Synovial Iron Deposition in Rheumatoid Arthritis Robert M. Bennett, E.D. Williams, S.M. Lewis and P.J.L. Holt An isotope ferrokinetic study has been used to study the rate of iron deposition in 13 rheumatoid knees. Iron accumulation occurred only after incorporation of labelled iron into circulating erythrocytes, suggesting that intermittent intraarticular hemorrhages were the source of the deposits. The mean iron accumulation was 1.25 mg/knee/day representing a mean intraarticular blood loss of 3.58 ml. There was no correlation between the degree of iron deposition and the hemoglobin, platelet count or serum iron. T h e presence of hemosiderin deposits in the synovial membrane of rheumatoid joints has long been recognized by pathologists (1). T h e mean concentration of iron in rheumatoid synovial membrane was found to be 347 pg/g dry tissue compared with 15.2 pg/g in normal synovium (2), and it has been suggested that these deposits are unavailable for erythropoiesis possibly contributing to the anemia of rheumatoid arthritis (3). Controversy has arisen over whether these deposits exert a beneficial effect on the course of the arthritis (4) or are deleterious (5), and theories on their origin include a direct uptake from the plasma (6), a result of chronic intraarticular bleeding (3) and phag- ocytosis of leukocytes containing iron bound to cytoplasmic lactoferrin (4). Using an isotope kinetic method we have studied the rate of iron deposition in synovial membrane, its mechanism and whether there is any relationship to development of anemia in patients with rheumatoid arthritis. From the Departments of Medicine, Medical Physics and Haematology, The Royal Postgraduate Medical School, Hammenmith, London, W.12. This work was supported by the Arthritis and Rheumatism Council for Research. ROBERT M . BENNETT, MRCP: Registrar and Tutor in Medicine, Department of Medicine, Royal Postgraduate Medical School; E. D. WILLIAMS, MA, MSC: Senior Physicist, Department of Medical Physics, Royal Postgraduate Medical School; s. M . LEWIS, MD, MRC (PATH): Reader in Haematology, Department of Haematology, Royal Postgraduate Medical School; P. J. L. HOLT, MRCP: Consultant and Lecturer in Medicine, Department of Medicine, Royal Postgraduate Medical School. Reprint requests should be addressed to: Dr. Robert M. Bennett, The University of Chicago, Department of Medicine, Box 404,950 East 59th Street, Chicago 111 60637. Submitted for publication July 11, 1972; accepted Dec 1, 1972. Isotope Administration. Ten microcuries of 59Fein the form of ferric citrate was incubated with the patients' plasma for 30 minutes at 37" C in order to label the transferrin. An accurately measured volume was injected intravenously. 298 MATERIALS AND METHODS Subjects. Patients with definite rheumatoid arthritis (7) involving the knees were studied. For comparison, 2 patients with osteoarthrosis of the knees were used as controls. All patients were ambulant within the hospital confines. All the rheumatoid patients had a full hematologic profile including bone marrow aspiration; they had not taken any iron preparation for at least 3 months. Counting. Blood samples were taken every 10 minutes for the first hour and every 3 to 10 days over the course of the ensuing 50 to 90 days. Surface counting was performed at similar intervals over the affected knees, the thighs and sacrum by means of a collimated scintillation counter. Sufficient counts were obtained to give an error of less than 3%. All counts were adjusted for background and decay. Calculations. These are largely based on the assumption that iron accumulation in a rheumatoid knee has a Arthritis and Rheumatism, Vol. 16, No. 3 (May-June 1973) SYNOVIAL IRON DEPOSITION IN RA 150 SACRUM 100 I 60 1w 50 0 KNE 1% 1W m THIGH 60 Fig 1. Ferrokinetic study in a rheumatoid patient showing surface counting and blood activity. do 03060w120 1 10 20 30 Fig 2. lntraarticular accumulation of iron after an intravenous injection of 59Fe-labelledtransferrin i n 2 patients-1 with rheumatoid arthritis (W) and the other with osteoarthrosis of the knees ( 0 ) . 40 M Days Minutes 1 Arthritis and Rheumatism, Vol. 16, No. 3 (May-June 1973) 10 15 w m 23 299 BENNETT ET AL monoexponential increase of the type y = 1 - e-x. Reference to Figure 1 (data from a rheumatoid patient) shows that iron rapidly leaves the plasma over the first 24 hours and appears in the bone marrow (sacral trace). T h e counts over the involved knee and thigh (representing blood flow) also decrease over the first 24 hours. Over the subsequent 50 days, as iron leaves the marrow and becomes incorporated into circulating red blood cells, there is an increased activity over the thigh and knee. However, with time the knee trace continues to increase while the thigh trace falls gradually. This is attributed to the deposition of iron within the knee. By equating the thigh counts (representative of blood flow) to the knee counts on Day 1 and using the same coefficient for the thigh counts on subsequent days, a correcfed count is obtained. Substracting this from the knee counts gives an excess count in the knee which is proportional to iron deposition within the knee. When plotted against time the excess knee counts (Figure 2) are seen to rise steeply and then level off to a plateau. By comparison, 2 osteoarthritic knees showed no accumulation of iron. A diagrammatic representation of the accumulation of iron in a rheumatoid knee is seen in Figure 3, from which the following mathematical analysis is derived: Ai = accumulated knee counts at time i days P = plateaucounts Ki = excess knee counts at i days (KO being 24 hours post isotope injection) r = fractional rate of accumulation of final activity in knee t = timeindays B = percentage of blood-borne iron accumulated per day C = percentage of total blood-borne iron in knee Then A;=Ki-Ko also ~i = P (1 - e - n ) or P - ~i = Pe-" therefore log 10 ( P - Ai) a -rt - - _ _ ___-- ---__-__ ---- 1 -ie, plotting log,,, (P - Ai) against t gives a line slope -r, using the least squares fit of a straight line. This slope was derived graphically from -,. - 0.693 %t knowing -r: B - r 100 P x c ----------2 KO As 1 g/lOO ml of hemoglobin (Hb) contains 0.335 g of iron the total blood-borne iron (T) = 0.355 x blood volume x H b 100 ----- 3 Thus absolute iron accumulation per day (F) = Xg ____________ 4 100 Assuming that all this iron is derived from red Excess Counts Fig 3. Diagramatic representation of iron accumulation in a rheumatoid knee. The curve is a monoexponential function of the type y = 1 = e-'. Arthritis and Rheumatism, Vol. 16, No. 3 (May-June 1973) SYNOVIAL IRON DEPOSITION IN RA Fig 4. Semilogarithmic plot of excess knee counts against time, in a rheumatoid patient (Case C). rheumatoid knees have a similar monoexponential increase, in that when plotted on a (I)= F X 1 0 0 100 ~ ml _ _ _ _ _ _ _ _ _ _ _ 5 logarithmic scale against time, the points lie on Hb x 0.335 a straight line, well within the conventional levels of statistical significance. T h e three osT h e value “C” was obtained by injecting 20 ml of autologous blood (with 100% erythrocyte incorporation of 59Fe) teoarthritic knees show no such monointo the knee joints of 2 patients undergoing synovectomy exponential increase. T h e coefficient of “x” in the next day. “B” was then directly calculated from the increase in count rate and thus, by substitution in Formula 2, the regression equations is the “r” of Formua value for “C” was obtained. T h e values for the two knees las 1 and 2. From this value the percentage of were 0.43% and 0.2570, the mean value of 0.34% was used blood-borne iron accumulated per day (B), the in all calculations. Total blood volume was calculated from absolute iron accumulation per day (F) and the the packed cell volume and plasma volume (derived from intraarticular blood loss (I) are calculated from the initial dilution of labelled transferrin). Formulas 2, 4 and 5. T h e values are seen in Table 2. T h e mean iron accumulation per day RESULTS for the 13 rheumatoid knees was 1.25 mg Accumulation of iron in the affected knees (+ 0.7 2 SD). If this were all derived from red only occurred after it had been completely blood cells, as suggested by the fact that accucleared from the plasma and had become incor- mulation only occurred after incorporation of iron into erythrocytes, it represents a mean porated into the circulating red cells (Figure 1). T h e results of plotting log,c(P - Ai) against daily intraarticular blood loss of 3.58 ml (k9.0 “t” for a rheumatoid knee is seen in Figure 4. 2 SD). There was no obvious correlation beT h e regression equations of the 13 rheumatoid tween iron deposition and the degree of inflamknees are given in Table 1. It is seen that all the mation in the individual patients, but in those blood cells the intraarticular blood loss per day Arthritis and Rheumatism, Vol. 16, No. 3 (May-June 1973) 301 BENNETT ET AL Table 1. RegressionEquationsfor iron Accumulation in 13 Rheumatoid Knees and 3 OsteoarthriticKnees Patient Diagnosis" RA RA RA RA E RA F RA RA OA OA Regression equation Coefficient of correlation ( I ) Significance + + y =- 2.6~ + 5272 y =- 3.9~ + 4266 y = - 4 . 8 ~+ 2630 y = - 4.6~ + 3467 v = - 3 . 2 ~+ 3890 y = -14.5~+ 4677 y =- 4.1~ + 3388 y =- 3 3 + ~ 5248 y =- 2 6+ ~ 3236 y =- 3 2+ ~ 3890 y =- 2 6 + ~ 3631 y = - 0 . 7 ~+ 613 y=- 0.7~ + 389 y =- 1.8~ + 1445 -0.97 -0.89 o.ooooo1 0.0006 -0.96 -0.94 0.0000001 0.000007 -0.95 -0.97 0.000007 0.000002 -0.95 0.0000006 -0.89 -0.6 0.001 0.05 -0.85 -0.73 0.03 0.04 -0.96 -0.94 0.003 0.007 -0.34 -0.49 0.13 0.20 -0.36 0.24 y = -10.3~ 1866 y = - 3 . 5 ~ 2630 (0 "RA = Rheumatoid arthritis OA = Osteoarthritis Table 2. iron Accumulation and Bleeding in 13 RheumatoidKnees Patient Knee Accumulation of iron/day (TO) A R L R L R L L R L R L R L 0.054 0.014 0.025 0.022 0.026 0.023 0.024 0.342 0.074 1.034 0.523 0.76 0.57 B C D E F G 302 Absolute accumulation of ironlday (mg) 0.32 0.08 0.18 0.16 0.18 0.16 0.12 1.61 0.35 4.60 2.30 3.5 2.7 Bleeding into knee/day (ml) 0.91 0.23 0.43 0.38 0.44 0.39 0.36 3.96 0.86 13.80 6.90 10.1 7.9 Arthritis and Rheumatism, Vol. 16, No. 3 (May-June 1973) SYNOVIAL IRON DEPOSITION IN RA patients showing a marked difference between the knees (Subjects E, F and G) the more inflamed knee showed the higher iron accumulation. All the rheumatoid patients were anemic with a low serum iron although total iron binding capacity was normal and all had plentiful iron stores in the bone marrow. There was no correlation between the degree of intraarticular bleeding and the hemoglobin: y = - 0 . 1 8 ~ + 11.3 ( r = 0.49, P > 0.1) or the serum iron y = - 0 . 0 5 ~ + 56 ( r = -0.2, P > 0.1). Similarly, no correlation was found between the platelet count and degree of intraarticular bleeding: y = 0.01~ + 385,000 ( T = 0.41, P > 0.1). DISCUSSION T h e validity of these results is based on two assumptions. First, that the vascularity of the knee remains unchanged throughout the study: in so far as these patients were all in hospital having supervised therapy it is likely that the vascularity would, if anything, regress slightly, a change that would lead to an underestimation of iron deposition in subsequent calculations. Second, that the percentage of blood-borne iron in the knee (0.34%),a figure derived from the mean values for 2 rheumatoid knees from different patients, was the same for all the knees studied. Neither of these assumptions is likely to be completely valid, although the errors involved are of a small magnitude and as such our results are probably a close approximation of intraarticular iron deposition. As significant iron accumulation in rheumatoid knees only occurs after it has become incorporated into circulating erythrocytes, it is reasonable to suppose that the synovial iron deposits accrue directly from hemoglobinbound iron rather than transferrin-bound iron. Conceivably this could result from small hemorrhages within the synovial membrane with subsequent incorporation of the iron into subintimal macrophages, or from intraarticular bleeding. We have previously shown in a patient with a popliteal synovial fistula (8) that intraarticular bleeding does occur and is directly related to exercise of the affected knee. T h e average blood loss in that particular patient, measured directly with 51Cr-labelled red blood cells, was 2.5 ml/day, a figure agreeing closely with our results derived from ferrokinetic studies. It is not surprising to find that there is no relationship between the degree of intraarticular bleeding and the anemia of rheumatoid arthritis. Although quite large amounts of iron may be sequestered in the synovial membrane of a rheumatoid patient, these patients are seldom iron deficient as judged by their iron marrow stores. Poor incorporation of iron into developing erythroblasts despite the fact that there are plentiful iron stores within the reticuloendothelial cells of the bone marrow has led to the concept of a recticuloendothelial cellerythroblast block in the anemia of chronic inflammation (9- 1 1 ). However, in rheumatoid patients with inadequate iron stores it is probable that intraarticular bleeding could tip the balance towards overt iron deficiency. Whether under such circumstances, synovial iron deposits could be utilized for subsequent erythropoiesis remains to be elucidated. T h e finding of a relationship between synovial iron deposits and the severity of joint destruction (3) has led to a belief that the accumulated iron has a cytotoxic effect in the joint. An equally acceptable explanation would be that the more severely involved joint will exhibit the most intraarticular bleeding and this, compounded by a possible longer duration of the disease in this situation, leads to the observed increase in iron deposits. ACKNOWLEDGMENTS We are grateful to Professor E.G.L. Bywaters for his advice and encouragement and Dr. J.M. Gumpel for allowing us to study several of his patients. REFERENCES 1 . Collins DH: Haemosiderosis and haemochro- Arthritis and Rheumatism, Vol. 16, No. 3 (May-June 1973) 303 BENNETT ET AL 2. 3. 4. 5. 6. matosis of synovial tissue. J Bone Joint Surg Br 33:436-441,1951 Senator GB, Muirden KD: Concentration of iron in synovial membrane, synovial fluid and serum in rheumatoid arthritis and other joint diseases. Ann Rheum Dis 27:49-53, 1968 Muirden KD: The anaemia of rheumatoid arthritis: the significance of iron deposits in the synovial membrane. Aust Ann Med 2:97-104, 1970 Mowat AG, Disney T F , Vaughan J H : Effect of iron dextran, gold thiosulphate and hydrocortisone acetate on experimental synovitis in the guinea pig. Ann Rheum Dis 30:187-193, 1971 Muirden KD, Senator CB: Iron in the synovial membrane in rheumatoid arihritis and other joint diseases. Ann Rheum Dis 27:38-48, 1968 Mowat AG, Hathersell T E : Nature of anaemia in rheumatoid arthritis: VIII. Iron content of 304 synovial tissue in patients with rheumatoid arthritis and in normal individuals. Ann Rheum Dis 27:345-350,1968 7. Ropes MW, Bennett GA, Cobb S, et al: Diagnostic criteria-1958 review. Ann Rheum Dis 18:49-53, 1959 8. Bennett RM, Williams ED, Lewis SM, et al: Studies of a popliteal synovial fistula. Ann Rheum Dis 31:482-486,1972 9. Freireich EJ, Miller A, Emerson EP, et al: The effect of inflammation in the utilization of erythrocyte and transferrin bound radioiron for red cell production. Blood 12:972-983, 1957 10. Cartwright LE, Lee GR: The anaemia of chronic disorders. Br J Haematol 2 1 :147- 152, 1971 11. Bennett RM, Holt PJL, Lewis SM: The kinetics of distribution, uptake and utilization of iron dextran in rheumatoid arthritis. (Unpublished data) Arthritis and Rheumatism, Vol. 16, No. 3 (May-June 1973)
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