Variations of Beta Glucuronidase Concentration in Abnormal Human Synovial Fluid

VARIATIONS OF BETA GLUCURONIDASE CONCENTRATION IN ABNORMAL HUMAN SYNOVIAL FLUID

Ralph F. Jacox, Alexandra Feldmahn

J Clin Invest. 1955;34(2):263-267. https://doi.org/10.1172/JCI103079.

Research Article

Find the latest version: https://jci.me/103079/pdf VARIATIONS OF BETA GLUCURONIDASE CONCENTRATION IN ABNORMAL HUMAN SYNOVIAL FLUID 1 By RALPH F. JACOX AND ALEXANDRA FELDMAHN2 (From the University of Rochester, School of Medicine and Dentistry, Rochester, N. Y.) (Submitted for publication September 1, 1954; accepted October 27, 1954)

Enzyme studies of human synovial fluid have the 370 C. waterbath for 16 to 24 hours. At the con- revealed the presence of amylase (1), a protease clusion of the incubation period, 5.0 millilitres of 0.1 M glycine buffer of pH 10.5 was pipetted into each tube. (2), and lipase (1), and a peroxidase (2). Al- Both were rapidly centrifuged to sediment particulate kaline phosphatase (3) has also been found and material. The supernatant fluid was then removed and more recently, the enzyme aminotripeptidase (4) assayed for phenolphthalein concentration. A Lumetron has been demonstrated. No studies have been colorimeter with a 540 filter was employed, with the made of the 8 glucuronidase content of human colorimeter set for the corresponding blank of each fluid to be analyzed. By interpolation from a previously pre- synovial fluid. In view of the significance of the pared standard curve, the concentration of phenolphtha- enzyme in mucin metabolism (5) it was felt that lein was then calculated. The concentration of P glu- an investigation of this problem would be of curonidase was then expressed as one microgram of interest. phenolphthalein liberated per millilitre per hour equal to The present report represents a study of the one unit. This method of glucuronidase assay is a modi- fication of that described by Talalay, Fishman, and Hug- p glucuronidase concentration of synovial fluids gins (6). obtained from patients with various pathologic joint diseases. In several instances, serial assays RESULTS were made in different patients suffering with de- Fifty-six patients with various pathologic joint generative joint disease, rheumatoid arthritis, or disorders have been studied. In some instances pyogenic arthritis. It will be shown that signifi- serial determinations have been made on the same cant variations of enzyme concentration are found patient over a period of several months. The ex- in these different types of joint disease. perimental results obtained from all assays of joint fluid are shown in Figure 1. It will be ob- METHOD served that patients with severe rheumatoid arthritis had considerable 8 glucuronidase in the Synovial fluid was obtained from human patients by aspiration of the knee, or elbow joint. The aspirated synovial fluid while those that had a mild and fluid was placed in a bottle containing sufficient potassium slowly progressive rheumatoid arthritis had less oxalate to delay clot formation. As soon as possible p glucuronidase. In contrast, the data obtained 1.0 millilitre aliquots of this material were pipetted into from patients with degenerative joint disease show each of two tests tubes. In one tube, serving as the con- trol, 4.0 millilitres of 0.1 M acetate buffer of pH 4.5 was that with a single exception, these synovial fluids added. Three and five-tenths millilitres of 0.1 M acetate had significantly less p glucuronidase than any of buffer was added to the second tube. The test tubes were the patients with rheumatoid arthritis. then placed in a 37° C. waterbath. After a sufficient time Three patients with pyogenic arthritis were had elapsed for temperature equilibration of the solu- tion of joint fluid, 0.5 millilitres of 0.005 M phenolph- studied. It will be noted that the enzyme concen- thalein glucuronide 8 was added to the second tube. A tration in these individuals was greatly elevated, few drops of chloroform were added to each tube to in- rising to values a thousand times those seen in de- hibit bacterial growth. Both tubes were incubated in generative joint disease. In patients with other 1 Research carried out under a grant for the study of types of joint disease (Figure 1), it will be ob- rheumatic fever from the Masonic Foundation for Medi- served that one individual with gout No. 5, one cal Research and Human Welfare. with psoriasis and arthritis No. 1, and one with a 2Research Fellow in Medicine, University of Rochester School of Medicine and Dentistry. Charcot joint No. 6 had values somewhat higher B Obtained from the Sigma Chemical Co., 4648 Easton than observed in patients with degenerative joint Avenue, St. Louis 13, Missouri. disease. Two patients with a traumatic joint ef- 263 264 RALPH F. JACOX AND ALEXANDRA FELDMAHN fusion, T, one with uremia No. 2 and one with an In the second patient, the data for /8 glucu- unexplained joint effusion No. 4 had low concen- ronidase determinations are illustrated succes- trations of /3 glucuronidase. One patient with a sively in a 68-year-old woman with an acute, se- proliferative synovitis No. 3 also had a low concen- vere rheumatoid arthritis. Initially this patient tration of synovial fluid /3 glucuronidase. had little relief with systemic cortisone therapy or Data on three patients on whom serial determi- by local instillation of Hydrocortone® into both nations of glucuronidase were made are shown in knee joints. Three months before her first clinic Figure 2. The first patient was a 72-year-old visit, and shortly after she sustained a hip fracture, man admitted to the Municipal Hospital with an the patient developed multi-articular pain, swelling acute febrile illness characterized by mild delirium, and increased limitation of joint function. Dur- an acute monoarticular arthritis, and cutaneous le- ing the next six months there was progressive, se- sions suggestive of meningococcal bacteremia. vere acute joint disease involving both knees and Cerebrospinal fluid examination was negative and the left wrist. several blood cultures showed no growth of bac- The first observation of the /3 glucuronidase teria. The left elbow was swollen, red and pain- concentration of the knee fluid (indicated by open ful and there was marked restriction of joint mo- circles, Figure 2) was made in July, 1953. At tion. An aspiration of the joint revealed purulent this time 50 mgm. of cortisone, administered by fluid, which contained 123,000 W. B. C. per mm.8, mouth, afforded moderate relief of joint symp- most of which were polymorphonuclear leucocytes. toms. However, the patient required occasional This fluid showed no bacterial growth after cul- supplementary injections of compound F into the ture aerobically, anaerobically, and under CO2. left knee during August. In September, 1953, the After therapy for several days with penicillin, a patient developed more pain and effusion of the gradual fall of fever and a slow subsidence of joint swelling and improvement of joint motion oc- 2.0 curred. However, the patient developed a severe, DEGENERATIVE RHEUMATOID OTHER JOINT JOINT ARTHRITIS DISEASE exfoliative dermatitis due to penicillin sensitivity. 1.8 DISEASE During this reaction, the left knee became swollen, IV? W SVERE MOPERAT red and tender. An aspiration of fluid from this TO PYOGENIC 1.6 o MILD ARTHRITIS joint revealed a cell count of 5,600 W. B. C. per 0 mm.8 After ACTH therapy, both the dermatitis 0 and arthritis improved. On cessation of ACTH 1.4 0 therapy, a recrudescence of joint swelling involving both knees, and wrists, and left elbow oc- -a 1.2 0 curred. 0 WVhile the etiology of the arthritis in this pa- z 1.0 0 tient is not unequivocally defined, it was felt that -J 0 0O the patient had an acute bacterial infection, pos- 0 * I-t 0.8 S sibly due to a meningococcus, which caused a sup- 00 0* 0 I purative arthritis of the left elbow. Subsequently z 0 0 this disease developed into multi-articular involve- a3- 0.6 0 ment which was more characteristic of a rheu- 0 00..0 Un 0.00 matoid arthritis. 4: 0.4 2 of a 0:-0. ,8 glucuronidase studies the elbow joint fluid 4c 0D 0 aspirations (indicated by the solid circle, Figure 0.2 0 . a 2) revealed very high levels of enzyme activity. 6 After chemotherapy, a decrease in enzyme con- 0 I Wa centration was found. With later recrudescence BETA GLUCURONIDASE OF JOINT FLUID of joint swelling, an increase of /3 glucuronidase FIG. 1. SYNOVIAL FLUID CONCENTRATION OF P GLucu- was observed. RONIDASE FOUND IN VARIOUS TYPES OF JOINT DISEASE BETA GLUCURONIDASE IN ABNORMAL HUMAN SYNOVIAL FLUID 265 wrist and knees and other joints became painful (180th day-Figure 2) when clinical improvement and swollen. After increasing the cortisone dos- was evident. age to 75 mgm., some improvement resulted, but The third patient is a 64-year-old woman with her symptoms again increased in November. In severe bilateral, degenerative joint disease in- November, Butazolidin@ therapy was instituted volving both knees. She had considerable sympto- at a time when cortisone was being gradually re- matic relief of pain with monthly injections of duced. This therapeutic change produced more Hydrocortoneg into the synovial space of each joint pain and disability. In early December, 50 knee. It will be noted that the /8 glucuronidase mgm. of cortisone each day was re-instituted and concentration in the synovial fluid of this patient Butazolidin® discontinued. By the end of De- was considerably less than in the patient with cember, while still taking 50 mgm. of cortisone rheumatoid arthritis. The enzyme remained con- each day, the patient began to improve. She was sistently low during the period of observation less depressed and all the previously swollen and (lower curve, Figure 2). tender joints, excepting the right knee, were less These data suggest that the concentration of /8 painful and swollen. By January, 1954, she was glucuronidase reflects the severity of inflammation better than she had been in months. within the synovium and joint space. In patients The /3 glucuronidase determinations made peri- with septic arthritis the enzyme is greatly in- odically throughout this six-month interval re- creased. In patients with degenerative joint dis- flected the severity of the arthritic process. Dur- ease or traumatic effusion, low concentrations are ing September (65th day of observation, Figure found. The rheumatoid arthritic patients were 2), when the peak of clinical activity was greatest, divided into mild and severe forms of disease by the highest concentration of enzyme was found. evaluation of the duration and degree of symptoms This was followed by a decrease in /8 glucuronidase of active inflammation, degree of progressive joint to a value of 0.42 units during January, 1954 destruction and by determination of the erythro-

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20 40 60 80 100 120 140 160 IOD DAYS BETA GLUCURONIDASE ASSAY OF JOINT FLUID FIG. 2. RESULTS OF SUCCESSIvE DETERMINATIONS OF 8 GLUCURONIDASE CONCENTRATION IN THE SYNOVIAL FLUIDS OF PATIENTS WITH A) PYOGENIC ARTHRITIS (SOLID CIRCLE), B) RHEUMATOID ARTHRITIS (OPEN CIRCLE), AND C) DEGENERATIVE JOINT DISEASE (SEMI-CLOSED CIRCLE) 266 RALPH F. JACOX AND ALEXANDRA FELDMAHN cyte sedimentation rate, plasma alpha globulin and in tears, and in gastric and bronchial secretions fibrinogen concentrations, degree of anemia and (5). extent of radiographic bone and joint alteration. The present investigation reveals that synovial With such a clinical estimation it was observed fluid obtained from human patients with various that higher concentrations of synovial fluid 8 glu- joint disorders also contains p glucuronidase. In- curonidase are usually found in the patient with dividuals with degenerative joint disease and those severe rheumatoid arthritis than in the mild, sub- with traumatic effusions or unexplained effusions acute disease (Figure 1). had enzyme concentrations ranging between 0.015 A comparison of p glucuronidase concentration to 0.080 units. Only one exception outside of this with the leucocyte count of joint fluid revealed range was observed. Patients with rheumatoid some correlation. If the synovial fluid leucocyte arthritis had values ranging from 0.190 to 1.0 units. In general, those patients with the most count was very elevated, high concentrations of severe rheumatoid arthritis had the highest values enzyme were found. If a few leucocytes were within this range, whereas the mild rheumatoid present, only small amounts of 8 glucuronidase patients had concentrations of 8 glucuronidase in were found. However, no absolute linear rela- the lower part of this range. Patients with septic tionship was observed. arthritis had concentrations of pt glucuronidase as Joint fluid obtained from patients with severe high as 19.7 units. rheumatoid arthritis and with septic arthritis was The concentration of 8 glucuronidase in syno- centrifuged to remove nearly all the formed ele- vial fluid appears to reflect the cellular response ments. The supernatant fluid was then analyzed to the inflammatory change in the synovial mem- for p glucuronidase activity. In one instance, the brane. Not only is the enzyme contained within untreated joint fluid had a pt glucuronidase ac- the leucocytes, but it is also found in the cell-free tivity of 3.09 units. The centrifuged supernatant fluid. Thus the contribution of intact leucocytes, fluid had an activity of 0.76 units or nearly ten disintegrated leucocytes and possible contribution times the concentration found in synovial fluid of by synovial membrane structures is measured degenerative joint disease. In another patient when unclotted synovial fluid is assayed for en- with a septic arthritis containing 216,000 polymor- zyme activity. phonuclear leucocytes, the p glucuronidase activity The data from this investigation suggest that was 19.7 units, while the centrifuged supernatant the estimation of p glucuronidase in synovial fluid fluid assayed 6.15 units. may afford another method of estimating severity It thus appears that enzyme, apart from that of inflammatory change within the joint structure contributed by leucocytes, may accumulate in syn- and may be of some aid in the differentiation of ovial fluid. This soluble enzyme is probably de- early rheumatoid arthritis from non-inflammatory rived from disintegrated leucocytes, although con- joint disease. tributions by synovial secretion cannot be ex- SUMMARY cluded. p8 glucuronidase has been demonstrated in hu- DISCUSSION man synovial fluid. In pathological states such as The enzyme p glucuronidase, which splits con- rheumatoid arthritis and pyogenic arthritis, there jugated glucuronides, is widely distributed within is as much as 200 to 1000-fold increase of enzyme the human body (5). It is found in high concen- over that found in synovial fluid from patients tration in liver and spleen and in lower concentra- with degenerative joint disease or those with a tions in other tissues. Polymorphonuclear leuco- traumatic joint effusion. While much of the 8 cytes contain p glucuronidase while lymphocytes, glucuronidase of the synovial fluid is contributed platelets, and red blood corpuscles contain none by the polymorphonuclear leucocytes, some of it (5). Pleural and ascitic effusions due to various is soluble. The soluble fraction may be derived pathologic disorders have been shown to contain from disintegrated leucocytes or from secretion by ,8 glucuronidase (5). This enzyme is also found the synovial membrane. BETA GLUCURONIDASE IN ABNORMAL HUMAN SYNOVIAL FLUID 267

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