80 ±6.20 ±3.40SerumNormal 3 8.13 11.67 40.22 48.06Individuals ±O.29 ±11.30 ±4.30 ±4.80The analysis by gel filtration with sephadex G·200 revealed that theproteins of the synovial fluid can be separated into three fractions as inthe case of serum (Table 2, Figs. 1, 2). Concerning the protein contentsof each fraction, percentage of protein contents in peak 1 showed a mark-ed high level, with a slight decrease in peak 3, while the protein in peak2 showed less significant change (Table 2). Ultracentrifugation testrevealed that the proteins in peak 1 contains 19 S r-globulin, peak 2 7S r-globulin, and peak 3,4 S or albumin (Fig. 3). Precipitin reaction tests on3Seno: Protein concentration of synovial fluid in chronic rheumatoidProduced by The Berkeley Electronic Press, 196854 T. SENO50401.51.0:::LE0coC\J'00.5cid30Fraction numberFig. 1 Fractionation pattern of the serum protein in rheumatoid arthritis by SephadexG-200 column and rheumatoid test of the protein at each peakHatched column: S. S. C. A. titer, Open column: L. F. T. titer,Method: see text502010:::LE0CDC\J'0 05('(30Fraction numberFig. 2 Fractionation pattern of the synovial protein in rheumatoid arthritis by SephadexG-200 column and rheumatoid test of the protein at each peakHatched column: S. S. C. A. titer, Open column: L. F. T. titerMethod: see text4Acta Medica Okayama, Vol. 22 [1968], Iss. 1, Art. 4http://escholarship.lib.okayama-u.ac.jp/amo/vol22/iss1/4Synovial Protein in Rheumatoid Arthritis40 min "'---- . -- -Peak I Peak 2 Peak 3Fig. 3 Ultracentrifugation patterns of the protein from 1st, 2nd and 3rd peaksappearing in Fig. 355~.:)•\('\ (...- f ••f,l, \ .• ..'\ )., .•) t;') ()... ~.t.15t pealeo." • ~ i~:ciPitln3rd peak Anti-hum"" 195 2nd peak0001 st peako PreclpltlDlineJ.'(5 AoU-cY) (5"Fig. 4 Double diffusion test made on the proteins from 1st, 2nd and 3rd peaksappearing in Fig. 1every protein by using the antibodies to 75, 195 and globulins reconfirm-ed the finding obtained by analytical centrifugation (Fig. 4). Waaler-Rose test and LFT on 3 peaks in synovial fluid and serum disclosed that5Seno: Protein concentration of synovial fluid in chronic rheumatoidProduced by The Berkeley Electronic Press, 196856 T. SENOthe factor gIvmg a positive rheumatoid arthritic reaction IS m peak 1(Figs. 1, 2).It is generally accepted that in allergic disease r-globulins 7S and 19Sglobulins increase in blood serum. This is also true in rheumatoid arthri-tis, i. e. percentage of the protein in the fraction appearing as peak 1 ingel filtration by Sephadex G-200, or 19 S globulin, showed a marked in-crease in rheumatoid arthritis, with a slight increase in 7S globulin,indicating that the tissue antibody is largely concerned to the developmentof this desease. Similar tendency has also been revealed between theprotein constituents of synovial fiuids from rheumatoid arthritis andgeneral arthritis other than rheumatism, though the increase in 7 S glubu-lin was predominated in the latter. Comparing the protein constituentof the synovial fluid of rheumatoid arthritis to there of the blood serumof the same disease some difference in the percentage of 19 S fraction butsomewhat similar values in 7 S globulin and 48 or albumin fractions.DISCUSSIONThe electropaperchromatographic analysis of the synovial fluid andblood serum of the chronic rheumatoid arthritis also indicates that theprotein constituents of the synovial fluid of the chronic rheumatoid arth-ritis are very similar to those of the serum protein, though slightly highin r-globulin in the synovial fluid. This fact suggests the possibilitythat most of the proteins of the synovial fluid are of serum proteinstransudated. But it does not mean the direct communication as there isalways some difference between protein constituents of synovial fluid andserum. The present study has demonstrated that in the case of chronicrheumatoid arthritis there occurs an increase of 78 component (mainly78 r·globulin by paper chromatography) in the synovial fluid, and thisincrease may be responsible for the increase in the protein concentration ofthe synovial fluid (Fig. 5). It still remains unc1arified whether the 78 t-globulin is derived from r-globulin of serum proteins or derived from thetissue cells (mainly plasmacytes) of inflammatory site of the joint. As theprotein contents of the serum are found always higher than those of thesynovial fluid, protein in serum is transudated into synovial cavity. How-ever, concerning the communication of synovial fluid with serum there isa paper12 reporting the translocation of pSI r-globulin from synovial fluidto serum. Therefore, it is probable that there is a mutual communicationbetween synovial fluid and serum protein. Rheumatoid tests also showeda positive reaction in 198 fraction of synovial protein as in the case of6Acta Medica Okayama, Vol. 22 [1968], Iss. 1, Art. 4http://escholarship.lib.okayama-u.ac.jp/amo/vol22/iss1/4Synovial Protein in Pheumatoid Arthritis/577060~ 50ic:0 40a-Su'0 30.2'00:20102 :3 4 5 6 7Total protein cone, ( 9/dl)Fig. 5 Correlation between total protein concentration of synovial fluid in R. A.and relative protein percentage of Peaks 1. 2. 3. respectivly by gel filtrationCrosses: Peak I, Filled Circles: Peak 2, Open Circles: Peak 3serum protein., Although we have no definitive answer to this problemat present, in view of the fact that the r-globulin percentage of synovialfluid surpasses the r-globulin of serum, it is probable that the r-globulinfrom the local tissue cells of a portion of it might have seeped into serum.For the solution of this point further study is required.CONCLUSIONFor the purpose to reveal the characteris6cs of the synovial fluid ofthe chronic rheumatoid arthritis the proteins of the synovial fluid andblood 'serum have been analysed by employing the methods of electro·phoresis, gel filtration on Sephadex G-200 column and ultracentrifugation.Waaler-Rose test and latex fixation test have also been made on eachprotein fraction, and the following results were obtained.1) The total protein level of synovial fluid, which is 3/5 of that ofthe serum, is slightly higher than that of control.2) Fractionation of the synovial proteins by electrophoresis revealed7Seno: Protein concentration of synovial fluid in chronic rheumatoidProduced by The Berkeley Electronic Press, 196858 T. SENDnearly the same protein contents in each fraction in percentage as that ofcomparable fraction of the serum protein, with a slight increase in r-globulin fraction.3) The fractionation by Sephadex column G-200 give three peaks bothin serum and synovial fluid, 19 S, 7Sand 4 S.4) 19S fraction of the synovial fluid, which is mainly of r-globulin,showed a higher level than that of the synovial fluid from the controls.5) Rheumatoid tests gave positive reaction in the 1st peak containing19 S r-globulin from the synovial fluid and blood serum.ACKNOWLEDGEMENTThe author wishes to thank Prof. M. OGATA and Prof. T. KODAMA of our MedicalSchool for their helpful advices.REFERENCES1. SVARTZ, N.: Macroglobulin in rheumatoid arthritis and other diseases. j. A. M. A. 194,516, 19652. HORIUCHI, Y. et al.: Abnormal serum protein. b) On the rheumatoid factors. Taisha(Metabolism) 1, 611, 1964 (in Japanese)3. SVARTZ, N., SCHATZ, H. and HEDMAN, S.: The influence of different methods of dis-sociating the rheumatoid factor, studied by ultracentrifugation, sheep cell tests andimmunoelectrophoresis. Acta Med. Scandi1l/lvia, 177, 213, 19654. SCHUR, P. H. and SANDSON, J.: Immunologic studies of the proteins of human synovialfluid. Arthritis and Rheumatism, 6, 115, 19635. HIRAYAMA, S. and TOMINAGA, K.: Gel filtration. Nih~n Rinsho 21,2637, 1963 (in Japanese)6. ABE, M. et al.: Paper-electrophoresis of serum and protein. Nihon Rinsho 17, 562, 1959(in Japanese)7. MASUYAMA, Y. et al.: Normal value of serum protein fractions by fractionatin~ elutionmethod. The Physico-Chemical Biology 6, 254, 19608. SUENAGA, A.: A study on synovial fluid an:! hydrarthrosis. Okayama Igakkai Zasshi, 76,725, 19649. HELLER, G., JACOBSON, A. S. and KOLODY, M. H.: A modification of the hemagglutinationtest for rheumatoid arthritis. Proc. Soc. Exp. Biol. and Med. 72, 316, 194910. PLOTZ, C. M. and SINGER, J. M.: Latex fixation test. I. Application to serologic diag-nosis of rheumatoid arthritis, 11. Result in rheumatoid arthritis. Am. j. Med. 21, 888,195611. BLAND, J. H. and CLARK, L.: Rheumatoid factor in serum and joint fluid. Ann. Intern.Med. 58, 829, 196312. YANa, R. et al.: Protein metabolism in rheumatoid arthritis. j. jap. Rheum. Assoc. 7, 292,1967 (in Japanese)8Acta Medica Okayama, Vol. 22 [1968], Iss. 1, Art. 4http://escholarship.lib.okayama-u.ac.jp/amo/vol22/iss1/4

A modification of the hemagglutination test for rheumatoid arthritis.

A study on synovial fluid an:! hydrarthrosis.

et al.: Abnormal serum protein. b) On the rheumatoid factors.

et al.: Normal value of serum protein fractions by fractionatin~ elution method.

et al.: Paper-electrophoresis of serum and protein.

Gel filtration. Nih~n Rinsho 21,2637,

Immunologic studies of the proteins of human synovial fluid.

Latex fixation test. I. Application to serologic diagnosis of rheumatoid arthritis, 11. Result in rheumatoid arthritis.

Macroglobulin in rheumatoid arthritis and other diseases.

Protein metabolism in rheumatoid arthritis.

Rheumatoid factor in serum and joint fluid.

The influence of different methods of dissociating the rheumatoid factor, studied by ultracentrifugation, sheep cell tests and immunoelectrophoresis.

Protein concentration of synovial fluid in chronic rheumatoid arthritis. Estimation of protein in the synovial fluid of chronic rheumatoid arthritis by gel filtration and paper electrophoresis

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Protein concentration of synovial fluid in chronic rheumatoid arthritis. Estimation of protein in the synovial fluid of chronic rheumatoid arthritis by gel filtration and paper electrophoresis

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