Oxidative Stress and Cardiovascular Risk in Patients With Rheumatoid Arthritis

Background and Current State of Knowledge Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease affecting approximately 1% of the global population and has a complex pathogenesis. It is characterised by synovial inflammation and cartilage destruction caused by marked cytokine release in the synovium by activated T cells and macrophages, together with an amplified B-cell response responsible for the production of autoantibodies such as rheumatoid factor and anti-citrullinated protein antibodies. Synovial fibroblasts also produce metalloproteinases that destroy cartilage and activate osteoclasts, thereby promoting bone resorption. Neutrophils are another key cell type; upon cytokine activation, they can increase joint destruction and amplify the inflammatory response through the synthesis of additional pro-inflammatory cytokines and chemokines.

These processes induce oxidative stress through the release of reactive oxygen species (ROS), including lipid peroxidation. Ferroptosis, a non-apoptotic form of cell death induced by lipid peroxidation, has recently been proposed as a key pathogenic mechanism in RA; modulation of this pathway may represent a therapeutic target.

ROS production and lipid peroxidation contribute to RA pathogenesis through multiple pathways. ROS released into synovial fluid disrupt the redox balance, counteract antioxidant defences, and may promote accelerated atherosclerosis in RA. They damage extracellular and intracellular matrix components, leading to chondrocyte injury. ROS can also oxidise low-density lipoproteins (LDL), increasing adhesion molecules, chemokines, and advanced glycation end products and thereby amplifying inflammation. Tumour necrosis factor alpha may further induce ROS generation and perpetuate oxidative stress, leading to hydroperoxides that fragment to form truncated phospholipids. These compounds may disrupt mitochondria and increase cytotoxicity through caspase activation. In addition to hydroperoxides, lipid peroxides and several aldehydes are generated, including malondialdehyde (MDA).

MDA is a terminal product of polyunsaturated fatty-acid peroxidation in cell membranes. It is considered a biomarker of oxidative stress and is used to assess ROS-related membrane lipid damage. MDA can be measured non-invasively in serum using the method described by Kikugawa et al. SOD catalyses the dismutation of superoxide into oxygen and hydrogen peroxide and is therefore an important antioxidant defence in oxygen-exposed cells. Similarly, GPX catalyses the oxidation of glutathione to glutathione disulphide using hydrogen peroxide; selenium is a cofactor. GPX protects the body endogenously against the damaging effects of hydroperoxides.

Several studies have reported higher MDA levels in patients with RA than in controls and have related increased MDA concentrations to altered sodium/potassium pump activity, a marker of membrane injury. The identification of lipid-peroxidation-mediated cell death and the role of MDA as a terminal lipid-peroxidation product further justify investigation of this biomarker. However, published series have included relatively few patients and have not assessed the relationship of MDA, SOD, and GPX with cardiovascular disease in RA.

The lipid profile in RA is clearly pro-oxidative. This pattern, together with evidence that specific cytokines can directly promote oxidative modification of LDL, suggests high concentrations of oxidised lipids in RA. During periods of high disease activity, higher concentrations of total cholesterol, triglycerides, LDL cholesterol, and lipoprotein(a), as well as lower HDL cholesterol concentrations, may be observed.

We aim to characterise MDA, a terminal product of lipid peroxidation, and its regulatory factors, SOD and GPX, in a large series of patients with RA. We will explore whether serum concentrations of these substances are related to subclinical atherosclerosis, including carotid intima-media thickness, carotid plaque, and carotid stiffness, as well as inflammatory dyslipidaemia and indices of insulin resistance.

Specific Objectives and Study Hypotheses Main Hypothesis Oxidative stress is independently associated with atherosclerotic burden and carotid stiffness in patients with RA. Oxidative stress can be estimated through measurement of serum MDA levels and SOD and GPX activity.

Secondary Hypothesis Oxidative stress is associated with other characteristics related to subclinical atherosclerosis in RA, including an abnormal lipid profile, reduced cholesterol efflux, insulin resistance, and beta-cell dysfunction.

Overall Objective To determine whether serum MDA levels and SOD and GPX activity are independently associated with carotid atherosclerotic burden and carotid endothelial dysfunction in patients with RA.

Specific Objectives To determine whether serum MDA levels and SOD and GPX activity are independently associated with carotid atherosclerotic burden or endothelial dysfunction, as assessed by carotid stiffness, in patients with RA.

To investigate whether serum MDA levels and SOD and GPX activity are associated with disease characteristics that are, in turn, related to subclinical atherosclerosis and cardiovascular disease, including an abnormal lipid profile, cholesterol efflux, insulin resistance, and pancreatic beta-cell function.

Methods Study Design This is a 24-month multicentre cross-sectional study in patients with RA, with one funding centre. It will measure serum MDA concentrations and plasma SOD and GPX activity.

Outcomes and Variables The primary outcome is lipid peroxidation, assessed by serum MDA concentrations and antioxidant enzyme activity (SOD and GPX).

Explanatory variables include disease activity assessed using the 28-joint Disease Activity Score (DAS28), Clinical Disease Activity Index (CDAI), Simple Disease Activity Index (SDAI), and Health Assessment Questionnaire (HAQ). Acute-phase reactants will include erythrocyte sedimentation rate and C-reactive protein. Rheumatoid factor, anti-citrullinated peptide antibodies, prednisone use (continuous and binary variables), anti-TNF therapy or other biologic therapies, and radiographic damage will be recorded. Radiographic damage will be assessed as the presence or absence of hand and foot erosions and continuously using the van der Heijde-modified Sharp score.

A complete lipid profile will be determined, including total cholesterol, HDL cholesterol, LDL cholesterol, apolipoproteins A1, B, and C-III, and lipoprotein(a). Glucose, insulin, and C-peptide levels will be measured, and indices of insulin resistance, including HOMA2, will be calculated. Carotid intima-media thickness, carotid plaque, and carotid stiffness will be assessed.

Potential confounders include sex, age, body mass index, blood pressure, antihypertensive treatment, fasting glucose, smoking, and statin use. Demographic data, surgical history, smoking status, vascular comorbidity, and current treatment will be collected. Weight and height will be measured to calculate body mass index (weight divided by height squared, kg/m²), and waist-to-hip ratio will be determined. Metabolic syndrome will be defined according to the 2005 National Cholesterol Education Program Adult Treatment Panel III criteria. Cardiovascular risk will be assessed using SCORE2 for the Spanish population and categorised as very high, high, or moderate/low.

Setting The reference population comprises patients with RA fulfilling the 2010 American College of Rheumatology classification criteria who are treated at Hospital Universitario de Canarias, Santa Cruz de Tenerife, and Hospital Universitario Doctor Negrín, Las Palmas de Gran Canaria, Spain, including patients attending hospital outpatient clinics and referral-area services.

Participant Selection Inclusion Criteria Men or women who are not pregnant or breastfeeding.

Age 18 years or older and younger than 70 years.

Treatment with any disease-modifying antirheumatic drug, including biologic therapies.

For patients receiving oral corticosteroids, a prednisone dose of 10 mg or less that has remained stable for at least one month before study inclusion.

Ability and willingness to provide written informed consent.

Exclusion criteria

Autoimmune rheumatic disease other than RA, including systemic lupus erythematosus, mixed connective tissue disease, systemic sclerosis, or polymyositis. Sjögren syndrome associated with RA will not be an exclusion criterion.

Functional class IV RA with complete or substantial disability, including confinement to bed or wheelchair that prevents personal self-care.

History or current presence of inflammatory joint disease other than RA, such as gout, reactive arthritis, psoriatic arthritis, seronegative spondyloarthropathy, or Lyme disease.

Estimated glomerular filtration rate below 60 mL/min/1.73 m² or active renal disease.

Pregnancy or breastfeeding.

Evidence of severe uncontrolled concomitant cardiovascular, neurological, pulmonary (including obstructive lung disease), renal, hepatic, endocrine (including diabetes mellitus), or gastrointestinal disease, or any condition considered by the investigator likely to alter the lipid profile.

Body weight greater than 150 kg.

History of alcoholism, drug abuse, or substance misuse within six months before the screening visit.

Statin use will not be an exclusion criterion.

Sample Size In a previous study conducted by our group in 28 healthy individuals, MDA levels were estimated at 1.53 ± 0.34. Previous RA studies from our group found an approximately 30% prevalence of carotid plaque. Assuming adjustment for 10 variables, 80% power to detect differences under the null-hypothesis test, a two-sided 5% significance level, and an estimated odds ratio of 1.5, 156 patients with RA will be required.

Recruitment Patients will be recruited consecutively as they attend the participating clinics.

Data Collection At the baseline study visit, participant data will be recorded in a paper case-report form (CRF). CRF entries will be verifiable against source documents, including medical records and laboratory data. Measured substance concentrations and standard curves from each experiment will be attached to each participant's CRF. Data will be entered into an electronic database to facilitate statistical management. Fasting blood samples will be obtained from the antecubital vein. MDA, SOD, and GPX measurement procedures are described below.

Statistical Analysis The associations between MDA and the described cardiovascular comorbidities will be analysed using multivariable logistic and linear regression models, adjusted for potential confounders. Analyses will be performed using Stata version 17. Statistical significance will be defined as a two-sided p value below 0.05.

Ethical Considerations The study will be conducted in accordance with the ethical principles for medical research involving human participants set out in the Declaration of Helsinki, Good Clinical Practice standards, Spanish Biomedical Research Law 14/2007, Royal Decree 1716/2011 on biobanks, Organic Law 3/2018, and Regulation (EU) 2016/679 on data protection. The project will be submitted to the Research Ethics Committees of Hospital Universitario de Canarias and Hospital Universitario Doctor Negrín for review and favourable opinion before any study-related procedure is initiated.

Study Limitations Other lipid-peroxidation products and antioxidant mechanisms contribute to redox homeostasis. Therefore, relevant components of these complex metabolic pathways lie beyond the scope of the present clinical approach. Nevertheless, MDA is a terminal product of lipid peroxidation and a biomarker that provides an estimate of pro-oxidant activity in RA and is involved in highly relevant processes, including cell damage.

Work Plan Timeline Study start: after ethics approval and availability of funding.

Recruitment period for the cross-sectional study: 18 months.

First participant included: January 2024.

Last participant included: May 2025.

Data analysis: second half of 2025.

Study report and manuscript submission: before December 2026.

Distribution of Responsibilities Resources required for molecule quantification and sample management are available at the Department of Internal Medicine, the Rheumatology Department laboratory at Hospital Universitario de Canarias, and the hospital's multidisciplinary day-care unit.

Iván Ferraz Amaro, Candelaria Martín González, and Juan Carlos Quevedo Abeledo: participant information and informed-consent procedures; completion of CRFs.

Iván Ferraz Amaro: interpretation of results, statistical analysis, and dissemination/publication of findings.

Antonia de Vera González, Clinical Biochemist, Central Laboratory: biochemical determinations, enzyme-activity studies, and ELISA techniques, performed in the Central Laboratory and the laboratory adjacent to the clinical research unit, which is equipped with ELISA readers and a cell-culture incubator.

Pedro Abreu González: measurement of serum MDA and plasma SOD and GPX activity.

The study will be conducted in the Rheumatology Departments of Hospital Universitario de Canarias and Hospital Universitario Doctor Negrín. Analytical determinations will be performed in the departmental laboratory and Central Laboratory using the resources funded through this project.

Suitability of the Principal Investigator and Research Group Experience in Lipid Metabolism and Chronic Inflammation Dr Iván Ferraz has published several studies on cardiovascular disease and lipid metabolism in RA, demonstrating the group's capacity to undertake and publish this type of research in high-impact specialty journals. Dr Ferraz has received three grants in competitively funded AES-ISCIII project calls, with the third project ending in December 2023. This demonstrates his ability to manage competitive research funding and to produce high-impact scientific publications.

Dr Candelaria Martín has previous experience in MDA analysis in patients with harmful alcohol consumption. In a study of 62 patients with severe acute alcoholic hepatitis, MDA and several cytokines (IL-6, IL-8, IL-4, TNF-alpha, and interferon-gamma) were measured at admission and one week later; increased MDA was associated with higher mortality. In another study of 184 patients with harmful alcohol consumption, alpha-klotho, a hormone with anti-inflammatory and antioxidant properties, was directly correlated with MDA, suggesting that alpha-klotho levels increase in response to oxidative stress.

Dr Juan Carlos Quevedo, rheumatologist at Hospital Doctor Negrín, has collaborated for several years with the principal investigator. The team's recruitment capacity and coordinated work are supported by multiple previous publications on cardiovascular disease in immune-mediated inflammatory diseases.

Dr Antonia de Vera has extensive experience in laboratory techniques, and Dr Pedro Abreu has focused his research for several years on oxidative-stress-related topics.

Experience in Sample Collection and Analysis The participating services have an adjacent laboratory for clinical trials and basic research, staffed by personnel trained in sample processing and analysis, including ELISA procedures. Dr Antonia de Vera has previously participated in clinical trials and group studies related to dyslipidaemia and is experienced in ELISA, immunohistochemistry, flow cytometry, real-time PCR, and cell culture. Dr Pedro Abreu is also experienced in measuring oxidative-stress products and has access to the techniques described in this protocol.

Overall Team Capacity During 2022, the Rheumatology Department at Hospital Universitario de Canarias conducted approximately 12,000 outpatient consultations and 4,500 consultations in primary-care centres in its catchment area. Therefore, the service cares for a substantial number of patients with inflammatory joint disease who may be eligible for this study.

Dr Iván Ferraz holds a Master's degree in Biostatistics, and Dr Candelaria Martín has completed a Master's degree in Clinical Research Methodology. This enables the clinical investigators themselves to perform the study's statistical analysis, facilitating efficient generation and interpretation of results.

Expected Applicability and Practical Value Cardiovascular disease is an important cause of morbidity and mortality in patients with RA. RA affects approximately 1% of the population, and myocardial infarction and cerebrovascular disease are among the most common cardiovascular events in this population. Cardiovascular risk in RA is estimated to be 1.5- to 3-fold higher than in the general population. Given the high prevalence of RA and the frequent cardiovascular involvement in these patients, research on endothelial damage and its relationship with chronic inflammation is warranted.

This project may generate new hypotheses concerning the complex relationships among inflammation, oxidative stress, lipids, and cardiovascular risk. The inclusion of a clinical biochemist and a chemistry researcher with laboratory expertise promotes synergy between clinical and laboratory research.

Finally, this project aligns with the priority research areas of the FIISC call, including hospital care and public health aimed at improving the health and well-being of the Canary Islands population and supporting the sustainability of the Canary Islands Health Service through the generation of scientific knowledge in a highly prevalent disease such as RA.

Study Sites, Facilities, Equipment, and Available Techniques The study will be conducted at the Rheumatology Laboratory of Hospital Universitario de Canarias, the laboratories of the Department of Pharmacology at the Faculty of Medicine of the University of La Laguna, and the multidisciplinary day-care unit of Hospital Universitario de Canarias, where patients will be recruited and followed. Laboratory procedures, including enzyme-linked immunosorbent assays (ELISA), DuoSet kits, enzymatic-activity measurements, and cholesterol-efflux assays, will be performed by Dr Antonia de Vera and Dr Pedro Abreu at the Hospital Universitario de Canarias and University of La Laguna facilities under the supervision of the principal investigator.

Available equipment includes:

ELISA reader, Rheumatology Department, Hospital Universitario de Canarias.

Fluorimeter, Rheumatology Department, Hospital Universitario de Canarias.

Inverted microscope, Department of Pharmacology, University of La Laguna.

Laminar-flow cabinet, Department of Pharmacology, University of La Laguna.

CO₂ incubator, Department of Pharmacology, University of La Laguna.

Liquid-nitrogen tank, Department of Pharmacology, University of La Laguna.

Refrigerated centrifuge, Department of Pharmacology, University of La Laguna.

Refrigerated microcentrifuge, Rheumatology Department, Hospital Universitario de Canarias.

Refrigerators and -80 °C freezer, Rheumatology Department, Hospital Universitario de Canarias.

Portable ultrasound system with software for carotid intima-media thickness measurement (Esaote RF-QIMT) and carotid stiffness measurement (Esaote QAS), Rheumatology Departments at Hospital Universitario de Canarias and Hospital Universitario Doctor Negrín.

Plasticware, culture media, buffers, and other consumables, Rheumatology Department, Hospital Universitario de Canarias.

Laboratory Procedures Measurement of Malondialdehyde The thiobarbituric acid-reactive substances (TBARS) assay will be used to detect lipid oxidation. This assay measures MDA, one of several terminal products generated through degradation of lipid-peroxidation products. Serum MDA levels will be measured according to the method described by Kikugawa et al., with modifications. A 0.2-mL sample will be added to 0.2 mL of 0.2 M H₃PO₄, and the colour reaction will be initiated by adding 25 μL of 0.11 M thiobarbituric acid (TBA). Samples will be heated at 90 °C for 50 minutes. After cooling, the pink TBARS complex will be extracted with 0.4 mL n-butanol. The butanol phase will be separated by centrifugation at 6,000 × g for 10 minutes. Samples will be placed in a 96-well plate and read at 535 nm using a microplate spectrophotometer (SpectraMAX-190; Molecular Devices, Sunnyvale, CA, USA). The calibration curve will be prepared using authentic MDA standards.

The assay detection limit is 0.079 μmol/L. The estimated intra-assay and inter-assay coefficients of variation are 1.82% and 4.01%, respectively. Serum MDA concentrations will be expressed in micromoles per litre. To avoid interference from compounds that react with or absorb at 532 nm, each sample will be tested in a blank tube without TBA, and the absorbance of the blank will be subtracted from that of the corresponding sample. Use of butanol as an extraction agent for the TBARS complex also reduces many of these interferences.

Measurement of Superoxide Dismutase and Glutathione Peroxidase The Cu/Zn SOD assay kit uses the auto-oxidation of 5,6,6a,11b-tetrahydro-3,9,19-trihydroxybenzofluorene, a chromophore with maximum absorbance at 525 nm (Calbiochem, San Diego, CA, USA). The GPX assay measures enzyme activity indirectly by monitoring oxidation of NADPH to NADP and absorbance at 340 nm (Calbiochem, San Diego, CA, USA).

Inclusion criteria

• Men or women who are not pregnant or breastfeeding.
• Age 18 years or older and younger than 70 years.
• Treatment with any disease-modifying antirheumatic drug, including biologic therapies.
• For patients receiving oral corticosteroids, a prednisone dose of 10 mg or less that has remained stable for at least one month before study inclusion.
• Ability and willingness to provide written informed consent.

Exclusion criteria

• Autoimmune rheumatic disease other than RA, including systemic lupus erythematosus, mixed connective tissue disease, systemic sclerosis, or polymyositis. Sjögren syndrome associated with RA will not be an exclusion criterion.
• Functional class IV RA with complete or substantial disability, including confinement to bed or wheelchair that prevents personal self-care.
• History or current presence of inflammatory joint disease other than RA, such as gout, reactive arthritis, psoriatic arthritis, seronegative spondyloarthropathy, or Lyme disease.
• Estimated glomerular filtration rate below 60 mL/min/1.73 m² or active renal disease.
• Pregnancy or breastfeeding.
• Evidence of severe uncontrolled concomitant cardiovascular, neurological, pulmonary (including obstructive lung disease), renal, hepatic, endocrine (including diabetes mellitus), or gastrointestinal disease, or any condition considered by the investigator likely to alter the lipid profile.
• Body weight greater than 150 kg.
• History of alcoholism, drug abuse, or substance misuse within six months before the screening visit.
• Statin use will not be an exclusion criterion.