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 Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 44  |  Issue : 1  |  Page : 12-15

Assessment of serum nitrite level in patients with rheumatoid arthritis


1 Department of Rheumatology and Rehabilitation, Faculty of Medicine, Tanta University, Tanta, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Tanta University, Tanta, Egypt

Date of Web Publication15-Apr-2016

Correspondence Address:
Dina H.M. Abdella
Department of Rheumatology and Rehabilitation, Tanta University, 1112 Tanta
Egypt
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DOI: 10.4103/1110-1415.180544

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  Abstract 

Context
Rheumatoid arthritis (RA) is an inflammatory autoimmune disease involving joints and other systems. Nitric oxide (NO) participation is recognized in numerous physiological and pathological processes. The role of NO in the pathogenesis of RA is still unknown.
Aim
The aim of this work was to evaluate serum nitrite as a disease marker in patients with RA and its correlation with different parameters of disease activity and severity.
Patients and methods
A total of 30 RA patients and 15 healthy controls were included. Clinical and functional assessments with routine laboratory investigations were carried out, and serum level of nitrite was measured using enzyme-linked immunosorbent assay. Plain radiographies of both hands and feet were performed for radiological status scoring using the Larsen score.
Statistical analysis
The collected data were coded, tabulated, and statistically analyzed using SPSS program, version 16.0. Student's t-test and Pearson's correlation coefficient were used for statistical analysis.
Results
Serum nitrite level in patients with RA was statistically significantly high compared with controls. A statistically significant positive correlation was found between serum nitrite levels and different clinical and laboratory markers of disease activity as well as radiographic joint status assessed using Larsen score.
Conclusion
There was an increase in serum nitrite levels in patients with RA that significantly correlated with the clinical and laboratory markers of disease activity and radiological joint status.

Keywords: disease activity, Larsen score, nitric oxide, nitrite, rheumatoid arthritis


How to cite this article:
Khallaf HA, Nosair NA, Alashkar DS, Abdella DH. Assessment of serum nitrite level in patients with rheumatoid arthritis . Tanta Med J 2016;44:12-5

How to cite this URL:
Khallaf HA, Nosair NA, Alashkar DS, Abdella DH. Assessment of serum nitrite level in patients with rheumatoid arthritis . Tanta Med J [serial online] 2016 [cited 2020 Feb 21];44:12-5. Available from: http://www.tdj.eg.net/text.asp?2016/44/1/12/180544


  Introduction Top


Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disorder that primarily targets the synovium, resulting in synovial inflammation and proliferation, loss of articular cartilage, erosions of periarticular surfaces, and juxta-articular osteopenia [1] . Although the precise etiology of RA remains unknown, studies have implicated a role for oxidative stress and redox signaling in its pathogenesis [2] .

Nitric oxide (NO) is a biological messenger that mediates many physiological functions and pathological processes [3] . NO is synthesized from l-arginine by the enzyme nitric oxide synthase (NOS), which exists in three isoforms, neuronal (nNOS or NOS1), inducible (iNOS or NOS2), and endothelial (eNOS or NOS3) isoforms [4] . NOS1 and NOS3 are calcium-dependent and generally produce low levels of NO involved in normal physiologic processes. In contrast, NOS2 is calcium-independent, and its expression is upregulated by interferon α, interferon g, interleukin-1, and tumor necrosis factor-α, as well as other proinflammatory mediators, resulting in sustained and high levels of NO output [5] .

NO has also emerged as an important mediator in collagen-induced arthritis and RA synovium, as it mediates many different cell functions at the site of synovial inflammation, including cytokine production, signal transduction, mitochondrial functions, and apoptosis. NO can induce tissue damage, especially after reaction with superoxide anion O2 - to form peroxyinitrite radical (ONOO - ), whose oxidant potential is greater than that of O2 - alone [6] . Moreover, NO can affect the production of angiogenic cytokines and the activation of matrix metalloproteinases (MMPs) [7] . Because NO has a very short half-life, plasma concentration of nitrate and nitrite are often used as a marker of NO production and NOS enzyme activity [8] . Several authors have proposed that the inhibition of NOS has beneficial effects in acute and chronic joint inflammation, as experimental arthritis was found to be suppressed after inhibition of NOS [9] .

Thus, the aim of this work was to evaluate serum nitrite as a disease marker in patients with RA and its correlation with different parameters of disease activity and severity.


  Patients and methods Top


The patient group (group I) included 30 RA patients who fulfilled the American College of Rheumatology/European League Against Rheumatism 2010 criteria for diagnosis of RA. Patients with other collagen diseases, malignancies, active infections, hypertension, and diabetes mellitus were excluded from the study. In addition, patients under treatment with prednisolone of more than 10 mg/day, or under treatment with anticytokine agents such as antitumor necrosis factor drug therapy, were excluded from the study. Patients taking other drugs and food that affect NO level (e.g. salicylates, indomethacin, arugula, and beet) were also excluded from the study. The control group (group II) included 15 apparently healthy volunteers matched in age and sex with group I. The patients were selected from the outpatient clinic of Physical Medicine, Rheumatology and Rehabilitation Department of Tanta University Hospitals. The study was approved by the Local Research Ethics Committee of Faculty of Medicine Tanta University. All tests were explained to patients and controls before obtaining written informed consent to participate in this study.

All patients were subjected to the following:

  1. Full medical history taking.
  2. Thorough clinical examination.
  3. Assessment of disease activity using Disease Activity Score 28 (DAS28).
  4. Functional assessment using the Modified Health Assessment Questionnaire.
  5. Laboratory assessment:


    1. Complete blood picture.
    2. Erythrocyte sedimentation rate first hour (using the Wintrobe method).
    3. C-reactive protein.
    4. Rheumatoid factor (using nephelometry).
    5. Anticyclic citrullinated peptide.
    6. Serum nitrite evaluation using the enzyme-linked immunosorbent assay technique with total NO and nitrate/nitrite assay kits supplied from R&D systems catalog number KGE001 (USA). This assay determines NO concentrations based on the enzymatic conversion of nitrate to nitrite by nitrate reductase. The reaction is followed by colorimetric detection of nitrite as an azo dye product of the Griess reaction. The Griess reaction is based on the two-step diazotization reaction in which acidified NO2 - produces a nitrosating agent, which reacts with sulfanilic acid to produce the diazonium ion. This ion is then coupled to N-(1-naphthyl) ethylenediamine to form the chromophoric azo-derivative, which absorbs light at 540-570 nm.
  6. Radiological assessment using the Larsen score to evaluate bone erosions in both hands and feet.
Statistical analysis

The collected data were coded, tabulated, and statistically analyzed using SPSS program, version 16.0. Qualitative variables were presented as number and percentage. The χ2 -test was used to compare qualitative variables. The unpaired t-test was used to compare two independent groups as regards quantitative variables. Pearson's correlation coefficient rank test was used to rank different variables against each other positively or inversely. A P-value greater than 0.05 was considered not significant and a P-value less than 0.05 was considered significant.


  Results Top


  1. Patients with RA had significantly higher levels of serum nitrite compared with controls, and abnormal serum nitrite levels were found in 80% of the RA patients [Table 1].
  2. There was a significant positive correlation between serum nitrite levels in RA patients and clinical parameters of disease activity (morning stiffness, number of tender joints, number of swollen joints, Visual Analog Scale, and DAS28 score) [Table 2].
  3. There was a significant positive correlation between serum nitrite levels in RA patients and laboratory markers of disease activity (hemoglobin levels, total platelet count, erythrocyte sedimentation rate, and C-reactive protein). These results support the important role of NO in inflammatory reactions in RA disease [Table 3].
  4. There was a significant positive correlation between serum nitrite levels and Larsen score in RA patients, and significantly higher levels were found in patients with bone erosions than in patients without bone erosions, which provide further evidence that it may be an important mediator of joint damage in RA patients [Table 4] and [Table 5].
  5. There was no significant correlation between serum nitrite levels in RA patients and age, sex, disease duration, rheumatoid factor, anticyclic citrullinated peptide, total leukocytic count, and functional status (assessed using Modified Health Assessment Questionnaire).
Table 1 Serum nitrite levels in RA patients and controls

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Table 2 Correlation of clinical data of RA patients with serum nitrite levels

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Table 3 Correlation of laboratory data of RA patients with serum nitrite levels

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Table 4 Correlation of Larsen score of RA patients with serum nitrite levels

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Table 5 Comparison of serum nitrite levels in RA patients with and without bone erosions (according to Larsen score)

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  Discussion Top


RA is a chronic autoimmune disease characterized by systemic inflammatory and destructive joint lesions [10] . Numerous studies carried out on RA revealed increased endogenous NO synthesis. Overproduction of NO, as a mediator of inflammation and/or tissue destruction, may be important in the pathogenesis of RA [11] .

This study assessed serum nitrite level in RA patients and demonstrated higher levels in RA patients than in controls, with a significant correlation with clinical and laboratory parameters of disease activity. In agreement with our view, Triveni et al. [12] estimated serum NO as a marker of oxidation in RA patients and apparently healthy controls and reported a highly significant increase in serum NO level in RA patients as compared with healthy controls. Borysewicz et al. [13] reported a significant elevation in serum nitrite in RA patients and osteoarthritis patients compared with controls, with higher levels in patients with RA compared with osteoarthritic patients. Pham et al. [14] quantified circulating NO levels and iNOS expression in peripheral blood monocyte-derived macrophages from patients with RA as a measure of disease activity. The findings showed significantly higher serum NO in RA patients than in controls or patients with osteoarthritis, and the highest levels of NO were seen in patients with severe inflammation. Veselinovic et al. [15] assessed the oxidative stress status in RA by measuring NO and systemic activity of disease, and significant differences were observed in serum levels of NO in patients with high disease activity. In contrast, Gόzel et al. [11] did not find significant correlation between serum nitrite and DAS28 score in RA patients. However, they found significantly higher synovial fluid NO in patients with moderate disease activity than in patients with low disease activity; this may be due to local upregulation of NOS expression in synovial lining cells, chondrocytes, and blood vessels in joint tissues of RA patients. Our results demonstrated that there was a significant positive correlation between serum nitrite level and radiographic joint damage measured using Larsen score in RA patients. We found significantly higher level in erosive RA patients when compared with nonerosive RA patients. Thus, NO may be an important mediator of joint damage in RA patients. This view is supported by Dubikov et al. [16] , who found significantly higher synovial nitrite level in erosive RA patients. Hirai et al. [17] examined whether NO modulates the production of MMPs, which degrade all components of extracellular matrix in rheumatoid synovial cells, by studying the effects of exogenously generated NO by a NO donor, S-nitroso-N-acetyl-dl-penicillamine (SNAP), on the MMP production by rheumatoid synovial cells. Incubation of synovial cells with SNAP resulted in gelatinase A production in a dose-dependent manner. Furthermore, MMP-2 mRNA expression was induced in SNAP-treated synovial cells. This study indicated that NO could modulate MMP production by rheumatoid synovial cells and therefore contribute to extracellular matrix degradation of articular components in RA.


  Conclusion Top


There was an increase in serum nitrite levels in patients with RA, and it significantly correlated with the clinical and laboratory markers of disease activity and radiological joint status.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

1.
Kundu S, Bala A, Ghosh P, Mukhopadhyay D, Mitra A, Sarkar A, et al. Attenuation of oxidative stress by allylpyrocatechol in synovial cellular infiltrate of patients with rheumatoid arthritis. Free Radic Res 2011; 45:518-526.  Back to cited text no. 1
    
2.
Mirshafiey A, Mohsenzadegan M. The role of reactive oxygen species in immunopathogenesis of rheumatoid arthritis. Iran J Allergy Asthma Immunol 2008; 7:195-202.  Back to cited text no. 2
    
3.
Antosova M, Plevkova J, Strapkova A, Buday T. Nitric oxide - Important messenger in human body. Open J Mol Integr Physiol 2012; 2:98-106.  Back to cited text no. 3
    
4.
Förstermann U, Sessa WC. Nitric oxide synthases: regulation and function. Eur Heart J 2012; 33:829-837.  Back to cited text no. 4
    
5.
Weinberg JB, Lang T, Wilkinson WE, Pisetsky DS, St Clair EW. Serum, urinary, and salivary nitric oxide in rheumatoid arthritis: complexities of interpreting nitric oxide measures. Arthritis Res Ther 2006; 8:R140.  Back to cited text no. 5
    
6.
Bala A, Haldar PK. Free radical biology in cellular inflammation related to rheumatoid arthritis. OA Arthritis 2013; 1:1-6.  Back to cited text no. 6
    
7.
Leibovich SJ, Polverini PJ, Fong TW, Harlow LA, Koch AE. Production of angiogenic activity by human monocytes requires an l-arginine/nitric oxide-synthase-dependent effector mechanism. Proc Natl Acad Sci USA 1994; 91:4190-4194.  Back to cited text no. 7
    
8.
Osipov AN, Borisenko GG, Vladimirov YA. Biological activity of hemoprotein nitrosyl complexes. Biochemistry (Mosc) 2007; 72: 1491-1504.  Back to cited text no. 8
    
9.
Veihelmann A, Hofbauer A, Krombach F, Dorger M, Maier M, Refior HJ, Messmer K. Differential function of nitric oxide in murine antigen-induced arthritis. Rheumatology (Oxford) 2002; 41:509-517.  Back to cited text no. 9
    
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Demidova NV, Guseva IA, Karateev DE. Clinical and immunological aspects of early-stage rheumatoid arthritis. Ter Arkh 2010; 82:71-77.  Back to cited text no. 10
    
11.
Güzel S, Seven A, Güzel EC, Hamuryudan V. Nitric oxide and superoxide dismutase in rheumatoid arthritis: correlation with disease activity. Turkish J Family Med Primary Care 2012; 6:7-12.  Back to cited text no. 11
    
12.
Triveni JA, Halyal SS, Murthy JD. Study of serum malondialdehyde, nitric oxide, vitamin E levels in patients with rheumatoid arthritis. Int J Med Res Health Sci 2014; 3:322-325.  Back to cited text no. 12
    
13.
Borysewicz K, Seweryn E, Szmyrka-Kaczmarek M, Szechiñski J, Bana? T. Serum and synovial fluid nitrite levels in patients with rheumatoid arthritis and osteoarthritis. Adv Clin Exp Med 2008; 17:395-398.  Back to cited text no. 13
    
14.
Pham TN, Rahman P, Tobin YM, Khraishi MM, Hamilton SF, Alderdice C, Richardson VJ. Elevated serum nitric oxide levels in patients with inflammatory arthritis associated with co-expression of inducible nitric oxide synthase and protein kinase C-eta in peripheral blood monocyte-derived macrophages. J Rheumatol 2003; 30:2529-2534.  Back to cited text no. 14
    
15.
Veselinovic M, Barudzic N, Vuletic M, Zivkovic V, Tomic-Lucic A, Djuric D, Jakovljevic V. Oxidative stress in rheumatoid arthritis patients: relationship to diseases activity. Mol Cell Biochem 2014; 391:225-232.  Back to cited text no. 15
    
16.
Dubikov AI, Medved EE, Belogolovykh LA. The effect of methotrexate and leflunomide on the cytokine profile and nitric oxide metabolism in rheumatoid arthritis patients. Rheumatology 2012; 2:1-4.  Back to cited text no. 16
    
17.
Hirai Y, Migita K, Honda S, Ueki Y, Yamasaki S, Urayama S, et al. Effects of nitric oxide on matrix metalloproteinase-2 production by rheumatoid synovial cells. Life Sci 2001; 68:913-920.  Back to cited text no. 17
    



 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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Introduction
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