Scandinavian Journal of Rheumatology
ISSN: 0300-9742 (Print) 1502-7732 (Online) Journal homepage: http://www.tandfonline.com/loi/irhe20
Plasma And Synovial Fluid cAMP In Patients With Rheumatoid Arthritis Svend Wadskov, Rene Donde & Jesper Sylvest To cite this article: Svend Wadskov, Rene Donde & Jesper Sylvest (1979) Plasma And Synovial Fluid cAMP In Patients With Rheumatoid Arthritis, Scandinavian Journal of Rheumatology, 8:3, 136-138, DOI: 10.3109/03009747909114444 To link to this article: http://dx.doi.org/10.3109/03009747909114444
Published online: 12 Jul 2009.
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Citing articles: 1 View citing articles
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Date: 22 March 2016, At: 15:34
Scand J Rheumatology 8: 136138, 1979
PLASMA AND SYNOVIAL FLUID cAMP IN PATIENTS WITH RHEUMATOID ARTHRITIS Svend Wadskov,' Rene Donde2 and Jesper Sylvest3
Downloaded by [ECU Libraries] at 15:34 22 March 2016
From the 'Departments oj*Dennutology and 2Rheirmatologyand Physical Medicine, Ht-idowe Hospital, Copenltccgen. curd the :'Departmentof Rherfmatofogy and Physical Medicine, Rigshospitalet , University of Copenlmgen, Copenhagen, Denmark
ABSTRACT. cAMP was measured in plasma and synovial Sampling fluid from 11 patiemts suffering from rheumatoid arthritis Synovial fluid was obtained by puncture of the knee joint with a sped& proteln-biadning assay. Plasma and synovial between 9 and I 1 a.m. Blood was taken simultaneously flukl values were 17.0f6.8 pmd/ml and 8.3k3.7 pmdld, from an a m vein. Synovial fluid and blood were collected range 5-28 pmd/ml and 5-16 pmd/ml, respectively. No into cooled centrifuge tubes containing 100 pl of 0.5 moUl correlationcould be established between plasma and syna- EDTA. pH 7.5, centrifugatedimmediately and then stored vial fluid levels, plasma and disease activity. or synovial at -20°C until assay. nuld and disease activity, as)udged by Lansbury's index. It is d u d e d that It seems unlikely that synovial fluid CAMP Preparation of specimens is derived sddy from plasma and that M) simple relation The alcohol extraction method was used for deproteinizaexists ltetwew~CAMPin plasma and synovial nuid and totnl tion in both plasma and synovial fluid. dkme activity. Two millilitres of ethanol was added to I ml of plasma
During the last decade, cyclic nucleotides have proved to play a major role in inflammatory processes. Preliminary results from the measuring of cyclic nucleotides in synovial fluids have recently been published (5.6). The aim of this study was to evaluate the levels of CAMPin plasma and synovial fluid in patients suffering from rheumatoid arthritis and to determine whether plasma and synovial fluid cAMP are correlated to each other andlor to disease activity.
MATERIAL AND METHODS Subjects
1 I consecutive patients with rheumatoid arthritis (RA),all women, with a mean age of 65 (range 28-86) years, were included in the study. All patients were classified according to American Rheumatism Association criteria (4) and clinical activity was evaluated by Lansbury's clinical index (3). No patient was being treated systemically with corticosteroids. Eight days before the investigation all non-steroid antiinflammatory drugs were withdrawn and only analgetics (dextropropoxiphene65 mg qid) were allowed. One patient (no. 5 ) was followed by daily puncture after treatment with Naproxen (Naprosyn3 250 mg twice daily was initiated.
or synovial fluid, then mixed and left at mom temperature for 5 minutes. The mixture was then centrifugatedat 3 500 g and the supernatant collected. The precipitates were washed with 1 ml ethanol :water (2 :1) and centrifugated at 3500 g . The supernatants were combined and lyophitized. The residue was dissolved in 0.5 ml of assay buffer, for direct use in the cAMP assay. Cyclic A M P assay
Cyclic AMP assay kits were obtained from the Radiochemical Centre, Amersham (RCA), England. The RCA assay kit is based on a competitive protein binding method. The assay was performed at 4°C in 7x 11 mm test tubes. 50 pl standard or sample, 50 pl Labelled cAMP (0.9 p m l SH-cAMP) and 100 pl binding protein were added to test tubes and mixed. Standards and samples were always run in duplicate and a complete standard curve was included in each assay. The test tubes covered with parafilm were then incubated for 120 min at 4°C. At the end of the incubation period, 100 pl of the charcoal suspension was added, the tubes were mixed for 10 sec and then immediately centrifugatedfor 5 min at 3 MO g at room temperature. Two hundred jd of the supernatant was transferred to counting vials and 10 ml scintillation fluid (Instagel from Packard Instruments Company, IIlinois, USA) added. The samples were subsequently counted at least twice for 5 minutes in a Beckman type CS 250 liquid scintillationcounter. Standard curves were plotted on semilogarithmic paper with percentage finding as a function of added CAMP.
RESULTS
The coeficience of intra- and interassay variation (S.D. in percentage of mean) was determined by
Plustilo and svnoiialjlirid cAMP
137
Table 1. For details, see text
Plasma
Intra-assay coefficient of variation (S.D. in percentage of mean)
Inter-assay coeffcient of variation (S.D. in percentage of mean)
Recovery of 0.9 pmol 3H-cAMP in (mean _+ S.D.)
Undiluted (pmol/ml)
Diluted 1 : 2 (pmol/ml)
5.2%
9.5 % (7.350.7 pmol/ml. n = 10) 10.3% (5.8k0.6pmol/ml,
101%f3.9% n=10
7.5f0.3, n=8
3.5f0.4, n=8
83 % f 3 . 9%, n =9
6.2k0.5, n=8
3.8k0.4, n =8
(7.6f0.4 pmol/ml, n=6)
Downloaded by [ECU Libraries] at 15:34 22 March 2016
Synovial fluid
8.0% (6.2f0.5 pmol/ml, n=9)
Dilution test (mean f S . D . )
n=5)
measuring cAMP in a number of samples from the same known plasma and synovial fluid in the same assay (intra-assay variations) or in consecutive assays (inter-assay variation). In order to evaluate the recovery, labelled cAMP was added to plasma or synovial fluid samples which were then processed as samples of unknown. The specificity of the binding protein and the sensitivity of the binding assay (detection limit) have been reported previously (8). For further confirmation, cAMP was measured in diluted plasma and synovial fluid. These results are given in Table I. The results obtained in plasma and synovial fluid in subjects suffering from RA are given in Table 11. Mean2S.D. values and ranges in plasma and synovial fluid were 17.0k6.8 pmol/l, 5-28 pmol; 8.3k3.7 pmol/ml, 5-16 pmol/ml, respectively. The results of cAMP measurement in plasma and synovial fluid taken on 5 consecutive days in the same patient are given in Table 111. No correlation between plasma and synovial fluid content of cAMP (v=0.2580), plasma and disease activity (Lansbury’s index), or synovial fluid and disease activity (Lansbury’s index) was revealed (r=-O.1515 and r=-0.0430, respectively). DISCUSSION cAMP is the intracellular mediator for various hormones and hormone-like substances, including in-
flammatory mediators. Although cAMP is present in all body fluids investigated hitherto no known function of extracellular cAMP has yet been established in human beings. However, altered levels of cyclic nucleotides have been observed in several body fluids (e.g. urine, gastric juice, saliva), reflecting alterations in tissue stimulation and function (for review, see ref. 1). As demonstrated in this investigation, plasma cAMP in patients suffering from rheumatoid arthritis was within the normal range (1, 7 ) . Since normal synovial fluid cannot be obtained, the normal (basal) level of cAMP in synovial fluid is unknown. The cAMP concentrations in most extracellular fluids are in the range 10-20 nM. Our finding in synovial fouid (5-16 nM) tallies closely with this. Data on cyclic nucleotides in biological fluids must be interpreted with extreme caution until the source of origin has been established. We failed to demonstrate any correlation between plasma and synovial fluid CAMP. Thus, it seems unlikely that the synovial fluid concentration is determined solely by clearance of cAMP from plasma. This view is supported by the finding of a higher level of cAMP in synovial fluid as compared with plasma in patients l and by the lack of correlation between plasma and synovial fluid when samples were taken on 5 consecutive days from patient 5.
Table 11. Levels of cAMP in synovialfluid and plasma in patients with rheumatoid arthritis Patient no. 1
Synovial fluid, pmol/ml Plasma, pmol/ml Lansbury index
6.0 5.0 89
2
8.0 10.0
67
3 5.0 20.0 81
4 16.0 20.0 66
5 6.0 18.0 %
6 6.4 14.0 48
7
5.5 11.0 106
8 6.0 28.0 58
9
10
10.0 22.0 122
20.0 85
14.5
11
7.5 20.0 96
138
S. Wiiilskor et id.
h Y 1
2
3
4
5
cAMP in plasma, PmoUmI
Downloaded by [ECU Libraries] at 15:34 22 March 2016
cAMP in synovial fluid, pmollml
18.0 22.0 6.0
2.0
11.0 8.0
16.0
8.0 2.0
2.0
A clear relation between deposits of complement components in skin biopsies and clinical activity as judged by Lansbury's index has previously been established (2). We have been unable to demonstrate any such simple relation between levels of CAMP in plasma or synovial fluid and disease activity.
REFERENCES 1. Broadus, A. E.: Clinical cyclic nucleotide research. I n
Advances in Cyclic Nucleotide Research (ed. Greengaard and Robison), vol. 8. p. 509. Raven Press, New York, 1977. 2. Donde, R., Permin, H., Juhl, F.. Wiik. A., Hansen. N. E. & Andenen, R. B.: Immune deposits in the der-
mo-epidermaljunction in rheumatoid arthritis. Scand J Rheumatol6: 57. 1977. Lansbury. J.: Methods for evaluating. In Arthritis and Allied Conditions (ed. J. L. Hollander). 8th ed., p. 256, 1972. 4. Ropes, M.W.. Bennett, G. A.. Cobb, S.,Jacox, R. & Jessar, R. A.: 1958: Revision of diagnostic criteria for rheumatoid arthritis. Bull Rheum Dis 9: 175-176, 1958. 5. Tateishi. H., Hiruhata. K. & Takeda, M.:Cyclic AMP in rheumatoid synovial fluid. Abstracts XIV International Rheumatology Congress, San Francisco, p. 835, 1V7. Trang. L. E.. Granstriim. E., Liivgren, 0..RocaNordlund. A. E., Horn, R. & Walaas. 0.: Joint fluid levels of prostaglandins (PGFsEE), thromboxanes (TXB,) and cyclic nucleotides (CAMP, cGMP) in Rheumatoid artiritis. Abstracts XIV International Rheumatology Congress, San Francisco, p. 1036, 1977. Wadskov. S., Kassis, V. & Ssndergaard, J.: Day to day variation in plasma cyclic AMP. IRCS Med Science 6: 244. 1978. 8. Wadskov, S. & Sendergaard, J.: Determination of cyclic AMP in heat separated human epidermal tissue. Acta Dermatovener (Stockholm)58: 191, 1978.
.,
Jitbmitiedfor prrblicution October 25. 1978 Svend Wadskov DeptofDermatology Hvidovre Hospital DK-2650 Hvidovre Denmark
ISSN: 0300-9742 (Print) 1502-7732 (Online) Journal homepage: http://www.tandfonline.com/loi/irhe20
Plasma And Synovial Fluid cAMP In Patients With Rheumatoid Arthritis Svend Wadskov, Rene Donde & Jesper Sylvest To cite this article: Svend Wadskov, Rene Donde & Jesper Sylvest (1979) Plasma And Synovial Fluid cAMP In Patients With Rheumatoid Arthritis, Scandinavian Journal of Rheumatology, 8:3, 136-138, DOI: 10.3109/03009747909114444 To link to this article: http://dx.doi.org/10.3109/03009747909114444
Published online: 12 Jul 2009.
Submit your article to this journal
View related articles
Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=irhe20 Download by: [ECU Libraries]
Date: 22 March 2016, At: 15:34
Scand J Rheumatology 8: 136138, 1979
PLASMA AND SYNOVIAL FLUID cAMP IN PATIENTS WITH RHEUMATOID ARTHRITIS Svend Wadskov,' Rene Donde2 and Jesper Sylvest3
Downloaded by [ECU Libraries] at 15:34 22 March 2016
From the 'Departments oj*Dennutology and 2Rheirmatologyand Physical Medicine, Ht-idowe Hospital, Copenltccgen. curd the :'Departmentof Rherfmatofogy and Physical Medicine, Rigshospitalet , University of Copenlmgen, Copenhagen, Denmark
ABSTRACT. cAMP was measured in plasma and synovial Sampling fluid from 11 patiemts suffering from rheumatoid arthritis Synovial fluid was obtained by puncture of the knee joint with a sped& proteln-biadning assay. Plasma and synovial between 9 and I 1 a.m. Blood was taken simultaneously flukl values were 17.0f6.8 pmd/ml and 8.3k3.7 pmdld, from an a m vein. Synovial fluid and blood were collected range 5-28 pmd/ml and 5-16 pmd/ml, respectively. No into cooled centrifuge tubes containing 100 pl of 0.5 moUl correlationcould be established between plasma and syna- EDTA. pH 7.5, centrifugatedimmediately and then stored vial fluid levels, plasma and disease activity. or synovial at -20°C until assay. nuld and disease activity, as)udged by Lansbury's index. It is d u d e d that It seems unlikely that synovial fluid CAMP Preparation of specimens is derived sddy from plasma and that M) simple relation The alcohol extraction method was used for deproteinizaexists ltetwew~CAMPin plasma and synovial nuid and totnl tion in both plasma and synovial fluid. dkme activity. Two millilitres of ethanol was added to I ml of plasma
During the last decade, cyclic nucleotides have proved to play a major role in inflammatory processes. Preliminary results from the measuring of cyclic nucleotides in synovial fluids have recently been published (5.6). The aim of this study was to evaluate the levels of CAMPin plasma and synovial fluid in patients suffering from rheumatoid arthritis and to determine whether plasma and synovial fluid cAMP are correlated to each other andlor to disease activity.
MATERIAL AND METHODS Subjects
1 I consecutive patients with rheumatoid arthritis (RA),all women, with a mean age of 65 (range 28-86) years, were included in the study. All patients were classified according to American Rheumatism Association criteria (4) and clinical activity was evaluated by Lansbury's clinical index (3). No patient was being treated systemically with corticosteroids. Eight days before the investigation all non-steroid antiinflammatory drugs were withdrawn and only analgetics (dextropropoxiphene65 mg qid) were allowed. One patient (no. 5 ) was followed by daily puncture after treatment with Naproxen (Naprosyn3 250 mg twice daily was initiated.
or synovial fluid, then mixed and left at mom temperature for 5 minutes. The mixture was then centrifugatedat 3 500 g and the supernatant collected. The precipitates were washed with 1 ml ethanol :water (2 :1) and centrifugated at 3500 g . The supernatants were combined and lyophitized. The residue was dissolved in 0.5 ml of assay buffer, for direct use in the cAMP assay. Cyclic A M P assay
Cyclic AMP assay kits were obtained from the Radiochemical Centre, Amersham (RCA), England. The RCA assay kit is based on a competitive protein binding method. The assay was performed at 4°C in 7x 11 mm test tubes. 50 pl standard or sample, 50 pl Labelled cAMP (0.9 p m l SH-cAMP) and 100 pl binding protein were added to test tubes and mixed. Standards and samples were always run in duplicate and a complete standard curve was included in each assay. The test tubes covered with parafilm were then incubated for 120 min at 4°C. At the end of the incubation period, 100 pl of the charcoal suspension was added, the tubes were mixed for 10 sec and then immediately centrifugatedfor 5 min at 3 MO g at room temperature. Two hundred jd of the supernatant was transferred to counting vials and 10 ml scintillation fluid (Instagel from Packard Instruments Company, IIlinois, USA) added. The samples were subsequently counted at least twice for 5 minutes in a Beckman type CS 250 liquid scintillationcounter. Standard curves were plotted on semilogarithmic paper with percentage finding as a function of added CAMP.
RESULTS
The coeficience of intra- and interassay variation (S.D. in percentage of mean) was determined by
Plustilo and svnoiialjlirid cAMP
137
Table 1. For details, see text
Plasma
Intra-assay coefficient of variation (S.D. in percentage of mean)
Inter-assay coeffcient of variation (S.D. in percentage of mean)
Recovery of 0.9 pmol 3H-cAMP in (mean _+ S.D.)
Undiluted (pmol/ml)
Diluted 1 : 2 (pmol/ml)
5.2%
9.5 % (7.350.7 pmol/ml. n = 10) 10.3% (5.8k0.6pmol/ml,
101%f3.9% n=10
7.5f0.3, n=8
3.5f0.4, n=8
83 % f 3 . 9%, n =9
6.2k0.5, n=8
3.8k0.4, n =8
(7.6f0.4 pmol/ml, n=6)
Downloaded by [ECU Libraries] at 15:34 22 March 2016
Synovial fluid
8.0% (6.2f0.5 pmol/ml, n=9)
Dilution test (mean f S . D . )
n=5)
measuring cAMP in a number of samples from the same known plasma and synovial fluid in the same assay (intra-assay variations) or in consecutive assays (inter-assay variation). In order to evaluate the recovery, labelled cAMP was added to plasma or synovial fluid samples which were then processed as samples of unknown. The specificity of the binding protein and the sensitivity of the binding assay (detection limit) have been reported previously (8). For further confirmation, cAMP was measured in diluted plasma and synovial fluid. These results are given in Table I. The results obtained in plasma and synovial fluid in subjects suffering from RA are given in Table 11. Mean2S.D. values and ranges in plasma and synovial fluid were 17.0k6.8 pmol/l, 5-28 pmol; 8.3k3.7 pmol/ml, 5-16 pmol/ml, respectively. The results of cAMP measurement in plasma and synovial fluid taken on 5 consecutive days in the same patient are given in Table 111. No correlation between plasma and synovial fluid content of cAMP (v=0.2580), plasma and disease activity (Lansbury’s index), or synovial fluid and disease activity (Lansbury’s index) was revealed (r=-O.1515 and r=-0.0430, respectively). DISCUSSION cAMP is the intracellular mediator for various hormones and hormone-like substances, including in-
flammatory mediators. Although cAMP is present in all body fluids investigated hitherto no known function of extracellular cAMP has yet been established in human beings. However, altered levels of cyclic nucleotides have been observed in several body fluids (e.g. urine, gastric juice, saliva), reflecting alterations in tissue stimulation and function (for review, see ref. 1). As demonstrated in this investigation, plasma cAMP in patients suffering from rheumatoid arthritis was within the normal range (1, 7 ) . Since normal synovial fluid cannot be obtained, the normal (basal) level of cAMP in synovial fluid is unknown. The cAMP concentrations in most extracellular fluids are in the range 10-20 nM. Our finding in synovial fouid (5-16 nM) tallies closely with this. Data on cyclic nucleotides in biological fluids must be interpreted with extreme caution until the source of origin has been established. We failed to demonstrate any correlation between plasma and synovial fluid CAMP. Thus, it seems unlikely that the synovial fluid concentration is determined solely by clearance of cAMP from plasma. This view is supported by the finding of a higher level of cAMP in synovial fluid as compared with plasma in patients l and by the lack of correlation between plasma and synovial fluid when samples were taken on 5 consecutive days from patient 5.
Table 11. Levels of cAMP in synovialfluid and plasma in patients with rheumatoid arthritis Patient no. 1
Synovial fluid, pmol/ml Plasma, pmol/ml Lansbury index
6.0 5.0 89
2
8.0 10.0
67
3 5.0 20.0 81
4 16.0 20.0 66
5 6.0 18.0 %
6 6.4 14.0 48
7
5.5 11.0 106
8 6.0 28.0 58
9
10
10.0 22.0 122
20.0 85
14.5
11
7.5 20.0 96
138
S. Wiiilskor et id.
h Y 1
2
3
4
5
cAMP in plasma, PmoUmI
Downloaded by [ECU Libraries] at 15:34 22 March 2016
cAMP in synovial fluid, pmollml
18.0 22.0 6.0
2.0
11.0 8.0
16.0
8.0 2.0
2.0
A clear relation between deposits of complement components in skin biopsies and clinical activity as judged by Lansbury's index has previously been established (2). We have been unable to demonstrate any such simple relation between levels of CAMP in plasma or synovial fluid and disease activity.
REFERENCES 1. Broadus, A. E.: Clinical cyclic nucleotide research. I n
Advances in Cyclic Nucleotide Research (ed. Greengaard and Robison), vol. 8. p. 509. Raven Press, New York, 1977. 2. Donde, R., Permin, H., Juhl, F.. Wiik. A., Hansen. N. E. & Andenen, R. B.: Immune deposits in the der-
mo-epidermaljunction in rheumatoid arthritis. Scand J Rheumatol6: 57. 1977. Lansbury. J.: Methods for evaluating. In Arthritis and Allied Conditions (ed. J. L. Hollander). 8th ed., p. 256, 1972. 4. Ropes, M.W.. Bennett, G. A.. Cobb, S.,Jacox, R. & Jessar, R. A.: 1958: Revision of diagnostic criteria for rheumatoid arthritis. Bull Rheum Dis 9: 175-176, 1958. 5. Tateishi. H., Hiruhata. K. & Takeda, M.:Cyclic AMP in rheumatoid synovial fluid. Abstracts XIV International Rheumatology Congress, San Francisco, p. 835, 1V7. Trang. L. E.. Granstriim. E., Liivgren, 0..RocaNordlund. A. E., Horn, R. & Walaas. 0.: Joint fluid levels of prostaglandins (PGFsEE), thromboxanes (TXB,) and cyclic nucleotides (CAMP, cGMP) in Rheumatoid artiritis. Abstracts XIV International Rheumatology Congress, San Francisco, p. 1036, 1977. Wadskov. S., Kassis, V. & Ssndergaard, J.: Day to day variation in plasma cyclic AMP. IRCS Med Science 6: 244. 1978. 8. Wadskov, S. & Sendergaard, J.: Determination of cyclic AMP in heat separated human epidermal tissue. Acta Dermatovener (Stockholm)58: 191, 1978.
.,
Jitbmitiedfor prrblicution October 25. 1978 Svend Wadskov DeptofDermatology Hvidovre Hospital DK-2650 Hvidovre Denmark
