Effects of Different Medical Treatments on Serum Copper, Selenium and Zinc Levels in Patients with Rheumatoid Arthritis
- 333 Downloads
The aim of the present study was to measure the changes in serum selenium, zinc, and copper in patients being treated for rheumatoid arthritis. Thirty-two patients and 52 healthy controls were included in the study. The copper level was higher and those of selenium and zinc were lower in the patients relative to controls. Treatment with methotrexate elevated the zinc levels, but not zinc and selenium. Treatments with salazopyrin, corticosteroids, chloroquine, and non-steroidal anti-inflammatory drugs did not change the levels of any of the elements studied. The decrease in zinc and selenium levels and elevation in copper levels observed in the patients probably resulted from the defense response of organism and are mediated by inflammatory-like substances.
KeywordsRheumatoid arthritis Selenium Zinc Copper Inflammation Methotrexate
Non-steroidal anti-inflammatory drugs
Reactive oxygen species
Tumor necrosis factor-α and
Rheumatoid arthritis (RA) is an autoimmune disorder of unknown etiology . In recent years, a few studies have investigated the possible role of trace elements in the etiology and pathogenesis of RA [2, 3, 4, 5]. The mechanism(s) by which cells play a role in RA pathogenesis has been the subject of intense research in recent years. The changes in trace element levels are part of immune defense system of organism and are induced by the hormone-like substances interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) . These substances are immunocytokines liberated in a dose-dependent mode, mostly by activated macrophages, in response to several stimuli, including trauma, stress, and infection . Known changes in many infections are alterations in ferric ion, zinc, and copper levels in sera associated with elevated levels of acute phase proteins such as ceruloplasmin [6, 7].
Free radicals produced by various processes in peripheral blood are inhibited by enzymatic and non-enzymatic antioxidants . These antioxidants protect DNA and other important molecules from oxidative damage which would otherwise induce apoptosis.
For example, zinc is a cofactor of several enzymes and plays critical roles in cell membrane stabilization, protein synthesis, growth of normal tissues, and nucleic acid metabolism . Joint antioxidant roles of zinc and copper involve Cu–Zn superoxide dismutase (SOD) and catalase (CAT) which catalyze the reduction of hydrogen peroxide to water [9, 10]. Selenium is a cofactor of glutathione peroxidase (GSH-Px), an important antioxidant enzyme for removal of lipid hydroperoxides and hydrogen peroxide . Recently, a few studies investigated the possible role of zinc, copper, and selenium in the etiology and pathogenesis of RA [2, 3, 4, 5], but the results are conflicting so further studies are needed on the subject.
There are many drugs available for the treatment of RA. These include gold compounds, non-steroidal anti-inflammatory drugs (NSAIDs), penicillamine, sulphasalasine, chloroquine, glucocorticoids, and methotrexate as an immunosuppressant . NSAIDs block the activity of cyclooxgenases (COX), namely COX-1 and COX-2, which catalyze the production of leukotrienes as arachidonic acid metabolites. COX-2 is mainly present in inflammatory cells, while COX-1 is expressed in most tissues and constitutive enzymes .
The action mechanism of sulphasalazine is not clearly known, but there is evidence that it can scavenge toxic oxygen metabolites produced by neutrophils . Chloroquine, an anti-malarial drug, inhibits mitogen-induced lymphocyte proliferation and decreases leucocyte chemotaxis, lysosomal enzyme release, and generation of toxic oxygen metabolites. Glucocorticoids are inhibitory agents for phospholipase A2, an enzyme that catalyzes the production of prostaglandins from arachidonic acid metabolites and has a large-scale effect on metabolism and immune system . Additionally, glucocorticoids also inhibit COX-2 enzyme. Methotrexate, an immunosuppressant and anti-neoplastic agent, is a folate antagonist.
All these drugs are used alone or in combination according to well-established clinical treatment protocols . It has been reported that most of these treatments changed serum trace element levels or CAT, SOD, and GSH-Px enzyme activities in different inflammatory diseases [6, 7, 14]. They may also have effects on serum copper, selenium, and zinc levels in patients with RA [13, 15], but there is no sufficient information on the effects of methotreaxate, salasopyrin, corticosteroids, and NSAIDs in patients with RA. In consequence, the present study was carried out to investigate levels of copper, selenium, and zinc in patients with RA in relation to established treatments.
Material and Methods
Patients and Controls
The study was approved by the Ethics Committee, Medical Faculty, Fırat University, Elazığ, Turkey. All participants gave written consent, confirming their acceptance for giving blood through vena brachialis and were informed about the whole experimental procedure. The patients were diagnosed and classified by clinicians of the Internal Medicine Clinic, and the control group was selected among healthy parents or siblings.
The study was performed in 32 RA patients (21 males and 11 females) aged 29 to 77 years; mean age, 50 years (48 years for men and 52 years for women). None of them had an alcohol abuse problem. The patients had not received any systemic therapy which might affect cellular immunity during the 3 weeks prior to sample collection. The control group consisted of 52 healthy volunteers precisely matched for age and sex. The women who were included in the study had not been taking oral contraceptives for at least 6 months before sample collection. All patients had active disease according to the criteria of the American Rheumatism Association,  and the median duration of illness was 7.36 years (range 1–15 years).
Serum copper, selenium, and zinc levels (Mean ± SD) in patients with rheumatoid arthritis before (baseline) and after the 30-day treatment
0.44 ± 0.20 (n = 21)
0.41 ± 0.22 (n = 11)
1.67 ± 0.29 (n = 21)
1.63 ± 0.34 (n = 11)
140.5 ± 41.9 (n = 21)
139.1 ± 31.1 (n = 11)
0.43 ± 0.21 (n = 20)
0.44 ± 0.22 (n = 12)
1.67 ± 0.31 (n = 20)
1.65 ± 0.30 (n = 12)
145.5 ± 40.7 (n = 20)
130.8 ± 32.6 (n = 12)
0.39 ± 0.21 (n = 22)
0.52 ± 0.18a (n = 10)
1.59 ± 0.32 (n = 22)
1.81 ± 0.19 (n = 10)
131.4 ± 36.7 (n = 22)
159.0 ± 35.4 (n = 10)
0.41 ± 0.23 (n = 29)
0.43 ± 0.21 (n = 3)
1.57 ± 0.45 (n = 29)
1.67 ± 0.29 (n = 3)
150.0 ± 10.0 (n = 29)
139.0 ± 39.7 (n = 3)
0.42 ± 0.22 (n = 28)
0.50 ± 0.06 (n = 4)
1.65 ± 0.31 (n = 28)
1.71 ± 0.28 (n = 4)
140.0 ± 36.4 (n = 28)
135.0 ± 54.5 (n = 4)
Preparation of Blood Samples
Fasting blood samples were collected by standard clinical procedures. EDTA was used to prevent coagulation. Whole blood (1 ml) was used for hematological analysis. Serum (2 ml) was used for the detection of C-reactive protein, rheumatoid factor, and anti-streptolysin O. The plasma used for biochemical analysis was separated by centrifugation for 10 min at 1,000×g at +4°C. The plasma and serum samples were stored at −30°C. Serum was stored for <3 months pending measurement of the trace element levels. Hematological and serum inflammatory markers were measured within 6 h of blood collection.
For each blood sample, two blank samples of highly purified water (element content <0.01) were collected using the same tools/equipment (e.g., gloves, syringes, etc.) and served to determine background noise (lower detection limit).
The trace element levels were determined by means of an Atomic Absorption Spectrophotometer with a graphite furnace GTA-96 (SpectrAA 250 Plus Zeeman, Varian, Australia) with deuterium background correction. Varian hollow cathode lamps were employed at the 196-nm wavelength with 1.0-nm bandwidth. Pyrolytically coated graphite tubes with pyrolytic graphite platforms (Varian, Australia) were used.
All reagents were of analytical reagent grade. Doubly distilled deionized (DI) water was used throughout. All glassware used was washed with 10% nitric acid for 1 day and rinsed with DI water before use. Stock solutions of copper selenium and zinc were prepared by taking appropriate amounts of standards in nitric acid solution. Working solutions were prepared immediately before use. Adjustment of pH was made with a buffer containing acetic acid, boric acids, and their potassium salts.
Copper and Zinc Analysis
The serum samples were slowly digested with 3 ml nitric acid for 12 h. Alternatively, they were digested in a microwave oven for 5–10 min. After cooling to room temperature for 1 h, 0.5-ml hydrogen peroxide was added to complete the destruction of organic matter. Aliquots were then analyzed by GFAAS.
Serum selenium was determined by an internal standard addition method, as previously described . For the analysis of selenium, sera were diluted four times in nitric acid (v/v, 0.125%) with the addition of Triton-x100 (v/v, 0.05%). All the samples were run in duplicate, and 10 μl of the sample in 2 ml of palladium chloride and ascorbic acid (w/v, 2%) solution were assessed in the system. The lowest threshold Se detection of instrument was 10 μg/dl. The accuracy and precision of the method were regularly checked with commercial standards (Seronorm serum, Nycomed AS, Oslo, Norway).
All results are expressed as means ± SD. Significant values were established with Student’s t test. The SPSS statistical program (version 9.05 software, SPSS Inc. Chicago, Illinois, USA) was used for statistical treatment of the data. P values of less than 0.05 were regarded as significant.
If the groups are compared according to treatment, patients treated with methotrexate had higher levels of serum zinc than untreated controls (p < 0.05, Table 1). The copper, selenium, and zinc levels did not change by 30-day administration of salasopyrin, corticosteroids, non-steroid anti-inflammatory, and chloroquine.
In patients with rheumatoid arthritis, the serum selenium and zinc levels were lower, and copper level was higher relative to controls. The zinc levels were higher in the patients treated with methotrexate.
Research efforts have shifted from experiments to describe the changes in mineral metabolism associated with immune response to investigations of the mediators responsible for these changes . The observations that host products are released from stimulated leukocytes could induce metabolic changes similar to an acute-phase response revealed an endocrine role for the immune system. Characteristic changes in trace mineral metabolism are an integral part of the acute-phase response. The changes are usually reflected in decreased serum zinc and increased serum copper concentrations, although in animal studies, there is some species specificity .
In the current study, we observed a decreased level of serum zinc in patients with RA. The role of certain inflammatory products in the regulation of the zinc balance has been well documented . Some interleukins released from leukocytes or activated phagocytes may lead to zinc deficiency by inhibiting its transport from plasma to the liver . Lower serum zinc levels apparently result from the synthesis of methallothionein in liver and other tissues. Methallothionein binds 7 g of atoms of zinc per mol and serves to draw zinc away from free circulating pools .
Increased copper may be due to inflammation associated with the disease. Inflammation is very important in RA . Zoli et al. observed erythrocyte sedimentation rate and acute-phase proteins correlated negatively with serum zinc levels of patients with RA and positively with serum copper levels. IL-α and TNF-α were also found to correlate negatively with zinc and positively with copper in patients with RA . They decided that lower levels of zinc might be due to accumulation of zinc-containing proteins in liver and in inflamed joints in patients with RA. The elevated serum copper level in the current study seems to be linked to increased synthesis of ceruloplasmin by the liver.
Reports on element levels in patients are conflicting. For example, Yazar et al.  reported that plasma copper was higher and synovial selenium was lower in patients with RA compared to controls. They also observed no significant changes on plasma and synovial zinc levels in the patients. Helgeland et al.  reported that serum zinc levels were lower in 14 patients with juvenile arthritis than in 22 healthy controls. Recently, Ala et al.  reported that plasma copper levels of patients with RA did not change in comparison with healthy controls although zinc levels were decreased. Amancio et al.  reported that serum copper, but not zinc, was decreased in juvenile RA. Tuncer et al.  reported a rise in erythrocyte zinc and plasma copper levels and a decrease in erythrocyte copper and plasma zinc levels in patients with RA relative to controls. The results reported in this study for zinc and copper are in agreement with the results of Yazar et al.  and Helgeland et al. .
Rheumatoid arthritis is an inflammatory disease , but little is known about the factors that may influence inflammation . It is known that moderate and severe inflammatory reactions increase oxidative stress and lead to a decrease in GSH-Px activities in the serum and erythrocytes of patients with RA [3, 7, 14].
It is also known that selenium is a cofactor of the antioxidant enzyme GSH-Px . In the present study, the serum selenium level in patients with RA was lower than in the healthy controls. Therefore, the inflammatory character of RA can explain the observed low selenium levels in the serum of patients with the disease. In a similar study, Pemberton et al.  recently reported low levels of blood selenium in 46 RA patients. Köse et al.  reported that plasma Se levels in 60 patients were lower than those of 60 healthy controls. Helgeland et al.  hypothesized that consumption of selenium was inversely associated with the risk of developing juvenile arthritis in patients.
Zinc is required for normal growth and development and tissue repair processes. It induces metallothionein synthesis, which has been shown to sequester free radicals . Methotrexate treatment increased Zn–SOD activity although it had no effect on GSH-Px activity in rats [24, 25]. In the present study, methotrexate increased serum zinc levels in the patients with RA enhancing the activity of Zn–SOD [24, 25]. No changes in serum copper and selenium levels were observed. These results are consistent with those of Al-Saleh et al.  who reported that serum copper and selenium levels did not change in pregnant rats treated with methotrexate. Armagan et al.  reported that methotrexate had no effect on GSH-Px activity in rat testis.
In conclusion, the serum levels of copper, selenium, and zinc were altered probably by some immunocytokines as a defense response against rheumatoid arthritis. The results of this study confirm that rheumatoid arthritis patients have reduced serum zinc and selenium levels. Patients may benefit from supplementation with these essential trace elements in cases where dietary intake does not reach the recommended dietary allowances.
M. N. formulated the present hypothesis and was responsible for writing the report. S.O. and M. Ç. were responsible for data analyses. V.B. and M.F.F-A. made critical revisions to the manuscript.
- 9.Kovacic P, Somanathan R (2008) Unifying mechanism for eye toxicity: electron transfer, reactive oxygen species, antioxidant benefits, cell signaling and cell membranes. Cell Membr Free Radic Res 2:56–69Google Scholar
- 22.Pemberton PW, Ahmad Y, Bodill H, Lokko D, Hider SL, Yates AP, Walker MG, Laing I, Bruce IN (2009) Biomarkers of oxidant stress, insulin sensitivity and endothelial activation in rheumatoid arthritis: a cross-sectional study of their association with accelerated atherosclerosis. BMC Res Notes 2:83PubMedCrossRefGoogle Scholar