The absorption of serum albumin and gamma globulin from the knee joint of man and rabbit.код для вставкиСкачать
The Absorption of Serum Albumin and Gamma Globulin from the Knee Joint of Man and Rabbit By GERALDP. RODNANAND MARGARET J. MACLACHLAN Studies of the rate of clearance of radioactivity from the knee joints of human subjects free of joint disease and of normal rabbits following the intra-articular injection of 1131-serum proteins indicate that the synovial cavity is readily and equally permeable to both serum albumin and gamma globulin. Studios deI rapiditate del clearance de radioactivitate ab le articulation del g e m in humanos sin morbo articular e in conilios normal post le injection intraarticular de proteinas seral marcate con 1191 indica gue le cavitate synovial es preste- e equalmente permeabile pro albumina seral e globulina gamma. B AUER, Short, and Bennett reported studies on the permeability of the synovium of the rabbit and dog in 1933, utilizing serologic technics for the detection of a variety of heterologous pr0teins.l They noted the rapid appearance of egg albumin and horse serum albumin in the joint fluid of rabbits after intravenous administration of these proteins, and, in the dog, their prompt removal via lymphatic channels following injection directly into the knee joint. They failed to detect horse serum globulin in thoracic duct lymph after intra-articular injection of this protein and concluded that globulins were removed from a normal joint with difficulty, if at all. After reviewing this matter some years later, and adding their own observations on the removal of colloidal and finely particulate matter from the joint cavity of the rabbit, Adkins and Davies2 concluded that particles considerably larger than those of serum globulin passed rapidly into the regional lymphatic glands. In the present experiments, we have sought to obtain information concerning the permeability of synovium to protein by examining the clearance of radioiodine ( 11")-labeled serum albumin and gamma globulin injected into the knee joints of rabbits and of human subjects free of joint disease. The use of serum proteins in this regard is supported by the studies of Schmid and MacNair which indicate that the proteins of normal synovial fluid are identical in character with those of plasma.3 Our findings confirm the observations of others on the rapid absorption of serum albumin and indicate similar ready permeability of synovium to serum gamma globulin. EXPERIMENTAL PROCEDURE Iodine (1131)-lubeledproteins.-The Il~l-r&bitserum albumin and gamma globulin used in this study were obtained from the Department of Pathology, University of Pittsburgh.' F r o m the Department of Medicine, Uniucrsity of Pittsburgh, Pittsburgh, Pa. This inuestigution wus supported by a reseurch grunt from the Western Penruylvaniu Chapter of the Arthritis and Rheumatism Foundation and by research grant B-1287, United States Public Health Service. Presented ut the Annual Meeting of the American Rheumatism Association, Washington, D. C., June, 1959. 'We wish to thank Miss Maria Deichmiller for supplies of these labeled proteins. 152 SERUM ALBUMIN AND Y-GLOBULIN ABSORPTION FROM KNEE 153 P l - h u m a n serum albumin and gamma globulin were purchased from Abbott Laboratories. The homogeneity of the 1131-hurnan serum protein fractions was checked by adding small quantities of the labeled albumin or gamma globulin to human serum and then separating the proteins by means of filter paper electrophoresis. When portions of the paper strip containing the individual protein fractions were assayed in a well-type scintillation counter, radioactivity was found to be confined, respectively, to the albumin or gamma globulin moiety. Determination of clearance of serum proteins from knee joint following intra-articular injectimN.-Hospitalized patients free of joint and circulatory disease each received 3 ,UC. of 1131-human serum albumin or gamma globulin. The radioactive protein was added to 3 to 5 ml. of either 0.9 per cent NaCl solution or the subject's own plasma, and the mixture, containing either relatively minute or relatively large doses of protein, was injected into a suprapatellar bursa. All patients remained at bed rest during the course of study. Mature New Zealand white rabbits received both homologous and heterologous serum albumin and gamma globulin. The II"-labeled proteins were diluted with either 0.9 per cent NaCl solution or rabbit plasma, and 1 ml. of the mixture, containing 1 pc. of radioactivity, was injected into the knee joint via a 25 gage needle inserted directly through the subpatellar tendon. The animals were confined to their cagcs during the period of study but were not otherwise restricted in activity. The gamma radiation of the knee joint was measured by means of a collimated scintillation probe kept at a fixed position immediateIy above the infrapatellar (in the case of the rabbits) or suprapatellar (in the case of the human subjects) ligament of the flexed joint. The area of detection of radioactivity encompassed by the probe at the level of the knee joint was 17 cm. in diameter. It thus included in the case of the rabbit the prolongation of the synovial membrane of the knee about the tendon of the extensor digitorurn Zongus. Duplicate counts were obtained a t intervals following the injection of radioactive protein, and the values corrected for background activity and natural decay of isotope. The results are expressed in figures 1 to 4 in terms of the change in radioactivity of the knee joint as a percentage of the amount present in the joint immediately following injection. Determination of appearance of serum proteins in peripheral blood following intraarticular injection of knee.-In 6 patients free of joint disease, the amount of Ilal-human serum albumin or gamma globulin injected into the knee joint was increased to 40 pc. Samples of serum and of the various ,protein fractions obtained by filter paper electrophoresis were assayed for radioactivity a t intervals beginning 15 minutes after injection. In a similar manner, the serum protein fractions of 5 rabbits were analyzed for radioactivity following the intra-articular administration of 12 pc. of 1131-rabbit serum albumin or gamma globulin. OBSERVATIONS Clearance of Serum Proteins from the Knee Joint of the Rabbit Serum albumin.-Both rabbit and human serum albumin, diluted in saline solution, and injected in the amount of 0.1 mg., were rapidly removed from the knee (table 1; fig. 1).An average of 25 per cent of the original radioactivity remained in the joint after 24 hours. At 48 hours, the clearance of radioactivity was nearly complete. After this time the low level of radioactivity of the joint and the circulation of radioactive albumin in the blood prevented any further accurate estimation of the amount of local, i.e., noncirculating, radioactivity. The peripheral blood was found to contain nondialyzable radioactivity as early as 15minutes after the intra-articular injection of 1131-serum albumin. This activity was present in the albumin fraction obtained by electrophoresis of the serum proteins. Serum gamma globulin.-The radioactivity of 0.03 to 0.6 mg. of salinediluted 1131-serumgamma globulin was absorbed from the knee joint of the 154 RODNAN AND MACLACHLAN Table 1.-The Clearance of Llsl-Serum Proteins from the Knee Joint following Intra-articular Iniection in Normal Rabbits % of original radioactivity of Type and amount of protein injected No. of animals studied RSA, 0.1 mg. HSA, 0.1 mg. 4 6 60 k 6* 81 2 6 25 + 5 28 ir 9 9+4 8t3 RSGG, 0.03 mg. HSGG, 0.6 mg. 5 5 74 46 70 I 3 24 t 4 34 t 4 10 -+ 2 16 c 2 RSGG, 10 mg. RSGG, 10 mg. 150 U. hyaluronidase 5 57 k 8 16 F 1 6-tl 5 58 t 8 16 F 4 8-+3 + 4 hr. joint, hours after injection 24 hr. 48 hr. RSA = rabbit serum albumin, HSA = human serum albumin, RSGG gamma globulin, HSGG = human serum gamma globulin. "Mean and standard deviation. 12 hr. 4311 = rabbit serum rabbit at a rate which did not differ significantly from that of albumin (table 1; fig. 2 ) . The clearance of the globulin was virtually complete at 72 hours. There was again little, if any, difference between the rate of removal of the heterologous and the homologous protein. The peripheral blood was found to contain increasing amounts of radioactivity beginning 15 minutes after injection of the labeled protein. This radioactivity was confined to the gamma globulin fraction. When the total amount of gamma globulin injected into the knee was sharply increased by administering the tracer dose of labeled protein in 1 inl. of rabbit plasma instead of saline solution, there was no significant clifference in the rate of removal of radioactivity from that observed followI'3'-SERUM ALBUMIN AMOUNT OF PROTEIN. 0.1 mg II3'-SERUM GAMMA GLOBULIN AMOUNT PROTEIN INJECTED VOLUME INJECTATE: I ml. 0A.m - RABBIT S A OQOoAV-HUMAN SA RABBIT S G G - 0 0 3 m q HUMAN S G G - 0 6 mg VOLUME INJECTATE OAT.4 OAPO 0 HOURS AFTER INTRA-ARTICULAR INJECTION OF 1 1 3 1 - s ~ I ml -RSGG -HSGG HOURS AFTER INTRA-ARTICULAR INJECTION OF 1131 - SGG Fig. 1.-The clearance of rabbit and human P1l-serum albumin from the knee joint of the rabbit, as determined by surface counting technic. Fig. 2.-The clearance of rabbit and human 1131-serumgamma globulin from the knee joint of the rabbit. 155 SEAUM ALBUMIN AND y-GLOBULIN ABSORPTION FROM KNEE ing the injection of more minute quantities of the protein (table 1; fig. 3). Inclusion of 150 U. of hyaluronidase ( Wydase) in the intra-articular injection of the gamma globulin appeared to have no effect upon the rate of clearance of the radioactive protein from the joint. the Human Serum albumin.-In seven subjects the tracer dose of P3l-human serum albumin was mixed in 3 to 5 ml. of each individual’s plasma, the approximate amount of serum albumin injected ranging from 90 to 160 mg. Five ather individuals were given minute quantities of I131-albumin, 0.1 to 0.25 mg., in 3 to 4 ml. of 0.9 per cent NaCl solution. Both the small and large doses of albumin were removed from the joint at a remarkably similar rate (table 2), averages of 37 and 40 per cent of original radioactivity being absorbed at 24 hours. Appreciable quantities of radioactivity remained at the site of injection after 72 hours, however. The serum albumin fraction of the peripheral blood of two patients was found to contain radioactivity within 15 minutes after injection of the I1”-protein into the suprapatellar bursa. Serum gamma globulin.-Six patients, all free of joint disease, received the tracer dose of 1131-humanserum gamma globulin mixed in 3 to 4 ml. of plasma. The total amount of gamma globulin given in the intra-articular injection was approximately 30 to 40 mg. The radioactivity of the knee declined at a rate remarkably similar to that following the injection of 11”albumin (table 3; fig. 4). At 24 hours, an average of 61 per cent remained in the joint; by 48 hours the average amount left was 43 per cent of the original dose. Approximately one-fourth of the radioactivity was still present at the site of injection after 96 hours. Radioactive gamma globulin was detected in the peripheral blood of four patients within 15 minutes after the intraarticular injection of the 1131-gamma globulin, and increased steadily in amount for a period of 48 hours after the intra-articular injection. Clearance of Serum Proteins from the Knee joint of Table 2.--The Clearance of Z131’-€ZumanSerum Albumin from the Knee Joint f o l l m i n g Intra-articular Znjection in Human Subjects Free of Joint Disease Subject number, age and sex % of original radioactivity of joint, hours after injection 4 hr. 24 hr. 48 hr. 72 hr. a. Injection of 0.1 to 0.25 mg. albumin in 3 t o 4 ml. of 0.9% NaCl solution 1,35, M 94 72 52 38 2,34, F 64 49 3,62, F 76 41 28 4,24, F 83 65 44 32 5,34, F 98 73 55 42 Mean and S.D. 88 4 7 63 2 1 1 46 rt 9 b. Injection of 90 to 160 ing. albumin in 3 to 5 ml. of plasma 1,41, F 56 29 2, 36, F 77 60 29 3,35, F 96 72 59 4,65, M 68 63 43 5,% F 70 48 33 28 6,58, M 93 61 7,60,F 92 62 45 36 Mean and S.D. 83 -t- 11 60 & 6 40 3z 10 156 RODNAN AND MACLACHLAN Table 3.-The Clearance of IIS1-HumanSerum Gamma Globulin from the Knee Joint following Intra-articular Injection in Human Subjects Free of Joint Disease % of original radioactivity of joint, hours after injection Subject number, age and sex 24 hr. 4 hr. 72 hr. 48 hr. 96 Injection of 30 to 40 mg. gamma globulin in 3 to 4 ml. of plasma 1, 35, F 90 74 54 45 2,19, F 82 65 52 34 3,26, F 82 63 43 31 82 60 41 28 4,74, M 5,40, M 80 52 39 27 6,39, F 71 50 30 20 Mean and S.D. 81 +- 5% 61 2 8% 43 -t 8% 31 & 7% RABBIT SERUM GAMMA GLOBULIN t3I- hr. 37 25 24 22 20 15 24 2 6% HUMAN SERUM GAMMA GLOBULIN AMOUNT OF PROTEIN 10 mq AMOUNT OF PROTEIN 30-40mg VOLUME INJECTATE VOLUME INJECTATE 3-4 ml *A789 OOAVO I ml - CONTROL STUDY - HYALURONIOASE (150 units Included in injection) Y ,I I O f 4 : 41.9 ;2 HOURS AFTER INTRA-ARTICULAR INJECTION OF -RSGG 1 Of; I 24 i 8 ;2 916 HOURS AFTER INTRA-ARTICULAR INJECTION OF 113'-HSGG Fig. 3.-The clearance of Pf-rabbit serum gamma globulin from the knee joint of the rabbit with and without simultaneous administration of hyaluronidase. Fig. 4.-The clearance of Pl-hurnan seruni gamma globulin from the knee joints of 6 patients free of rheumatic disease. DISCUSSION It would appear from this study that the synovium of the knee joint of man and of rabbit is as readily permeable to serum gamma globulin as it is to serum albumin. The discrepancy in the rate of clearance of these proteins observed between rabbit and man may be more apparent than real, however, and can be accounted for by a difference in intra-articular hydrostatic pressure resulting from the injection of the 1131-protein-containingfluid. Edlund has shown that high initial pressures following intra-articular injection lead to an increased absorption of fluid and of hem~globin.~ In the present experiments, the injection of 1 ml. into the knee joint of the rabbit produced visible distention and considerabIe increase in intra-articular pressure, whereas in- SERUM ALBUMIN AND Y-GLOBULIN ABSORPTION FROM KNEE 157 jection of 3 to 5 ml. of fluid into the human knee had relatively little effect. The rate of clearance of 1131-serum albumin from the joints of normal subjects reported herein is less rapid than that observed by Ahlstrom and Kuipers and their respective co-workers5p6in studies of individuals with active rheumatoid arthritis. There is relatively little difference, however, when the absorption of the protein is compared to that noted in patients with inactive joint disease or those with active arthritis who have received an intra-articular injection of hydrocortisone,j circumstances in which the permeability of the synovium may be considered as having been restored to a more normal state. SUMMARY 1. The synovium of the knee of man and of rabbit is readily permeable to homologous I1"-serurn aIbumin and gamma globulin, and there appears to be little difference in the rate of absorption of these two proteins following their intra-articular injection. 2. Radioactively labeled albumin and gamma globulin appear in the peripheral blood within 15 minutes after intra-articular injection of the respective proteins. 3. Under the conditions of the present experiments, absorption of both serum albumin and gamma globulin from the knee joint of the rabbit is virtually complete within 72 hours after intra-articular injection. In the case of absorption from the joints of human subjects free of joint disease, appreciable quantities of radioactivity (approximately one-third of the amount injected) remain in sits as long as 72 hours after intra-articular injection. 4. The simultaneous intra-articular administration of hyaluronidase appears to have had no effect upon the rate of clearance of gamma globulin from the knee joint of the rabbit. REFERENCES 1. Bauer, W., Short, C. L., and Bennett, G. A,: The manner of removal of proteins from normal joints. J.ExPer*Md. 57:419, 1933. 2. Adkins, E. W. D., and Davies, D. V.: Absorption from the joint cavity. Quart. J.Exper.Physio1. 30~147, 1940. 3. Schmid, K., and MacNair, M. B.: Characterization of he proteins of certain postmortem human synovial fluids. J. Clin.Invest. .37:708, 1958. 4. Edlund, T.: Studies on absorption of colloids and fluid from rabbit knee joints. Acta physiol. scandinav. Supplementum 62, 1949. 5. Ahlstrom, Gedda p. o.,and &&erg, H.: Disappearance of radioactive serum albumin from joints in rheumatoid arthritis. Acta Rheum. scandinav. 2~129, 1956. 6. K ~ i ~ e r sR., K. W., F r a n k C., and Robert, W. N.: Effects of antirheuinatic drugs on the resorption of 1131labeled albumin from the synovial fluid. Acta Rheum. scandinav. 231, 1956. Gerald P . Rodnan, M.D.,Assistant Professor of Medicine, University of Pittsburgh, School of Medicine, Pittsburgh, Pa. Margaret J . Maclachlan, M.D.,lnstructor in Medicine, University of Pittsburgh, School of Medicine, Pittsburgh, Pa.