Rheumatoid arthritis (RA) is an autoimmune inflammatory disease whose pathogenic mechanisms remain to be elucidated. The oxidative stress and antioxidants play an important role in the disease process of RA. The study of oxidants and antioxidants biomarkers in RA patients could improve our understanding of disease pathogenesis; likely determining the oxidative stress levels in these patients could prove helpful in assessing disease activity and might also have prognostic implications. To date, the usefulness of oxidative stress biomarkers in RA patients is unclear and the evidence supporting them is heterogeneous. In order to resume and update the information in the status of oxidants and antioxidants and their connection as biomarkers in RA, we performed a systematic literature search in the PubMed database, including clinical trials published in the last five years using the word combination “rheumatoid arthritis oxidative stress”. In conclusion, this review supports the fact that the oxidative stress is an active process in RA pathogenesis interrelated to other better known pathogenic elements. However, some controversial results preclude a definite conclusion.
Rheumatoid arthritis (RA) is an autoimmune disease affecting diarthrodial joints. It is characterized by erosive synovitis, which causes cartilage and bone destruction and systemic complications including cardiovascular, pulmonary, psychological, and other skeletal disorders [
ROS are the most important class of radicals generated in living systems. They are oxygen-derived radicals and include the superoxide radical (
Generation of oxygen and nitrogen reactive species (ROS and RNS). CAT: catalase, ETC: electron transport chain, H2O: water, H2O2: hydrogen peroxide, HOCl: hypochlorous acid, HOONO: peroxynitrous acid, GPx: glutathione peroxidase, GR: glutathione reductase, GSH: reduced glutathione, GSSH: oxidized glutathione, MPO: myeloperoxidase, NADPH: reduced nicotinamide adenine dinucleotide phosphate, NOS: nitric oxide synthase, NH2Cl: chloramine, NH3: ammonia, NO•: nitric oxide,
Under physiological conditions, ROS are required to maintain the cell redox state and play a role in cell signaling, differentiation, proliferation, growth, apoptosis, cytoskeletal regulation, and phagocytosis. However if the concentrations of ROS are increased beyond physiological conditions they can damage cellular components, such as the lipids in the cell membranes, and also proteins and nucleic acids. If a given condition induces an imbalance between oxidants and antioxidants, where oxidants are favored, a disruption of redox signaling is produced, and a control and/or molecular damage occurs. This cellular state termed oxidative stress [
The damaging effect of free radicals is counteracted by the action of antioxidants. An antioxidant is any substance or compound capable to scavenge free radicals or inhibiting the oxidation process in the cell [
RA is one of the conditions that induce oxidative stress. A fivefold increase in mitochondrial ROS production in whole blood and monocytes of RA patients—compared with healthy subjects—suggests that oxidative stress is a pathogenic hallmark in RA. Free radicals are indirectly implicated in joint damage because they also play an important role as secondary messengers in inflammatory and immunological cellular response in RA. T-cell exposure to increased oxidative stress becomes refractory to several stimuli including those for growth and death and may perpetuate the abnormal immune response [
The chronic oxidative stress in the RA synovium has been explained by the elevated intra-articular pressure in RA joints, which increases ROS production in the cellular oxidative phosphorylation and induces repetitive cycles of hypoxia/reoxygenation. The hypoxia is an event observed in RA joints whose origin has been explained to be a consequence of the rapid cellular proliferation induced by the inflammatory response; however, according Jeon et al. [
The association between oxidative stress and RA has been explored using various oxidant or antioxidant biomarkers. These biomarkers include lipids, proteins, and DNA oxidation markers and also levels of enzymatic activities, antioxidants agents, and even the direct measurement of free radicals. The aim of this review is to resume and update the available evidence in regard to the potential role of oxidants and antioxidants in RA patients and the findings related to these biomarkers in RA. A systematic literature search was performed including studies published in the last 5 years. The findings are presented in a comparative way between studies included.
A systematic literature search was performed including studies published between May 2010 and May 2015. These studies assessed oxidant and/or antioxidant biomarkers in RA patients. The search was conducted in the PubMed database. The word combination used for the search was “
This review included original articles published in English within the last 5 years. The studies were clinical trials, which assessed oxidant and/or antioxidant biomarkers in RA patients. Even if the main purpose of any of the selected articles was to compare different diseases, we selected only the results obtained from RA. Studies with experimental interventions and those with oxidative stress induced by causes other than RA (periodontitis, smoking, nutritional status, and genetic polymorphisms in RA) were excluded.
We assessed the methodological quality of the included studies with the Newcastle-Ottawa Quality Assessment Scale (NOS). Every study received a score consisting in a number of stars. The NOS include three domains: (a) selection (maximum 4 stars), (b) comparability (maximum 2 stars), and (c) exposure (maximum 3 stars). The highest score possible was 9 stars. Studies with scores of 6 stars or above were considered to be of moderate to good study quality. The score was not an exclusion criterion. The quality of the selected articles was assessed by one reviewer and checked by a second reviewer.
We selected the following information from every included article: age, female/male ratio, sample size, disease activity score (DAS-28), disease duration, type of biological sample, oxidant and antioxidant biomarkers levels, and the findings related to these biomarkers. This information was organized in comparative tables.
The process of article selection is described in Figure
Flow chart of study selection.
The characteristics of the included studies are summarized in Table
Demographic and clinical characteristics of RA patients and control groups.
Author and year | Country | Sample size (% women/% men) | Age in years (mean or Min–Max) | DAS-28 (mean) | Duration of disease (mean or Min–Max) | ||
---|---|---|---|---|---|---|---|
Cases | Controls | Cases | Controls | ||||
García-González et al., 2015 [ |
Mexico | 10 (90/10) A, 19 (84/16) I |
41 (90/10) | 48 A, 48.5 I | 38.0 | 4.3 A, 2.1 I | 7.0 y A, 2.0 y I |
Thiele et al., 2015 [ |
USA | 1720 (9.1/90.9) | 80 (NS) | 63.4 | NS | 3.9 | 12.4 y |
Datta et al., 2014 [ |
India | 36 (77.7/22.3) | NS | 40 | NS | 5.6 | 11 m–24 y |
Nakajima et al., 2014 [ |
Japan | 152 (67.7/32.3) | 80 (42.5/57.5) | 63.1 | 59.2 | 3.5 | 14.3 y |
Nzeusseu Toukap et al., 2014 [ |
Belgium | 33 URA (NS), 33 TRA (NS) |
39 (NS) | NS | NS | 4.8 URA, 4.9 TRA | NS |
Veselinovic et al., 2014 [ |
Serbia | 52 (63.5/36.5) | 30 (63.2/36.8) | 52.4 | 54.2 | 3.6 | 5.7 y |
Wang et al., 2014 [ |
China | 100 (62/38) | 50 (68/32) | 55.7 | 52.5 | 5.3 | 7.0 y |
Jacobson et al., 2012 [ |
Australia | 35 (62.9/37.1) | 39 (61.5/38.5) | 62.9 | 62.8 | NS | NS |
Kundu et al., 2012 [ |
India | 25 (80/20) | 10 (80/20) | 40.0 | 26.5 | 5.7 | 11 m–25 y |
Kwaśny-Krochin et al., 2012 [ |
Poland | 46 (85/15) | 50 (86/14) | 57.0 | 56.0 | 5.2 | 8.1 y |
Mishra et al., 2012 [ |
India | 36 (61.1/38.9) | 36 (69.4/30.6) | 49.7 | 49.6 | NS | NS |
Stamp et al., 2012 [ |
New Zealand | 77 (71.4/28.6) | 120 (NS) | 54.7 | NS | 3.8 | NS |
Staroń et al., 2012 [ |
Poland | 25 (84/16) | 35 (NS) | NS | NS | NS | NS |
Alver et al., 2011 [ |
Turkey | 52 (76.9/23.1) | 42 (73.8/26.2) | 49.2 | 48.5 | NS | NS |
Aryaeian et al., 2011 [ |
Iran | 59 (64.4/35.6) | 59 (64.4/35.6) | 41.9 | 39.1 | NS | 8.2 y |
Ediz et al., 2011 [ |
Turkey | 25 (72/28) ACPA (+) | 24 (76/24) ACPA (−) | 54.4 | 56.2 | 4.1 ACPA (+), 3.4 ACPA (−) | 9.6 y ACPA (+), 8.1 y ACPA (−) |
Hassan et al., 2011 [ |
Egypt | 30 (100) | 30 (100) | 35.8 | 32.3 | 4.0 | 6.5 y |
Karaman et al., 2011 [ |
Turkey | 43 (74.4/25.6) | 30 (56.7/43.3) | 39.8 | 37.2 | NS | 4.2 y |
Desai et al., 2010 [ |
India | 40 (50/50) | 40 (NS) | 40–60 | 40–60 | NS | NS |
Rho et al., 2010 [ |
USA | 169 (NS) | 92 (NS) | >18 | >18 | NS | NS |
Shah et al., 2011 [ |
India | 30 (83.3/16.7) | 30 (90/10) | 24.2 | 26.7 | 4.5 | 5.0 y |
Tetik et al., 2010 [ |
Turkey | 20 (NS) | 20 (NS) | 48 | 25 | NS | 11 y |
ACPA: anti-citrullinated protein antibodies, DAS-28: Disease Activity Score, m: months, NS: not specified, y: years.
In regard to the country of the populations included, 5 studies were conducted in India, 4 in Turkey, 2 in Poland, 1 in New Zealand, 1 in Iran, 2 in USA, 1 in Serbia, 1 in Egypt, 1 in Mexico, 1 in Australia, 1 in Japan, 1 in Belgium, and 1 in China.
Thirty different oxidant and/or antioxidant markers were analyzed among the selected studies. They were classified in seven groups: (1) lipid peroxidation (4 markers: malondialdehyde [MDA], thiobarbituric acid reactive substances [TBARS], isoprostane [F2-I], and, malondialdehyde-acetaldehyde [MAA], adducts), (2) protein oxidation (4 markers: protein carbonyls [PC], 3-chlorotyrosine [CT], advanced oxidation of protein products [AOPP], and nitrosothiols [RSNO]), (3) DNA damage (2 markers: micronucleus [MN] and DNA stand breaks [DNA sb]), (4) urate oxidation (1 marker: allantoin [ALLA]), (5) enzymatic activity (7 markers: CAT, SOD, GR, GPx, myeloperoxidase [MPO], NADPH oxidase [NADPH ox], and arylesterase [AE]), (6) antioxidants (6 markers: GSH, oxidized glutathione [GSSG],
Summary of reported oxidant and antioxidant markers in RA patients compared to control group.
Author | Lipid oxidation | Protein oxidation | DNA oxidation | Urate oxidation | Enzymatic activity | Antioxidants | Free radicals/anions | Main findings |
---|---|---|---|---|---|---|---|---|
García-González et al., 2015 [ |
TBARS: |
PC: |
NE | NE | SOD: |
GSH: |
NE | Oxidative damage was elevated in RA patients. Antioxidants enzyme activities, GSH levels, and GSH/GSSG ratio were higher in RA than in control group; however they were insufficient to prevent oxidative damage. |
|
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Thiele et al., 2015 [ |
MAA: |
NE | NE | NE | NE | NE | NE | MAA adduct formation is increased in RA and colocalized with citrullinated proteins. MAA antibodies are associated with ACPA production. This suggests that MAA formation may drive tolerance loss to autoantibody formation in RA. |
|
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Datta et al., 2014 [ |
MDA: sf❖ | PC: sf❖; |
NE | NE | NE | NE | Total ROS: sf❖; |
All oxidative damage markers correlated positively with DAS-28; therefore the measurement of oxidative stress could serve as a biomarker for monitoring disease severity in RA. |
|
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Nakajima et al., 2014 [ |
NE | NE | NE | NE | NE | NE | ROM: |
Serum level of ROM was associated with CRP and DAS-28 suggesting that ROM may be able to be used as a disease marker to evaluate the disease activity. |
|
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Nzeusseu Toukap et al., 2014 [ |
NE | CT: |
NE | NE | MPO: |
NE | NE | MPO activity, MPO, and CT levels were significantly higher in synovial fluid of RA patients than OA patients. MPO activity and concentration were correlated with IL-8 and IL-18 in untreated but not in treated RA patients. |
|
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Veselinovic et al., 2014 [ |
TBARS: |
NE | NE | NE | CAT: |
GSH: |
H2O2: |
Higher levels of prooxidants in RA compared to control group. Stronger response in samples with higher diseases activity suggests that oxidative stress markers may be useful in evaluating the progression of RA. |
|
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Wang et al., 2014 [ |
NE | NE | NE | NE | MPO: |
NE | NE | Serum levels of MPO higher in RA than control group. Moderate positive correlations between MPO levels and CRP, DAS-28. These results support a role for MPO in the inflammatory process of RA. |
|
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Jacobson et al., 2012 [ |
MDA: |
NE | NE | NE | GPx: |
Anti-Cap: |
NE | There were no differences between RA cases and control group for oxidative stress and antioxidant capacity; however, GPx level was markedly elevated in RA. GPx levels were not associated with severity disease or CRP. |
|
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Kundu et al., 2012 [ |
NE | NE | NE | NE | NADPH ox: |
NE | Total ROS: |
ROS generated in both peripheral blood and synovial infiltrate correlated positively with both DAS-28 and CRP/ACPA levels; its measurement can serve as an indirect measure of the degree of inflammation in patients with RA. |
|
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Kwaśny-Krochin et al., 2012 [ |
F2-I: |
NE | NE | NE | NE | NE | NE | ADMA levels are significantly higher in RA than in control group. Positive associations between plasma ADMA levels and the production of 8-isoprostanes and CRP in RA. |
|
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Mishra et al., 2012 [ |
MDA: s |
NE | NE | NE | NE | NE | NE | LDL, total lipid, cholesterol, MDA, CRP, and triglycerides are elevated and HDL levels decreased in RA compared with control group. |
|
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Stamp et al., 2012 [ |
NE | PC: |
NE | ALLA: |
MPO: |
NE | NE | MPO protein concentration is elevated in RA and promotes oxidative stress through the production of hypochlorous acid. There is a significant relationship between plasma MPO concentration and DAS-28. |
|
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Staroń et al., 2012 [ |
TBARS: |
NE | NE | NE | CAT: |
GSH: |
NE | There are no significant differences in CAT and GPx activities. SOD activity is lower and lipid peroxidation is increased in RA. GSH and -SH groups are significantly lower in RA. |
|
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Alver et al., 2011 [ |
MDA: |
NE | NE | NE | CAT: |
GSH: |
NE | The CAII autoantibody titers were significantly higher in RA. The increased erythrocyte oxidative stress in RA may be effective in the mechanism of CA II autoantibody production. |
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Aryaeian et al., 2011 [ |
MDA: |
NE | NE | NE | GR: |
|
NE | There is an increased oxidative stress (MDA elevated) and a low antioxidant status (vitamin E, |
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Ediz et al., 2011 [ |
MDA: |
NE | NE | NE | CAT: |
NE | NE | There was positive correlation between ACPA levels and synovial MDA and MPO in ACCP (+) group. ACPA positivity seems to be associated with increased synovial fluid oxidant activity in RA. |
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Hassan et al., 2011 [ |
MDA: |
NE | NE | NE | GPx: |
GSH: |
NE | Oxidative stress was increased in RA. DAS-28 significantly correlated with MDA levels and negatively with GSH. |
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Karaman et al., 2011 [ |
MDA: |
NE | DNA sb: |
NE | SOD: |
NE | NE | Elevated degree of oxidative stress in RA patients associated with DNA damage. |
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Shah et al., 2011 [ |
MDA: |
NE | NE | NE | CAT: |
GSH: |
NE | Elevated ROS production disturbs redox status and can modulate the expression of inflammatory chemokines leading to inflammatory processes, exacerbating inflammation, and affecting tissue damage. |
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Desai et al., 2010 [ |
MDA: |
NE | NE | NE | SOD: |
NE | NE | There is an increased oxidative stress and a decreased antioxidant defense in patients with RA. |
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Rho et al., 2010 [ |
F2-I: |
NE | NE | NE | NE | NE | NE | F2-isoprostanes were higher in RA and they significantly modified the protective effect of HDL cholesterol against coronary calcification. |
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Tetik et al., 2010 [ |
NE | PC: |
NE | NE | NE | -SH: |
NE | Protein carbonyls content was higher in RA as compared to controls while the plasma -SH levels in RA was significantly lower than control. CRP levels were higher in RA. |
ACPA: anti-citrullinated protein antibodies, ADMA: asymmetric dimethylarginine, AE: arylesterase, ALLA: allantoin, Anti-cap: total antioxidant capacity, AOPP: advanced oxidation protein products, CAII: carbonic anhydrase II autoantibody, CAT: catalase, CRP: C-Reactive Protein, CT: 3-chlorotyrosine, DAS-28: disease activity score, DNA sb: DNA strand breaks, e: erythrocyte, F2-I: F2-isoprostane, GPx: glutathione peroxidase, GR: glutathione reductase, GSH: reduced glutathione, GSSG: oxidized glutathione, H2O2: hydrogen peroxide, HDL: high density lipoprotein, IL: interleukin, L: lymphocytes, LDL: low density lipoprotein, MAA: malondialdehyde-acetaldehyde, MDA: malondialdehyde, MN: micronucleus, MPO: myeloperoxidase, n-b: neutrophils isolated from blood, n-sf: neutrophils isolated from synovial fluid, NADPH ox: nicotinamide adenine dinucleotide phosphate oxidase, NE: not evaluated,
Sixteen of the 22 studies assessed lipid oxidation biomarkers (MDA, TBARS, F2-I, and MAA adducts). Ten of them measured MDA levels. Most of them observed a statistically significant increase in MDA blood levels in RA patients. A significant difference in MDA blood concentration between RA and control patients was not reported by Jacobson et al. [
The F2-I levels were reported in two studies. A significantly higher F2-I excretion in patients with RA than control subjects was found by Rho et al. [
The MAA adducts expression in RA synovial tissue was evaluated only in one study [
Protein oxidation was evaluated through different biomarkers (PC, RSNO, AOPP, and CT) in 5 studies [
Only one study assessed the DNA damage by MN and DNAsb (comet assay). Karaman et al. [
The ALLA plasma concentration as a measure of oxidation of urate was assessed by Stamp et al. [
Enzymatic activity was evaluated in 15 of the included studies (GPx, SOD, CAT, GR, AE, NADPH ox, and MPO); the results were heterogeneous. The enzyme most commonly assessed was GPx (8 studies); it was measured in red blood cells [
The SOD activity was also evaluated and gave conflicting results. In 5 studies, the SOD activity from RA patients was found lower than in controls in plasma and erythrocytes [
CAT activity was evaluated in 5 studies. Four of these studies [
Evidence regarding GR, AE, and NADPH ox activity in RA was limited in the included studies [
MPO activity was reported in 4 of the included studies. A decrease in the MPO activity in RA plasma was reported by Stamp et al. [
Nine studies assessed different antioxidants molecules. GSH concentrations were measured in 6 studies. With respect to control group, this biomarker was found diminished in 3 studies [
Total ROS, ROM, H2O2,
The aim of this review was to show and update the available evidence in regard to oxidants and antioxidants in RA patients and to highlight the findings related to biomarkers. Several oxidative stress molecules have been explored as potential biomarkers to monitor the disease progression and explore their role in the RA pathogenesis. Therefore, we consider that it was relevant to review the information in the last 5 years in a systematized approach. To our knowledge, no systematic review that analyzes this set of biomarkers has been published within the last 5 years.
In our revision, the information connecting free radical biomarkers with the oxidative damage was very consistent (Figure
Oxidants/antioxidants biomarkers and oxidative damage found in joints and blood of RA patients. The literature references pertaining to the indicated phenomena are provided in the scheme. Under oxidative stress conditions, the joints and blood of patients with RA show high concentrations of free radicals, mainly ROS (gray), which induce DNA, proteins, and lipids damage through different mechanisms. The nonenzymatic antioxidant response (blue square), in general, is diminished. The enzymatic activity (including enzymatic antioxidant response) shows variability. AE: arylesterase, ANTI-CAP: total antioxidant capacity, AOPP: advanced oxidation protein products, CAT: catalase, CT: 3-chlorotyrosine, DNA sb: DNA strand breaks, F2-I: F2-isoprostane, GPx: glutathione peroxidase, GR: glutathione reductase, GSH: reduced glutathione, H2O2: hydrogen peroxide, MAA: malondialdehyde-acetaldehyde, MDA: malondialdehyde, MN: micronucleus, MPO: myeloperoxidase, NADPH ox: reduced nicotinamide adenine dinucleotide phosphate oxidase,
Other free radicals biomarkers were analyzed in the included studies. Veselinovic et al. [
The oxidative damage biomarkers (lipid, proteins, uric acid, and DNA oxidation) were also analyzed, aside from the free radicals. In these studies it was shown that the oxidative damage biomarkers are consistently and significantly higher in RA patients if compared to control individuals; this increment was observed in any sample analyzed (serum, plasma, erythrocytes, urine, synovial fluid, and whole blood). An oxidative stress environment prevails in RA, which results in the oxidation of biomolecules in this disease.
Lipid peroxidation is one of the major consequences of oxidative stress. It alters the fluidity and permeability of cell membranes and impairs the activity of membrane-bound enzymes. Lipid peroxidation leads to the production of conjugated diene hydroperoxides and unstable substances, which disintegrate into various bioactive aldehydes such as MDA, 4-hydroxynonenal (HNE), and TBARS [
Additionally, in the included studies, the anti-MAA antibodies correlated closely with ACPA [
The effect of oxidative stress on lipids was analyzed in 3 of the included studies. Kwaśny-Krochin et al. [
Another way to assess oxidative damage in RA is throughout its effect on proteins. Free radicals can modify both their structure and functions. The presence of elevated protein carbonyls in RA samples, produced either by direct oxidation of certain amino acids, by a secondary reaction with HNE, or by a glycoxidation reaction [
Stamp et al. [
Additionally, in one of the included studies, AOPP, protein carbonyls, and RSNO levels were found elevated in RA synovial fluid and correlated positively with DAS-28. Proteins carbonyls also correlated positively with ROS and •OH radical [
The use of antioxidant molecules as indirect biomarkers of oxidative stress was also included in our revision. Of the antioxidants analyzed in the included studies, the
Finally, the activity of antioxidant enzymes (SOD, CAT, GPx, and GR) was evaluated in some of the included studies [
Gathering our results, we can conclude that oxidative stress is a dynamic and complex phenomenon occurring in RA and that is involved in the disease pathogenesis in a complex fashion. Unfortunately, the actual evidence is discrepant in the role of some oxidative stress related molecules. These discrepancies complicate our understanding of the mechanism of oxidative stress implication in RA. The variability and complexity of the regulating mechanisms of oxidative stress in humans, which are associated with genetic, epigenetic, age, gender, and dietary factors, can explain these discrepancies. The results of the present revision suggest the plausibility of several oxidative stress related compounds as potential biomarkers to assess the disease activity and probably prognosis. However, the biomarkers need to be validated in prospective clinical studies. This process implicates the compliance with certain requirements which include (a) a stable product of oxidative stress, not susceptible to artificial induction or loss during storage, (b) that this product can be detectable in the target tissue or a valid surrogate tissue where it causes oxidative modification and damage, (c) that it is present in sufficient and measurable concentrations, (d) that it could be determined by an assay that is specific, sensitive, reproducible, and robust, (e) that this compound should be free of confounding factors from dietary intake, and (f) that it could be measurable within a detection limit of a reliable analytical procedure. Additionally, it is essential to consider relevant clinical factors that could lead to misinterpretation of results such as disease duration, disease activity, its treatment, and even the patient status at the moment of the sample collection are important confounding factors that could affect the interpretation of the assays.
Although our study indicates important aspects of the status of oxidative stress in RA, it is important to highlight some of its limitations. The search strategy used in this review was limited to the findings of the last five years. Also, the search was conducted in a single database using one set of keywords. Expanding the search criteria the number of articles would increase and possibly allow stronger conclusions. A meta-analysis also would be appropriate.
The authors declare that there are no competing interests regarding the publication of this paper.
The authors thank Dr. Luz Helena-Sanin and Angelica Hance for their support in this investigation.