http://informahealthcare.com/mor ISSN 1439-7595 (print), 1439-7609 (online) Mod Rheumatol, 2014; Early Online: 1–5 © 2014 Japan College of Rheumatology DOI: 10.3109/14397595.2014.891495
ORIGINAL ARTICLE
Identification of clinical parameters associated with serum oxidative stress in patients with rheumatoid arthritis Arata Nakajima1,2, Yasuchika Aoki1, Yoshifumi Shibata1, Masato Sonobe1, Fumiaki Terajima1, Hiroshi Takahashi1, Masahiko Saito1, Shinji Taniguchi1, Manabu Yamada1, and Koichi Nakagawa1 Mod Rheumatol Downloaded from informahealthcare.com by University of Maastricht on 06/16/14 For personal use only.
1Department of Orthopedics, Toho University Sakura Medical Center, Sakura, Chiba, Japan and 2Department of Rheumatology,
Toho University Sakura Medical Center, Sakura, Chiba, Japan Abstract
Keywords
Objectives. Reactive oxygen species (ROS) are considered to be involved in the pathobiology of rheumatoid arthritis (RA); however, their association with disease activity has not been elucidated. In this study, we measured reactive oxygen metabolites (ROM) in patients with RA using a new Free Radical Analytical System and determined clinical parameters associated with ROM. Methods. One hundred and fifty-two patients with RA and 80 patients with diabetes mellitus (DM) were included in this observational study. To measure ROM, the d-ROM test was performed on blood samples drawn from all subjects. The correlation between ROM and biomarkers, disease activity, doses of methotrexate (MTX), and prednisolone (PSL) were investigated. Results. There were significant, positive correlations between ROM and CRP, matrix metalloproteinase 3 (MMP3), Disease Activity Score 28–erythrocyte sedimentation rate (DAS28-ESR), Clinical Disease Activity Index (CDAI), and the Simplified Disease Activity Index (SDAI). Multiple regression analysis revealed that CRP and DAS28-ESR were correlated with ROM. Conclusions. The serum level of ROM was associated with CRP and DAS28-ESR, suggesting that ROM, in conjunction with CRP and MMP3, may be able to be used as a new biological disease marker to evaluate the disease activity of RA.
Introduction Rheumatoid arthritis (RA) is a chronic inflammatory disease of unknown etiology and affects synovial joints, leading to the development of cartilage and subchondral bone erosion [1]. Regarding the pathobiology of RA, oxidative stress induced by reactive oxygen species (ROS) is thought to be an important mechanism that underlies joint destruction and synovial proliferation [2–5]. ROS are produced during oxidative phosphorylation. When the production of ROS exceeds the physiological border, it induces oxidative stress and damages proteins, lipids, and nucleic acids. Abundant amounts of ROS have been detected in the synovial fluid of inflamed rheumatoid joints [6], where they can act as intracellular signaling molecules in the cells of the immune system that amplify the synovial inflammatory, proliferative response [3]. T-cells are exposed to increased oxidative stress and become refractory to growth and death stimuli, which further contribute to the perpetuation of the immune response [7]. Thus, ROS are considered to be involved in the pathobiology of RA; however, their association with RA disease activity has not been elucidated. Recently, a method of measuring reactive oxygen metabolites (ROM) in blood has been developed. This method is the d-ROM test, which uses a Free Radical Analytical System (FRAS, Wismarl, Italy) [8,9]. Hydroperoxides are the main components of ROM. Correspondence to: Arata Nakajima, MD, PhD, Department of Orthopedics and Rheumatology, Toho University Sakura Medical Center, 564-1 Shimoshizu, Sakura, Chiba 285-8741, Japan. Tel: ⫹ 81-43-462-8811. Fax: ⫹ 81-43-462-8820. E-mail: [email protected]
Disease activity, Oxidative stress, Reactive oxygen metabolites, Rheumatoid arthritis History Received 7 October 2013 Accepted 4 February 2014
Despite their fair oxidant power, hydroperoxides in the blood are relatively stable compared with their parent free radicals; therefore, the d-ROM test can generate more reliable data compared with direct measurements of ROS. The d-ROM test has already been recognized as useful for the evaluation of oxidative stress in human acute and chronic diseases such as obesity and metabolic syndrome [10], renal insufficiency [11], pulmonary sarcoidosis [12], asthma [13], hypercholesterolemia [14], and influenza virusassociated encephalopathy [15]. In addition, using this method, it has been demonstrated that serum level of oxidative stress marker is dramatically low in patients with RA treated with tocilizumab, an interleukin-6-blocking agent [16]. In this study, we measured the serum level of ROM in patients with RA by means of a FRAS4, and determined clinical parameters associated with ROM.
Patients and methods Patients In this observational study, 152 patients with RA were evaluated for ROM and compared with 80 patients with diabetes mellitus (DM). We chose individuals with DM as comparative subjects because DM is a non-inflammatory disease known to be associated with oxidative stress [17,18]. Approval for the study was received from the Institutional Review Board at Toho University Sakura Medical Center and all activities were performed in accordance with the ethical standards set forth in the Declaration of Helsinki. Blood samples were collected from all patients with RA or DM who visited Toho University Sakura Medical Center from March
2
A. Nakajima et al.
2011 to October 2012 and provided consent. Venous blood samples were collected in tubes and analyzed for serum C-reactive protein (CRP) and matrix metalloproteinase 3 (MMP3). In our hospital, the normal reference value for CRP is 0.3 mg/dL. ROS were also measured as described below. To evaluate the disease activity of RA, measurements were obtained for the Disease Activity Score (DAS) based on the examination of 28 joints (DAS28), the Simplified Disease Activity Index (SDAI), and the Clinical Disease Activity Index (CDAI) during the same visit the blood samples were collected. The patients with RA also completed the Health Assessment Questionnaire (HAQ).
Mod Rheumatol Downloaded from informahealthcare.com by University of Maastricht on 06/16/14 For personal use only.
Measurement of oxidative stress markers in serum As previously mentioned, in standard laboratories direct measurement of ROS and free radicals is difficult because of their biochemical instability. Recently, a method for measuring ROM in blood has been developed. To measure ROM, the d-ROM test was performed using the FRAS4 analyzer (Wismarl, Italy) in accordance with the manufacture’s analytical procedures. In brief, a 20 μL blood sample and 1 mL of buffered solution (R2 reagent of kit, pH 4.8) were gently mixed in a cuvette before adding 10 μL of chromogenic substrate (R1 reagent of kit). After mixing, the cuvette was centrifuged for 60 s at 37°C and immediately incubated in the thermostatic block of this analyzer for 5 min at 37°C. The absorbance at 505 nm was recorded. The measurement unit was expressed as U.CARR (Carratelli units). It has been established that 1 U.CARR corresponds to 0.08 mg/dl hydrogen peroxide (H2O2). Reference values indicated by the manufacturer (Wismarl) range up to 300 U.CARR; values higher than 300 U.CARR suggest the presence of oxidative stress [8,9]. Statistical analysis Results are expressed as the mean ⫾ standard deviation (SD). Between-group differences were assessed by Mann–Whitney U-tests and P values of ⬍ 0.05 were considered to indicate statistical significance. All statistical analysis was performed using SPSS (ver. 19) software (SPSS, IL, USA).
Results
Mod Rheumatol, 2014; Early Online: 1–5
Table 1. Demographic and disease characteristics of patients with RA and DM. RA (n ⫽ 152) Age, years (range) Sex (male/female) Body weight (kg) BMI Disease duration, years (range) Stage (I, II, III, IV) RF (IU/mL) CRP (mg/dL) MMP3 (ng/mL) eGFR HbA1c (%) DAS28-ESR SDAI CDAI HAQ MTX, mg/week (% usage) PSL, mg/day (% usage)
63.1 ⫾ 13.1 (24–85) 49/103 54.1 ⫾ 10.3 22.5 ⫾ 3.34 14.3 ⫾ 12.9 (1–44) 22, 42, 35, 53 184.9 ⫾ 320.1 1.14 ⫾ 1.91 168.5 ⫾ 171.1 76.1 ⫾ 30.8 3.52 ⫾ 1.38 9.89 ⫾ 10.26 8.77 ⫾ 9.13 0.632 ⫾ 0.728 7.86 ⫾ 1.95 (79.0)
DM (n ⫽ 80) 59.2 ⫾ 13.4 (26–85)
p value 0.035
46/34 67.8 ⫾ 17.0 25.7 ⫾ 4.65
⬍ 0.01 ⬍ 0.01
0.053 ⫾ 0.053
⬍ 0.01
77.0 ⫾ 22.4 7.77 ⫾ 2.18
0.269
4.13 ⫾ 1.91 (53.9)
Values are expressed as the mean ⫾ SD. Between-group differences were assessed by Mann–Whitney U-tests. RA, rheumatoid arthritis; DM, diabetes mellitus; RF, rheumatoid factor; CRP, C-reactive protein; MMP3, matrix metalloproteinase 3; eGFR, estimated glomerular filtration rate; HbA1c, hemoglobin A1c; DAS28-ESR, disease activity score 28-erythrocyte sedimentation rate; SDAI, simplified disease activity index; CDAI, clinical disease activity index; HAQ, health assessment questionnaire; MTX, methotrexate; PSL, prednisolone.
biomarkers, disease activity, and doses of MTX, and PSL. There were significant, positive correlations between ROM and CRP (r ⫽ 0.676), MMP3 (r ⫽ 0.435), DAS28-ESR (r ⫽ 0.553), CDAI (r ⫽ 0.412), and SDAI (r ⫽ 0.473) (Table 2, Figure 2). To further identify the clinical factors affecting ROM levels, multiple regression analysis was performed. As a result, CRP and DAS28-ESR were determined to be parameters strongly affecting ROM (Table 3). The regression coefficients for CRP and DAS28ESR were 0.430 and 0.576, respectively, with the Durbin–Watson
Background characteristics Background characteristics of patients included in this study are shown in Table 1. Of the 152 patients with RA, 21 were classified as having high disease activity, 60 had moderate disease activity, 27 displayed low disease activity, and 44 patients were in remission. Seventy-nine percent of patients were treated with methotrexate (7.86 ⫾ 1.95 mg/week) and 53.9% with prednisolone (PSL; 4.13 ⫾ 1.91 mg/day). Forty-six patients (30.3%) received biological agents (infliximab, etanercept, adalimumab, tocilizumab, or abatacept). Serum levels of oxidative stress in patients with RA and DM The level of ROM in the RA group was 455.9 ⫾ 115.9 U. CARR compared with 390.7 ⫾ 72.6 U.CARR in the DM group. Although the level of ROM in the DM group exceeded the normal limit (⬍ 300 U.CARR), the serum level of oxidative stress was significantly increased in the RA group compared with the DM group (P ⬍ 0.01) (Figure 1). Identification of clinical factors associated with ROM in patients with RA To determine the clinical factors associated with ROM in patients with RA, we investigated the correlation between ROM and
Figure 1. Serum levels of ROM in patients with RA and DM. Mean serum levels of ROM in the RA and DM group was 455.9 and 390.7 U.CARR, respectively. The level of ROM was significantly increased in patients with RA (p ⬍ 0.001). Values are expressed as the mean ⫾ SD. Between-group differences were assessed by Mann–Whitney U-tests.
Serum oxidative stress in RA 3
DOI 10.3109/14397595.2014.891495
Table 2. Inter-correlation of clinical parameters in patients with RA. Age
ROM Age – 0.144 Disease duration RF 0.151 0.164 CRP 0.016 0.676∗∗ MMP3 0.053 0.435∗∗ eGFR ⫺ 0.047 ⫺ 0.155 DAS28 0.035 0.553∗∗ -ESR SDAI 0.473∗∗ ⫺ 0.122 CDAI 0.412∗∗ ⫺ 0.128 HAQ 0.165 0.207∗ MTX ⫺ 0.043 ⫺ 0.092 dosage PSL 0.066 0.154 dosage
Disease duration
RF
CRP
MMP3
eGFR
DAS28 -ESR
SDAI
CDAI
HAQ
MTX dosage
– ⫺ 0.071 ⫺ 0.012 ⫺ 0.204 0.126 ⫺ 0.243
– 0.07 – 0.049 – 0.547∗∗ – ⫺ 0.051 ⫺ 0.082 ⫺ 0.261 0.1 0.578∗∗ 0.431∗∗ ⫺ 0.2
0.646∗∗ 0.386∗∗ ⫺ 0.13 ⫺ 0.149 ⫺ 0.013 0.525∗∗ 0.329∗∗ ⫺ 0.132 ⫺ 0.164 ⫺ 0.023 0.183 0.086 0.251∗ 0.313∗∗ 0.23∗ 0.107 ⫺ 0.187 ⫺ 0.011 ⫺ 0.081 ⫺ 0.037 ⫺ 0.021
0.148
0.263∗
0.159
– – 0.875∗∗ – 0.867∗∗ 0.988∗∗ 0.316∗∗ 0.364∗∗ 0.348∗∗ ⫺ 0.03 ⫺ 0.076 ⫺ 0.069
⫺ 0.176
0.148
0.101
– ⫺ 0.126
0.086
–
0.093 0.004
Single regression analysis among ROM, age, disease duration, RF, CRP, MMP3, eGFR, DAS28-ESR, SDAI, CDAI, HAQ, MTX dosage, and PSL dosage was performed and statistical significance of correlation was determined using Pearson’s correlation tests. See text for results of multiple regression analysis. RA, rheumatoid arthritis; ROM, reactive oxygen metabolities; RF, rheumatoid factor; CRP, C-reactive protein; MMP3, matrix metalloproteinase 3; eGFR, estimated glomerular filtration rate; DAS28-ESR, disease activity score 28-erythrocyte sedimentation rate; SDAI, simplified disease activity index; CDAI, clinical disease activity index; HAQ, health assessment questionnaire; MTX, methotrexate; PSL, prednisolone. ∗P ⬍ 0.01, ∗∗P ⬍ 0.001.
ratio of 1.908. The multiple regression formula determining ROM is as follows:
studies. In this study, ROM was used as an oxidative stress parameter because they are relatively stable in the blood. Considering our improved method for measuring oxidative stress and the larger number of patients included in the present study, it is conceivable that oxidative stress is associated with disease activity in patients with RA. Oxidative stress induced by ROS is not only an important mechanism that underlies joint destruction and synovial proliferation [2–5] but also a very important mediator of extra-articular diseases in individuals with RA. Patients with RA are known to have a higher risk of cardiovascular events not explained entirely by traditional cardiovascular risk factors [21], implying that cardiovascular disease is an extra-articular manifestation of RA [22]. In fact, RA has been shown to be associated with increased
ROM⫽290.58(constant)⫹26.09⫻CRP⫹48.44⫻DAS28-ESR.
Discussion This paper is the first report showing a relationship between the serum level of ROM and disease activity and biomarkers associated with RA. Previous reports have demonstrated that total ROS, superoxide and hydroxyl radicals, and malondialdehyde (MDA) levels (an oxidative stress parameter) are correlated with DAS28 [19,20]; however, direct measurements of ROS or MDA seem to lack reliability due to their biochemical instability. Furthermore, a small number of patients (n ⫽ 30) were investigated in these
(A) 1000
(B) 1000
900
900 y = 46.488x + 292
y = 52.274x + 400.3 800
800
R2 = 0.458
R2 = 0.306
700 ROM (U.CARR)
700 ROM (U.CARR)
Mod Rheumatol Downloaded from informahealthcare.com by University of Maastricht on 06/16/14 For personal use only.
ROM – 0.121 ⫺ 0.113
600 500 400
600 500 400
300
300
200
200
100
100 0
0 0
1
2
3
4
CRP (mg/dL)
5
6
7
0
1
2
3
4
5
6
7
DAS28-ESR
Figure 2. Correlation between ROM and CRP (A), and ROM and DAS28-ESR (B). Significant, positive correlations were detected between ROM and CRP (R2 ⫽ 0.458), and ROM and DAS28-ESR (R2 ⫽ 0.306).
4
A. Nakajima et al.
Mod Rheumatol, 2014; Early Online: 1–5
Table 3. Multiple regression analysis to identify the clinical factors affecting ROM.
Constant CRP DAS28-ESR
Partial regression coefficient 290.58 26.09 48.44
Standardized regression coefficient 0.430 0.576
95% confidence interval 236.93–344.24 17.09–35.09 27.16–69.72
P value ⬍ 0.01 ⬍ 0.01 ⬍ 0.01
CRP and DAS28-ESR were finally determined to be parameters strongly affecting ROM. The multiple regression formula determining ROM is as follows: ROM⫽290.58(constant)⫹26.09⫻CRP⫹48.44⫻DAS28-ESR. ROM, reactive oxygen metabolities; CRP, C-reactive protein; MMP3, DAS28-ESR, disease activity score 28-erythrocyte sedimentation rate.
biology of RA is still unclear, further studies are required to establish the clinical significance of oxidative stress in RA. In summary, when ROM was used as a parameter for oxidative stress in this study, it was shown to be significantly increased in patients with RA. The serum level of ROM was associated with CRP and DAS28-ESR, but not with renal function or daily doses of PSL. Although ROM in RA patients could also be influenced by cardiovascular abnormalities, these results suggest that ROM, in conjunction with CRP and MMP3, may be able to be used as a new biological disease marker to evaluate the disease activity of RA. In the future, longitudinal studies designed to explore the association of therapeutic effects with decreases in ROM may further elucidate the clinical significance of oxidative stress in RA.
Mod Rheumatol Downloaded from informahealthcare.com by University of Maastricht on 06/16/14 For personal use only.
Acknowledgements cardiovascular morbidity and mortality [23]. In the pathobiology of cardiovascular events, arterial atherosclerosis exists and increased arterial stiffness, increased carotid intima media thickness, and endothelial dysfunction have been demonstrated in patients at increased risk of and with known cardiovascular disease [24–26], and these pathological changes in the cardiovascular system have also been reported in patients with RA [27]. While the levels of ROM in patients with DM in this study were increased from the normal value, the increase was significantly higher in patients with RA compared with those with DM. This suggests that ROM in RA patients may be influenced not only by cardiovascular abnormalities, but also by the disease activity that occurs as a result of inflammatory arthritis. Recently, there have been several papers reporting the relationship between oxidative stress and the disease activity of RA. Hassan et al. showed that markers of increased oxidative stress and impaired antioxidant capacity were profound and significantly reflected disease activity in RA [19]. Furthermore, Kundu et al. demonstrated that ROS generated in both peripheral blood and synovial infiltrates correlated positively with both DAS28 and CRP/anti-cyclic citrullinated peptide (CCP) levels [20]. Hayashi et al., by means of univariate analysis, showed that serum ROM levels were associated with disease activity in patients with RA [28]. In this study, by means of multiple regression analysis, we showed that the level of ROM was correlated with CRP and DAS28-ESR. This implies that ROM may be a novel and useful marker for evaluating the disease activity associated with RA. In the practical treatment of RA, serum levels of CRP and MMP3 are one of the items often measured to evaluate the severity of systemic inflammation and joint destruction. However, CRP reflects systemic, non-specific inflammation, and easily drops to negative values by administration of certain biologic agents, especially tocilizumab, the IL-6-blocking agent [29]. Also, MMP3 is influenced by doses of PSL and renal function [30]. Indeed, correlation analyses among laboratory parameters showed that MMP3 but not ROM was associated with the daily dose of PSL. Taking these findings into consideration, ROM, in conjunction with CRP and MMP3, may be able to be used as a biomarker to evaluate the disease activity of RA. Our concern was that the ratio of male and female in RA and DM was quite different as shown in Table 1, which could affect differences in serum ROM levels between RA and DM patients. Actually, the mean ROM of male and female was 416.0 and 445.2, respectively, and there was a statistically significant difference (p ⬍ 0.01) (data not shown). To verify the influence of gender difference on ROM, we performed multivariate analysis (multiple logistic regression analysis) and confirmed that gender difference was not a significant factor affecting ROM (p ⫽ 0.08) (data not shown). Limitations of this study included the variable timing of blood draws among patients and the measurement of ROM at a single time-point. Also, because the role of oxidative stress in the patho-
The authors are grateful to Dr Atsuhito Saiki (Department of Internal Medicine, Toho University Sakura Medical Center) for collecting blood samples from the DM patients.
Conflict of interest None.
References 1. Pap T, Muller-Ladner U, Gay RE, Gay S. Fibroblast biology: role of synovial fibroblasts in the pathogenesis of rheumatoid arthritis. Arthritis Res. 2009;2(5):361–7. 2. Mapp PI, Grootveld MC, Blake DR. Hypoxia, oxidative stress, and rheumatoid arthritis. Br Med Bull. 1995;51(2):419–36. 3. Hitchon CA, El-Gabalawy H. Oxidation in rheumatoid arthritis. Arthritis Res Ther. 2004;6:265–78. 4. Halliwell B. Oxygen radicals, nitric oxide, and human inflammatory joint disease. Ann Rheum Dis. 1995;54(6):505–10. 5. Tak PP, Zvaifler NJ, Grenn DR, Firestein GS. Rheumatoid arthritis and p53: how oxidative stress might alter the course of inflammatory diseases. Immunol Today. 2000;21(2):78–82. 6. Biemond P, Swaak AJ, Koster JF. Protective factors against oxygen free radicals and hydrogen peroxide in rheumatoid arthritis synovial fluid. Arthritis Rheum. 1984;27(7):760–5. 7. Griffiths HR. ROS as signaling molecules in T cells – evidence for abnormal redox signaling in the autoimmune disease, rheumatoid arthritis. Redox Rep. 2005;10(6):273–80. 8. Carratelli M, Porcaro L, Ruscica M, De Simone E, Bertelli AA, Corsi MM. Reactive oxygen metabolites and prooxidant status in children with Down’s syndrome. Int J Clin Pharm Res. 2001;21(2):79–84. 9. Trotti R, Carratelli M, Barbieri M, Micieli G, Bosone D, Rondanelli M, Bo P. Oxidative stress and a thrombophilic condition in alcoholics without severe liver disease. Haematologica. 2001;86(1):85–91. 10. Faienza MF, Francavilla R, Goffredo R, Ventura A, Marzano F, Panzarino G, et al. Oxidative stress in obesity and metabolic syndrome in children and adolescents. Horm Res Paediatr. 2012;78(3): 158–64. 11. Coaccioli S, Standoli ML, Biondi R, Panaccione A, Landucci P, Del Giorno R, et al. Assessment of the oxidative stress markers in patients with chronic renal insufficiency undergoing dialysis treatment. Clin Ter. 2010;161(5):441–4. 12. Koutsokera A, Papaioannou AI, Malli F, Kiropoulos TS, Katsabeki A, Kerenidi T, et al. Systemic oxidative stress in patients with pulmonary sarcoidosis. Pulm Pharmacol Ther. 2009;22(6):603–7. 13. Suzuki S, Matsukura S, Takeuchi H, Kawaguchi M, Ieki K, Odaka M, et al. Increase in reactive oxygen metabolite level in acute exacerbations of asthma. Int Arch Allergy Immunol. 2008;146 Suppl 1:67–72. 14. Kotani K, Koibuchi H, Miyamoto M, Yamada T, Taniguchi N. Relationship between reactive oxygen metabolites and carotid intimamedia thickness in subjects with hypercholesterolemia. Med Princ Pract. 2010;19(6):496–8. 15. Yamanaka G, Kawashima H, Suganami Y, Watanabe C, Watanabe Y, Miyajima T, et al. Diagnostic and predictive value of CSF d-ROM level in influenza virus-associated encephalopathy. J Neurol Sci. 2006;243(1–2):71–5. 16. Hirao M, Yamasaki N, Oze H, Ebina K, Nampei A, Kawato Y, et al. Serum level of oxidative stress marker is dramatically low in patients
Serum oxidative stress in RA 5
DOI 10.3109/14397595.2014.891495
17. 18. 19.
20.
21.
Mod Rheumatol Downloaded from informahealthcare.com by University of Maastricht on 06/16/14 For personal use only.
22. 23.
24.
with rheumatoid arthritis treated with tocilizumab. Rheumatol Int. 2011;32(12):4041–5. Yang H, Jin X, Kei Lam CW, Yan SK. Oxidative stress and diabetes mellitus. Clin Chem Lab Med. 2011;49:1773–82. Amaral S, Oliveria PJ, Ramalho-Santos J. Diabetes and impairment of reproductive function: possible role of mitochondria and reactive oxygen species. Curr Diabetes Rev. 2008;4(1):46–54. Hassan SZ, Gheita TA, Kenawy SA, Fahim AT, El-Sorougy IM, Abdou MS. Oxidative stress in systemic lupus erythematosus and rheumatoid arthritis patients: relationship to disease manifestations and activity. Int J Rheum Dis. 2011;14(4):325–31. Kundu S, Ghosh P, Datta S, Ghosh A, Chattopadhyay S, Chatterjee M. Oxidative stress as a potential biomarker for determining disease activity in patients with rheumatoid arthritis. Free Radic Res. 2012;46(12):1482–9. Del Rincon I, Williams K, Stern MP, Freeman GL, Escalante A. High incidence of cardiovascular events in a rheumatoid arthritis cohort not explained by traditional cardiac risk factors. Arthritis Rheum. 2001;44(12):2737–45. Van Doornum S, McColl G, Wicks IP. Accelerated atherosclerosis: an extraarticular feature of rheumatoid arthritis? Arthritis Rheum. 2002;46(4):862–73. Jacobsson LT, Turesson C, Hanson RL, Pillemer S, Sievers ML, Pettitt DJ, et al. Joint swelling as a predictor of death from cardiovascular disease in a population study of Pima Indians. Arthritis Rheum. 2001;44(5):1170–6. Kobayashi K, Akishita M, Yu W, Hashimoto M, Ohni M, Toba K. Interrelationship between non-invasive measurements of
25.
26.
27.
28.
29.
30.
atherosclerosis: flow-mediated dilation of brachial artery, carotid, intima-media thickness, and pulse wave velocity. Atherosclerosis. 2004;173(1):13–8. O’Leary DH, Polak JF, Kronmal RA, Manolio TA, Burke GL, Wolfson SK Jr. Cardiovascular Health Study Collaborative Research Group. Carotid-artery intima and media thickness as a risk factor for myocardial infarction and stroke in older adults. N Engl J Med. 1999;340(1):14–22. Boutouyrie P, Tropeano AI, Asmar R, Gautier I, Benetos A, Lacolley P, Laurent S. Aortic stiffness is an independent predictor of primary coronary events in hypertensive patients: a longitudinal study. Hypertension. 2002;39(1):10–5. Del Porto F, Lagana B, Lai S, Nofroni I, Tinti F, Vitale M, et al. Response to anti-tumour necrosis factor alpha blockade is associated with reduction of carotid intima-media thickness in patients with active rheumatoid arthritis. Rheumatology. 2007;46(7):1111–5. Hayashi H, Satoi K, Sato-Mito N, Kaburagi T, Yoshino H, Higaki M, et al. Nutritional status in relation to adipokines and oxidative stress is associated with disease activity in patients with rheumatoid arthritis. Nutrition. 2012;28(11–12):1109–14. Funahashi K, Koyano S, Miura T, Hagiwara T, Okuda K, Matsubara T. Efficacy of tocilizumab and evaluation of clinical remission as determined by CDAI and MMP-3 level. Mod Rheumatol. 2009;19(5):507–12. Ribbens C, Martin y Porras M, Franchimont N, Kaiser MJ, Jaspar JM, Damas P, et al. Increased metalloproteinase-3 serum levels in rheumatic diseases: relationship with synovitis and steroid treatment. Ann Rheum Dis. 2002;61(2):161–6.
ORIGINAL ARTICLE
Identification of clinical parameters associated with serum oxidative stress in patients with rheumatoid arthritis Arata Nakajima1,2, Yasuchika Aoki1, Yoshifumi Shibata1, Masato Sonobe1, Fumiaki Terajima1, Hiroshi Takahashi1, Masahiko Saito1, Shinji Taniguchi1, Manabu Yamada1, and Koichi Nakagawa1 Mod Rheumatol Downloaded from informahealthcare.com by University of Maastricht on 06/16/14 For personal use only.
1Department of Orthopedics, Toho University Sakura Medical Center, Sakura, Chiba, Japan and 2Department of Rheumatology,
Toho University Sakura Medical Center, Sakura, Chiba, Japan Abstract
Keywords
Objectives. Reactive oxygen species (ROS) are considered to be involved in the pathobiology of rheumatoid arthritis (RA); however, their association with disease activity has not been elucidated. In this study, we measured reactive oxygen metabolites (ROM) in patients with RA using a new Free Radical Analytical System and determined clinical parameters associated with ROM. Methods. One hundred and fifty-two patients with RA and 80 patients with diabetes mellitus (DM) were included in this observational study. To measure ROM, the d-ROM test was performed on blood samples drawn from all subjects. The correlation between ROM and biomarkers, disease activity, doses of methotrexate (MTX), and prednisolone (PSL) were investigated. Results. There were significant, positive correlations between ROM and CRP, matrix metalloproteinase 3 (MMP3), Disease Activity Score 28–erythrocyte sedimentation rate (DAS28-ESR), Clinical Disease Activity Index (CDAI), and the Simplified Disease Activity Index (SDAI). Multiple regression analysis revealed that CRP and DAS28-ESR were correlated with ROM. Conclusions. The serum level of ROM was associated with CRP and DAS28-ESR, suggesting that ROM, in conjunction with CRP and MMP3, may be able to be used as a new biological disease marker to evaluate the disease activity of RA.
Introduction Rheumatoid arthritis (RA) is a chronic inflammatory disease of unknown etiology and affects synovial joints, leading to the development of cartilage and subchondral bone erosion [1]. Regarding the pathobiology of RA, oxidative stress induced by reactive oxygen species (ROS) is thought to be an important mechanism that underlies joint destruction and synovial proliferation [2–5]. ROS are produced during oxidative phosphorylation. When the production of ROS exceeds the physiological border, it induces oxidative stress and damages proteins, lipids, and nucleic acids. Abundant amounts of ROS have been detected in the synovial fluid of inflamed rheumatoid joints [6], where they can act as intracellular signaling molecules in the cells of the immune system that amplify the synovial inflammatory, proliferative response [3]. T-cells are exposed to increased oxidative stress and become refractory to growth and death stimuli, which further contribute to the perpetuation of the immune response [7]. Thus, ROS are considered to be involved in the pathobiology of RA; however, their association with RA disease activity has not been elucidated. Recently, a method of measuring reactive oxygen metabolites (ROM) in blood has been developed. This method is the d-ROM test, which uses a Free Radical Analytical System (FRAS, Wismarl, Italy) [8,9]. Hydroperoxides are the main components of ROM. Correspondence to: Arata Nakajima, MD, PhD, Department of Orthopedics and Rheumatology, Toho University Sakura Medical Center, 564-1 Shimoshizu, Sakura, Chiba 285-8741, Japan. Tel: ⫹ 81-43-462-8811. Fax: ⫹ 81-43-462-8820. E-mail: [email protected]
Disease activity, Oxidative stress, Reactive oxygen metabolites, Rheumatoid arthritis History Received 7 October 2013 Accepted 4 February 2014
Despite their fair oxidant power, hydroperoxides in the blood are relatively stable compared with their parent free radicals; therefore, the d-ROM test can generate more reliable data compared with direct measurements of ROS. The d-ROM test has already been recognized as useful for the evaluation of oxidative stress in human acute and chronic diseases such as obesity and metabolic syndrome [10], renal insufficiency [11], pulmonary sarcoidosis [12], asthma [13], hypercholesterolemia [14], and influenza virusassociated encephalopathy [15]. In addition, using this method, it has been demonstrated that serum level of oxidative stress marker is dramatically low in patients with RA treated with tocilizumab, an interleukin-6-blocking agent [16]. In this study, we measured the serum level of ROM in patients with RA by means of a FRAS4, and determined clinical parameters associated with ROM.
Patients and methods Patients In this observational study, 152 patients with RA were evaluated for ROM and compared with 80 patients with diabetes mellitus (DM). We chose individuals with DM as comparative subjects because DM is a non-inflammatory disease known to be associated with oxidative stress [17,18]. Approval for the study was received from the Institutional Review Board at Toho University Sakura Medical Center and all activities were performed in accordance with the ethical standards set forth in the Declaration of Helsinki. Blood samples were collected from all patients with RA or DM who visited Toho University Sakura Medical Center from March
2
A. Nakajima et al.
2011 to October 2012 and provided consent. Venous blood samples were collected in tubes and analyzed for serum C-reactive protein (CRP) and matrix metalloproteinase 3 (MMP3). In our hospital, the normal reference value for CRP is 0.3 mg/dL. ROS were also measured as described below. To evaluate the disease activity of RA, measurements were obtained for the Disease Activity Score (DAS) based on the examination of 28 joints (DAS28), the Simplified Disease Activity Index (SDAI), and the Clinical Disease Activity Index (CDAI) during the same visit the blood samples were collected. The patients with RA also completed the Health Assessment Questionnaire (HAQ).
Mod Rheumatol Downloaded from informahealthcare.com by University of Maastricht on 06/16/14 For personal use only.
Measurement of oxidative stress markers in serum As previously mentioned, in standard laboratories direct measurement of ROS and free radicals is difficult because of their biochemical instability. Recently, a method for measuring ROM in blood has been developed. To measure ROM, the d-ROM test was performed using the FRAS4 analyzer (Wismarl, Italy) in accordance with the manufacture’s analytical procedures. In brief, a 20 μL blood sample and 1 mL of buffered solution (R2 reagent of kit, pH 4.8) were gently mixed in a cuvette before adding 10 μL of chromogenic substrate (R1 reagent of kit). After mixing, the cuvette was centrifuged for 60 s at 37°C and immediately incubated in the thermostatic block of this analyzer for 5 min at 37°C. The absorbance at 505 nm was recorded. The measurement unit was expressed as U.CARR (Carratelli units). It has been established that 1 U.CARR corresponds to 0.08 mg/dl hydrogen peroxide (H2O2). Reference values indicated by the manufacturer (Wismarl) range up to 300 U.CARR; values higher than 300 U.CARR suggest the presence of oxidative stress [8,9]. Statistical analysis Results are expressed as the mean ⫾ standard deviation (SD). Between-group differences were assessed by Mann–Whitney U-tests and P values of ⬍ 0.05 were considered to indicate statistical significance. All statistical analysis was performed using SPSS (ver. 19) software (SPSS, IL, USA).
Results
Mod Rheumatol, 2014; Early Online: 1–5
Table 1. Demographic and disease characteristics of patients with RA and DM. RA (n ⫽ 152) Age, years (range) Sex (male/female) Body weight (kg) BMI Disease duration, years (range) Stage (I, II, III, IV) RF (IU/mL) CRP (mg/dL) MMP3 (ng/mL) eGFR HbA1c (%) DAS28-ESR SDAI CDAI HAQ MTX, mg/week (% usage) PSL, mg/day (% usage)
63.1 ⫾ 13.1 (24–85) 49/103 54.1 ⫾ 10.3 22.5 ⫾ 3.34 14.3 ⫾ 12.9 (1–44) 22, 42, 35, 53 184.9 ⫾ 320.1 1.14 ⫾ 1.91 168.5 ⫾ 171.1 76.1 ⫾ 30.8 3.52 ⫾ 1.38 9.89 ⫾ 10.26 8.77 ⫾ 9.13 0.632 ⫾ 0.728 7.86 ⫾ 1.95 (79.0)
DM (n ⫽ 80) 59.2 ⫾ 13.4 (26–85)
p value 0.035
46/34 67.8 ⫾ 17.0 25.7 ⫾ 4.65
⬍ 0.01 ⬍ 0.01
0.053 ⫾ 0.053
⬍ 0.01
77.0 ⫾ 22.4 7.77 ⫾ 2.18
0.269
4.13 ⫾ 1.91 (53.9)
Values are expressed as the mean ⫾ SD. Between-group differences were assessed by Mann–Whitney U-tests. RA, rheumatoid arthritis; DM, diabetes mellitus; RF, rheumatoid factor; CRP, C-reactive protein; MMP3, matrix metalloproteinase 3; eGFR, estimated glomerular filtration rate; HbA1c, hemoglobin A1c; DAS28-ESR, disease activity score 28-erythrocyte sedimentation rate; SDAI, simplified disease activity index; CDAI, clinical disease activity index; HAQ, health assessment questionnaire; MTX, methotrexate; PSL, prednisolone.
biomarkers, disease activity, and doses of MTX, and PSL. There were significant, positive correlations between ROM and CRP (r ⫽ 0.676), MMP3 (r ⫽ 0.435), DAS28-ESR (r ⫽ 0.553), CDAI (r ⫽ 0.412), and SDAI (r ⫽ 0.473) (Table 2, Figure 2). To further identify the clinical factors affecting ROM levels, multiple regression analysis was performed. As a result, CRP and DAS28-ESR were determined to be parameters strongly affecting ROM (Table 3). The regression coefficients for CRP and DAS28ESR were 0.430 and 0.576, respectively, with the Durbin–Watson
Background characteristics Background characteristics of patients included in this study are shown in Table 1. Of the 152 patients with RA, 21 were classified as having high disease activity, 60 had moderate disease activity, 27 displayed low disease activity, and 44 patients were in remission. Seventy-nine percent of patients were treated with methotrexate (7.86 ⫾ 1.95 mg/week) and 53.9% with prednisolone (PSL; 4.13 ⫾ 1.91 mg/day). Forty-six patients (30.3%) received biological agents (infliximab, etanercept, adalimumab, tocilizumab, or abatacept). Serum levels of oxidative stress in patients with RA and DM The level of ROM in the RA group was 455.9 ⫾ 115.9 U. CARR compared with 390.7 ⫾ 72.6 U.CARR in the DM group. Although the level of ROM in the DM group exceeded the normal limit (⬍ 300 U.CARR), the serum level of oxidative stress was significantly increased in the RA group compared with the DM group (P ⬍ 0.01) (Figure 1). Identification of clinical factors associated with ROM in patients with RA To determine the clinical factors associated with ROM in patients with RA, we investigated the correlation between ROM and
Figure 1. Serum levels of ROM in patients with RA and DM. Mean serum levels of ROM in the RA and DM group was 455.9 and 390.7 U.CARR, respectively. The level of ROM was significantly increased in patients with RA (p ⬍ 0.001). Values are expressed as the mean ⫾ SD. Between-group differences were assessed by Mann–Whitney U-tests.
Serum oxidative stress in RA 3
DOI 10.3109/14397595.2014.891495
Table 2. Inter-correlation of clinical parameters in patients with RA. Age
ROM Age – 0.144 Disease duration RF 0.151 0.164 CRP 0.016 0.676∗∗ MMP3 0.053 0.435∗∗ eGFR ⫺ 0.047 ⫺ 0.155 DAS28 0.035 0.553∗∗ -ESR SDAI 0.473∗∗ ⫺ 0.122 CDAI 0.412∗∗ ⫺ 0.128 HAQ 0.165 0.207∗ MTX ⫺ 0.043 ⫺ 0.092 dosage PSL 0.066 0.154 dosage
Disease duration
RF
CRP
MMP3
eGFR
DAS28 -ESR
SDAI
CDAI
HAQ
MTX dosage
– ⫺ 0.071 ⫺ 0.012 ⫺ 0.204 0.126 ⫺ 0.243
– 0.07 – 0.049 – 0.547∗∗ – ⫺ 0.051 ⫺ 0.082 ⫺ 0.261 0.1 0.578∗∗ 0.431∗∗ ⫺ 0.2
0.646∗∗ 0.386∗∗ ⫺ 0.13 ⫺ 0.149 ⫺ 0.013 0.525∗∗ 0.329∗∗ ⫺ 0.132 ⫺ 0.164 ⫺ 0.023 0.183 0.086 0.251∗ 0.313∗∗ 0.23∗ 0.107 ⫺ 0.187 ⫺ 0.011 ⫺ 0.081 ⫺ 0.037 ⫺ 0.021
0.148
0.263∗
0.159
– – 0.875∗∗ – 0.867∗∗ 0.988∗∗ 0.316∗∗ 0.364∗∗ 0.348∗∗ ⫺ 0.03 ⫺ 0.076 ⫺ 0.069
⫺ 0.176
0.148
0.101
– ⫺ 0.126
0.086
–
0.093 0.004
Single regression analysis among ROM, age, disease duration, RF, CRP, MMP3, eGFR, DAS28-ESR, SDAI, CDAI, HAQ, MTX dosage, and PSL dosage was performed and statistical significance of correlation was determined using Pearson’s correlation tests. See text for results of multiple regression analysis. RA, rheumatoid arthritis; ROM, reactive oxygen metabolities; RF, rheumatoid factor; CRP, C-reactive protein; MMP3, matrix metalloproteinase 3; eGFR, estimated glomerular filtration rate; DAS28-ESR, disease activity score 28-erythrocyte sedimentation rate; SDAI, simplified disease activity index; CDAI, clinical disease activity index; HAQ, health assessment questionnaire; MTX, methotrexate; PSL, prednisolone. ∗P ⬍ 0.01, ∗∗P ⬍ 0.001.
ratio of 1.908. The multiple regression formula determining ROM is as follows:
studies. In this study, ROM was used as an oxidative stress parameter because they are relatively stable in the blood. Considering our improved method for measuring oxidative stress and the larger number of patients included in the present study, it is conceivable that oxidative stress is associated with disease activity in patients with RA. Oxidative stress induced by ROS is not only an important mechanism that underlies joint destruction and synovial proliferation [2–5] but also a very important mediator of extra-articular diseases in individuals with RA. Patients with RA are known to have a higher risk of cardiovascular events not explained entirely by traditional cardiovascular risk factors [21], implying that cardiovascular disease is an extra-articular manifestation of RA [22]. In fact, RA has been shown to be associated with increased
ROM⫽290.58(constant)⫹26.09⫻CRP⫹48.44⫻DAS28-ESR.
Discussion This paper is the first report showing a relationship between the serum level of ROM and disease activity and biomarkers associated with RA. Previous reports have demonstrated that total ROS, superoxide and hydroxyl radicals, and malondialdehyde (MDA) levels (an oxidative stress parameter) are correlated with DAS28 [19,20]; however, direct measurements of ROS or MDA seem to lack reliability due to their biochemical instability. Furthermore, a small number of patients (n ⫽ 30) were investigated in these
(A) 1000
(B) 1000
900
900 y = 46.488x + 292
y = 52.274x + 400.3 800
800
R2 = 0.458
R2 = 0.306
700 ROM (U.CARR)
700 ROM (U.CARR)
Mod Rheumatol Downloaded from informahealthcare.com by University of Maastricht on 06/16/14 For personal use only.
ROM – 0.121 ⫺ 0.113
600 500 400
600 500 400
300
300
200
200
100
100 0
0 0
1
2
3
4
CRP (mg/dL)
5
6
7
0
1
2
3
4
5
6
7
DAS28-ESR
Figure 2. Correlation between ROM and CRP (A), and ROM and DAS28-ESR (B). Significant, positive correlations were detected between ROM and CRP (R2 ⫽ 0.458), and ROM and DAS28-ESR (R2 ⫽ 0.306).
4
A. Nakajima et al.
Mod Rheumatol, 2014; Early Online: 1–5
Table 3. Multiple regression analysis to identify the clinical factors affecting ROM.
Constant CRP DAS28-ESR
Partial regression coefficient 290.58 26.09 48.44
Standardized regression coefficient 0.430 0.576
95% confidence interval 236.93–344.24 17.09–35.09 27.16–69.72
P value ⬍ 0.01 ⬍ 0.01 ⬍ 0.01
CRP and DAS28-ESR were finally determined to be parameters strongly affecting ROM. The multiple regression formula determining ROM is as follows: ROM⫽290.58(constant)⫹26.09⫻CRP⫹48.44⫻DAS28-ESR. ROM, reactive oxygen metabolities; CRP, C-reactive protein; MMP3, DAS28-ESR, disease activity score 28-erythrocyte sedimentation rate.
biology of RA is still unclear, further studies are required to establish the clinical significance of oxidative stress in RA. In summary, when ROM was used as a parameter for oxidative stress in this study, it was shown to be significantly increased in patients with RA. The serum level of ROM was associated with CRP and DAS28-ESR, but not with renal function or daily doses of PSL. Although ROM in RA patients could also be influenced by cardiovascular abnormalities, these results suggest that ROM, in conjunction with CRP and MMP3, may be able to be used as a new biological disease marker to evaluate the disease activity of RA. In the future, longitudinal studies designed to explore the association of therapeutic effects with decreases in ROM may further elucidate the clinical significance of oxidative stress in RA.
Mod Rheumatol Downloaded from informahealthcare.com by University of Maastricht on 06/16/14 For personal use only.
Acknowledgements cardiovascular morbidity and mortality [23]. In the pathobiology of cardiovascular events, arterial atherosclerosis exists and increased arterial stiffness, increased carotid intima media thickness, and endothelial dysfunction have been demonstrated in patients at increased risk of and with known cardiovascular disease [24–26], and these pathological changes in the cardiovascular system have also been reported in patients with RA [27]. While the levels of ROM in patients with DM in this study were increased from the normal value, the increase was significantly higher in patients with RA compared with those with DM. This suggests that ROM in RA patients may be influenced not only by cardiovascular abnormalities, but also by the disease activity that occurs as a result of inflammatory arthritis. Recently, there have been several papers reporting the relationship between oxidative stress and the disease activity of RA. Hassan et al. showed that markers of increased oxidative stress and impaired antioxidant capacity were profound and significantly reflected disease activity in RA [19]. Furthermore, Kundu et al. demonstrated that ROS generated in both peripheral blood and synovial infiltrates correlated positively with both DAS28 and CRP/anti-cyclic citrullinated peptide (CCP) levels [20]. Hayashi et al., by means of univariate analysis, showed that serum ROM levels were associated with disease activity in patients with RA [28]. In this study, by means of multiple regression analysis, we showed that the level of ROM was correlated with CRP and DAS28-ESR. This implies that ROM may be a novel and useful marker for evaluating the disease activity associated with RA. In the practical treatment of RA, serum levels of CRP and MMP3 are one of the items often measured to evaluate the severity of systemic inflammation and joint destruction. However, CRP reflects systemic, non-specific inflammation, and easily drops to negative values by administration of certain biologic agents, especially tocilizumab, the IL-6-blocking agent [29]. Also, MMP3 is influenced by doses of PSL and renal function [30]. Indeed, correlation analyses among laboratory parameters showed that MMP3 but not ROM was associated with the daily dose of PSL. Taking these findings into consideration, ROM, in conjunction with CRP and MMP3, may be able to be used as a biomarker to evaluate the disease activity of RA. Our concern was that the ratio of male and female in RA and DM was quite different as shown in Table 1, which could affect differences in serum ROM levels between RA and DM patients. Actually, the mean ROM of male and female was 416.0 and 445.2, respectively, and there was a statistically significant difference (p ⬍ 0.01) (data not shown). To verify the influence of gender difference on ROM, we performed multivariate analysis (multiple logistic regression analysis) and confirmed that gender difference was not a significant factor affecting ROM (p ⫽ 0.08) (data not shown). Limitations of this study included the variable timing of blood draws among patients and the measurement of ROM at a single time-point. Also, because the role of oxidative stress in the patho-
The authors are grateful to Dr Atsuhito Saiki (Department of Internal Medicine, Toho University Sakura Medical Center) for collecting blood samples from the DM patients.
Conflict of interest None.
References 1. Pap T, Muller-Ladner U, Gay RE, Gay S. Fibroblast biology: role of synovial fibroblasts in the pathogenesis of rheumatoid arthritis. Arthritis Res. 2009;2(5):361–7. 2. Mapp PI, Grootveld MC, Blake DR. Hypoxia, oxidative stress, and rheumatoid arthritis. Br Med Bull. 1995;51(2):419–36. 3. Hitchon CA, El-Gabalawy H. Oxidation in rheumatoid arthritis. Arthritis Res Ther. 2004;6:265–78. 4. Halliwell B. Oxygen radicals, nitric oxide, and human inflammatory joint disease. Ann Rheum Dis. 1995;54(6):505–10. 5. Tak PP, Zvaifler NJ, Grenn DR, Firestein GS. Rheumatoid arthritis and p53: how oxidative stress might alter the course of inflammatory diseases. Immunol Today. 2000;21(2):78–82. 6. Biemond P, Swaak AJ, Koster JF. Protective factors against oxygen free radicals and hydrogen peroxide in rheumatoid arthritis synovial fluid. Arthritis Rheum. 1984;27(7):760–5. 7. Griffiths HR. ROS as signaling molecules in T cells – evidence for abnormal redox signaling in the autoimmune disease, rheumatoid arthritis. Redox Rep. 2005;10(6):273–80. 8. Carratelli M, Porcaro L, Ruscica M, De Simone E, Bertelli AA, Corsi MM. Reactive oxygen metabolites and prooxidant status in children with Down’s syndrome. Int J Clin Pharm Res. 2001;21(2):79–84. 9. Trotti R, Carratelli M, Barbieri M, Micieli G, Bosone D, Rondanelli M, Bo P. Oxidative stress and a thrombophilic condition in alcoholics without severe liver disease. Haematologica. 2001;86(1):85–91. 10. Faienza MF, Francavilla R, Goffredo R, Ventura A, Marzano F, Panzarino G, et al. Oxidative stress in obesity and metabolic syndrome in children and adolescents. Horm Res Paediatr. 2012;78(3): 158–64. 11. Coaccioli S, Standoli ML, Biondi R, Panaccione A, Landucci P, Del Giorno R, et al. Assessment of the oxidative stress markers in patients with chronic renal insufficiency undergoing dialysis treatment. Clin Ter. 2010;161(5):441–4. 12. Koutsokera A, Papaioannou AI, Malli F, Kiropoulos TS, Katsabeki A, Kerenidi T, et al. Systemic oxidative stress in patients with pulmonary sarcoidosis. Pulm Pharmacol Ther. 2009;22(6):603–7. 13. Suzuki S, Matsukura S, Takeuchi H, Kawaguchi M, Ieki K, Odaka M, et al. Increase in reactive oxygen metabolite level in acute exacerbations of asthma. Int Arch Allergy Immunol. 2008;146 Suppl 1:67–72. 14. Kotani K, Koibuchi H, Miyamoto M, Yamada T, Taniguchi N. Relationship between reactive oxygen metabolites and carotid intimamedia thickness in subjects with hypercholesterolemia. Med Princ Pract. 2010;19(6):496–8. 15. Yamanaka G, Kawashima H, Suganami Y, Watanabe C, Watanabe Y, Miyajima T, et al. Diagnostic and predictive value of CSF d-ROM level in influenza virus-associated encephalopathy. J Neurol Sci. 2006;243(1–2):71–5. 16. Hirao M, Yamasaki N, Oze H, Ebina K, Nampei A, Kawato Y, et al. Serum level of oxidative stress marker is dramatically low in patients
Serum oxidative stress in RA 5
DOI 10.3109/14397595.2014.891495
17. 18. 19.
20.
21.
Mod Rheumatol Downloaded from informahealthcare.com by University of Maastricht on 06/16/14 For personal use only.
22. 23.
24.
with rheumatoid arthritis treated with tocilizumab. Rheumatol Int. 2011;32(12):4041–5. Yang H, Jin X, Kei Lam CW, Yan SK. Oxidative stress and diabetes mellitus. Clin Chem Lab Med. 2011;49:1773–82. Amaral S, Oliveria PJ, Ramalho-Santos J. Diabetes and impairment of reproductive function: possible role of mitochondria and reactive oxygen species. Curr Diabetes Rev. 2008;4(1):46–54. Hassan SZ, Gheita TA, Kenawy SA, Fahim AT, El-Sorougy IM, Abdou MS. Oxidative stress in systemic lupus erythematosus and rheumatoid arthritis patients: relationship to disease manifestations and activity. Int J Rheum Dis. 2011;14(4):325–31. Kundu S, Ghosh P, Datta S, Ghosh A, Chattopadhyay S, Chatterjee M. Oxidative stress as a potential biomarker for determining disease activity in patients with rheumatoid arthritis. Free Radic Res. 2012;46(12):1482–9. Del Rincon I, Williams K, Stern MP, Freeman GL, Escalante A. High incidence of cardiovascular events in a rheumatoid arthritis cohort not explained by traditional cardiac risk factors. Arthritis Rheum. 2001;44(12):2737–45. Van Doornum S, McColl G, Wicks IP. Accelerated atherosclerosis: an extraarticular feature of rheumatoid arthritis? Arthritis Rheum. 2002;46(4):862–73. Jacobsson LT, Turesson C, Hanson RL, Pillemer S, Sievers ML, Pettitt DJ, et al. Joint swelling as a predictor of death from cardiovascular disease in a population study of Pima Indians. Arthritis Rheum. 2001;44(5):1170–6. Kobayashi K, Akishita M, Yu W, Hashimoto M, Ohni M, Toba K. Interrelationship between non-invasive measurements of
25.
26.
27.
28.
29.
30.
atherosclerosis: flow-mediated dilation of brachial artery, carotid, intima-media thickness, and pulse wave velocity. Atherosclerosis. 2004;173(1):13–8. O’Leary DH, Polak JF, Kronmal RA, Manolio TA, Burke GL, Wolfson SK Jr. Cardiovascular Health Study Collaborative Research Group. Carotid-artery intima and media thickness as a risk factor for myocardial infarction and stroke in older adults. N Engl J Med. 1999;340(1):14–22. Boutouyrie P, Tropeano AI, Asmar R, Gautier I, Benetos A, Lacolley P, Laurent S. Aortic stiffness is an independent predictor of primary coronary events in hypertensive patients: a longitudinal study. Hypertension. 2002;39(1):10–5. Del Porto F, Lagana B, Lai S, Nofroni I, Tinti F, Vitale M, et al. Response to anti-tumour necrosis factor alpha blockade is associated with reduction of carotid intima-media thickness in patients with active rheumatoid arthritis. Rheumatology. 2007;46(7):1111–5. Hayashi H, Satoi K, Sato-Mito N, Kaburagi T, Yoshino H, Higaki M, et al. Nutritional status in relation to adipokines and oxidative stress is associated with disease activity in patients with rheumatoid arthritis. Nutrition. 2012;28(11–12):1109–14. Funahashi K, Koyano S, Miura T, Hagiwara T, Okuda K, Matsubara T. Efficacy of tocilizumab and evaluation of clinical remission as determined by CDAI and MMP-3 level. Mod Rheumatol. 2009;19(5):507–12. Ribbens C, Martin y Porras M, Franchimont N, Kaiser MJ, Jaspar JM, Damas P, et al. Increased metalloproteinase-3 serum levels in rheumatic diseases: relationship with synovitis and steroid treatment. Ann Rheum Dis. 2002;61(2):161–6.
