Arthritis and Rheumatism

Transcription

1 Arthritis and Rheumatism VOL. V, O. 5 OCTOBER, 1962 Trace etal Composition of Synovial Fluid from Patients with Rheumatoid Arthritis By WILLIA IEDEREIER, ELLIS E. CREITZ AD HOWARD L. HOLLEY Synovial fluid and blood serum from patients with rheumatoid arthritis and from subjects free of symptoms of connective tissue disease were analyzed by emission spectrography. Synovial fluid of patients with rheumatoid arthritis contained increased concentrations of copper, iron and aluminum. Blood serum from these patients contained increased concentrations of copper and rubidium and decreased concentrations of iron, aluminum and chromium. Specimens de liquid0 synovial e de sero sanguinee ab patientes con arthritis rheumatoide e ab subjectos libere de symptomas de morbo de tissu conjunctive esseva analysate per spectrographia emissionic. Liquid0 synovial ab patientes con arthritis rheumatoide contineva augmentate concentrationes de cupro, ferro, e aluminium. Sero sanguinee ab tal patientes contineva augmentate concentrationes de cupro e de rubidium e reducite concentrationes de ferro, aluminium, e chromo. HE DECREASED VISCOSITY of synovial fluid obtained from patients T with rheumatoid arthritis is thought to be due, at least in part, to a decreased polymerization of the hyaluronic acid present in the fluid.l Previous in vitro studies in this laboratory have demonstrated that, in the presence of certain inorganic ions, hyaluronic acid, isolated from bovine synovial fluid, is rapidly depolymerized.2 The present studies were undertaken to determine whether or not the trace-metal composition of synovial fluid was altered from normal in patients with rheumatoid arthritis. Collection of Specimens ATERIALS AD ETHODS Synovial hid was aspirated from the knee joints of 13 patients with rheumatoid arthritis with effusion and from 33 patients seen at post-mortem whose death was due to various causes, but who did not have evidence of connective tissue disease. Diagnosis of rheumatoid arthritis was made on the basis of criteria established by the American Rheumatism Association.3 The age, sex and medication which the patients were receiving at the time the From the Division of Rheumatic Diseases, Department of edicine, edical Colleg@ of Alabama, Birmingham, Ah. This study was supported by grants from the ational lmtitutes of Health (USPH A and USPH A-3555). 439 ARTHRITIS & RHEUATIS, VOL. 5, o. 5 (OCTOBER), 1962

2 440 IEDEREIER, CREITZ AD HOLLEY Table 1.-Clinical Data on Patients with Rheumatoid Arthritis from Whom Sqnovial Fluid Was Collected Patient O. Sex Age Race F 50 W 38 W 58 W 40 W 48 W 53 W 73 C 19 W 18 C 67 W 48 W 32 W 40 W edication at Time of Svnovial Fluid Collection steroids gold, vitamin B,, steroids steroids, plaquenil synovial fluid was collected are indicated in table 1. The specimens were centrifuged and the clear supernatant fluid removed for analysis. Blood specimens were collected simultaneously from nine of the patients with rheumatoid arthritis and from five other patients with this disease who did not have effusion. Blood specimens were also collected from volunteers who had no history or clinical evidence of connective tissue disease. After coagulation, the blood was centrifuged and the serum promptly removed from the clot for trace-metal analysis. Preparation of Specimens All glassware was acid-cleaned, followed by treatment with EDTA and final rinsing in doubly distilled water. Standard stainless steel needles were used for collection of the specimens. Approximately 1.5 Gm. of the specimen to be analyzed were weighed into a fused silica crucible, and evaporated to dryness on a steam cone. The crucibles were transferred to a cold muffle furnace and heated to 450 C. for 4 hours. After cooling, the ash was moistened with nitric acid, evaporated to dryness on the steam cone and the crucibles returned to the muffle furnace for additional ashing at 450 C. for 16 hours. The crucibles were cooled, weighed and approximately 11 mg. of ash scraped from them with the aid of a quartz rod. An equal mass of spectroscopic quality graphite was mixed with the ash, and 20 mg. of the mixture packed into 3 mm. thin-walled, deep crater graphite electrodes for spectrographic analysis. Preparation of Staiu?urds A series of 10 spectrographic standards was prepared from Spex ix" that contained Ag, Al, As, B, Ba, Be, Bi, Ca, Cd, Ce, Co, Cr., Cs., Cu, F. Fe, Ge, Hg, K, Li, g, n, o, a, b, i, P, Pb, Rb, Sb, Si, Sn, Sr, Ta, Te, Th, Ti, T1, U., V., W, Zn, Zr, in a matrix that consisted of 98.9 per cent specpure acl and 1.1 per cent Ca3P0,. Each standard contained all of the above 43 elements in concentrations of per cent, per cent, per cent, per cent, per cent, per cent, per cent, per cent, per cent, and per cent, respectively. The standards were mixed with an equal mass of spectroscopic quality graphite prior to use. Twenty mg. of each mixture was packed into 3 mm. thin-walled, deep crater graphite electrodes for spectrographic analysis. "Spex Industries, Scotch Plains,. J.

3 TCE ETAL I SYOVIAL FLUID 441 Spectrographic Analysis In order to minimize interference in the spectrum due to cyanogen bands, and in an effort to stabilize the arc and thus improve quantitative reproducibility, all specimens were analyzed in a semi-controlled atmosphere consisting of 70 per cent argon and 30 per cent oxygen at a rate of flow of 2 liters/minute. This was achieved by use of a Stallwood jet.4 The spectra from 3130A to 8087A were recorded on 2-5/8 x 10 inch Eastman -1 spectrographic plates in a large quartz Littrow spectrograph by burning the specimen to completion in a D-C arc at a potential of 250 volts and a current of 7.5 amps. The spectra of all 10 standards were recorded in sequenre on a single photographic plate in order to determine the response of the emulsion to increasing concentrations of each of the elements in the standards. This series of spectra served as a comparator plate. Semiquantitative estimation of the concentration of the elements was made by visually matching the intensity of the line appearing in the spectrum of the specimen with the comparable line of similar intensity appearing in the series of standard spectra.5 For the determination of barium, silver and iron, the photographic plates were also evaluated with the aid of a microdensitometer, using the 5150A line of sodium to obtain an exposure index for each spectrum. The results were in good agreement with those obtained by the visual comparison method. The results were calculated and expressed in pg. per 100 Gm. of specimen. Sylzovial Fluid RESULTS The elements detected in synovial fluid are tabulated in table 2. Copper, aluminum, rubidium, lithium and strontium were present in all specimens analyzed. Iron and chromium were found in all specimens of synovial fluid from patients with rheumatoid arthritis, but in only 12 of 33 and of 33 specimens of postmortem synovial fluid, respectively. Barium, silver and manganese were detected with about equal incidence in post-mortem synovial fluid and in that from patients with rheumatoid arthritis. Although the spectra were inspected for nickel, cobalt and cadmium, none was found in any of the specimens analyzed. The range and mean concentrations of the elements listed in table 2 were calculated from only those samples in which the element was detected. The mean concentrations of copper, iron and aluminum were elevated in the specimens from patients with rheumatoid arthritis. Although small mean differences were observed for the other elements, they are of questionable significance. Blood Serum The elements detected in blood serum are tabulated in table 3. Copper, iron, aluminum, rubidium, lithium and strontium were present in all the specimens analyzed. anganese was found in about half of the specimens of both groups, but chromium, barium, silver and nickel were detected, respectively, in per cent, 58 per cent, 33 per cent and 29 per cent fewer specimens of blood serum from patients with rheumatoid arthritis than in those from the normal subjects. Cesium was detected in two specimens of blood serum from patients with rheumatoid arthritis but in none of those from normal subjects. Although the spectra were inspected for lines of cobalt and

4 442 IEDEREIER, CREITZ AD HOLLEY Table 2.-Trace-metal Composition of Svnovial Fluid Concentration (pg. per 100 Gm.) o. of o. of Specimens Source of Specimens Containing ean Element Specimens* Analyzed Element Range ean diff. P. (%)t Copper P x Iron P Aluminum P Chromium P Rubidium P Barium P Silver P anganese P ickel P Cesium P Lithium P t Strontium P t ~ - _ - ~ ~ - - _ ~ P = synovial fluid collected from patients without evidence of connective tissue disease seen at post-mortem. = synovial fluid collected from patients with rheumatoid arthritis. tp = probability (per cent). Element detectable in concentrations below limit of quantitative estimation. cadmium, these elements, if present, occurred in concentrations below the limit of detection. The range and mean concentrations of the elements listed in table 3 were aalculated from only those samples in which the element was detected. The mean concentrations of copper and rubidium in blood serum from patients with rheumatoid arthritis were 77 per cent and 87 per cent higher, respectively, than the normal. The mean concentrations of iron, aluminum and chromium in blood serum from patients with rheumatoid arthritis were 52 per cent, 55 per cent and 41 per cent lower, respectively, than the normal. It was difficult to compare the mean differences of barium, silver, manganese, nickel and cesium observed in the two groups of specimens because of the inconsistency with which these elements were detected. Although lithium and strontium were detected in all the specimens analyzed, they were present in such small amounts that the concentration could not be estimated. The ratio of the mean concentration of some of the elements in synovial fluid to the mean concentration in blood serum is shown in table 4. In patients with rheumatoid arthritis, the ratio is higher than the control for every element listed except rubidium, and is greater than 1.0 for aluminum. t

5 FtACJ3 ETAL IS SYOVIAL FLUID 443 Table 3.-Trace-metal Composition of Blood Serum o. of Concentration (fig. per 100 Gm.) - o. of Specimens Source of Specimens Containing ean Element Specimens Analyzed Element Range ean diff. P. (%) t ~- Copper Iron Aluminum Chromium Rubidium Barium Silver anganese ickel Cesium Lithium t < 0.5 <0.5 <0.5 < Strontium t L blood serum from normal controls. = blood serum from patients with rheumatoid arthritis. tp = probability (per cent). IElement detectable in concentrations below limit of quantitative estimation. Table 4.-The Ratio of the ean Concentration of Trace Elements in Synovial Fluid to the ean Concentration in Blood Serum Ratio S.F. - B.S. Element Controls Rheumatoid Arthritis Copper Iron Aluminum Chromium Rubidium DISCUSSIO The results of this study confirm previous reports that the concentration of copper in blood serum from patients with rheumatoid arthritis is elevated.6 They further demonstrate that the concentration of copper in synovial fluid from these patients is higher than that from patients without evidence of connective tissue disease. The data also indicate that synovial fluid from patients with rheumatoid arthritis has increased concentrations of iron and aluminum.

6 444 IEDERWIER, CREIlZ AD HOLLEY The emission spectrograph offers a relatively sensitive method of analysis and is well suited for qualitative studies. It lacks quantitative precision, however, and yields data that are essentially semiquantitative. The results suggest that in patients with rheumatoid arthritis, the metabolism of a number of trace elements is abnormal, and provide an impetus for the development and application of procedures that will yield more accurate quantitative data. COCLUSIOS Blood serum and synovial fluid specimens from patients with rheumatoid arthritis were analyzed for trace-element composition by emission spectrography and compared with specimens from subjects without evidence of connective tissue disease. The synovial fluid of patients with rheumatoid arthritis contained increased concentrations of copper, iron and aluminum. The blood serum of patients with rheumatoid arthritis contained increased concentrations of copper and rubidium and decreased concentrations of iron, aluminum and chromium. ACKOWLEDGET The authors wish to thank r. Carl Rampacek, Research Director, U. S. B.. etallurgy Research Center, Tuscaloosa, Ala., for his cooperation in making available the spectrographic equipment required for this study. REFERECES 1. Sundblad, L.: Studies on hyaluronic acids 4. Stallwood, B. J.: Air-cooled electrodes in synovial fluids, Acta SOC. med. up- for the spectrochemical analysis of sallen 38:113, powders. J. Optic. SOC. Amer. 44: 2, 2. Pigman, W., Hawkins, W., Gramling, E., Rizvi, S., and Holley, H. L.: Factors 5. itteldorf, A. J.: Semiquantitative specaffecting the viscosity of hyaluronic trochemical analysis. In Clark, G. L.: acid and synovial fluid. Arch. Biochem. The Encyclopedia of Spectroscopy, 89: 184, ew York, Reinhold Publ. Corp., 1960, 3. Ropes,. W., Bennett, G. A., Cobb, S., pp Jacox, R., and Jessar, R. A.: Proposed 6. Evers, A.: Behavior of serum copper in diagnostic criteria for rheumatoid arth- rheumatoid arthritis. Ztschr. Rheumaritis. J. Chron. Dis. 5620, forsch 11:164, William iedemeier, Ph.D., Assistant Professor, Department of edicine, University of Alabama 0dical Center* Bimingham, Ah. Ellis E. Creitz, B.S., Supervisory Analytical Chemist, U. S. Bureau of ines, University of Alahma, Tuscaloosa, Ala. Howard L. Holley,.D., Director, Diuision of Rhmmtic Diseases, Professor of edicine, University of Alabama edical Center, Birmingham, Ah.