Introduction 1 2 3 4 5 6 6 7 Against this background, an open (uncontrolled) pilot study was designed to assess if effects of antioxidants on the clinical parameters of patients with RA could be confirmed and if these effects would associate with changes in a selected number of molecular markers related to human antioxidant defence system status. Further, a mix of antioxidants was to be tested rather than a single high-dosed compound. Materials and methods The open study was approved by the ethical committee. Eight patients with rheumatoid factor + RA, as defined by the ARA revised criteria were enrolled in the study. To avoid influences of gender, only female patients participated. Inflammatory disease activity was defined as a Disease Activity Score (DAS) higher than 2.5. Inclusion criteria included non-smoking and no serious co-morbidity. Patients had to be receiving stable non-steroidal anti-inflammatory drug (NSAID) treatment and/or ‘second line’ medication for at least 3 months. Those taking antioxidant supplements were excluded from the study. During a period of 10 weeks, patients consumed 20 g of antioxidant-enriched margarine daily. The spread contained a mix of a-tocopherol (400 mg), lycopene (10 mg), palm oil carotenoids (5 mg; mainly α-carotene) and lutein (10 mg). Further, patients received vitamin C (200 mg daily) as a supplement.  t t t t t t t t t t t t Results t 1 2 Fig. 1 Clinical condition by Disease Activity Score  Fig. 2 Mean Disease Activity Score  3 Fig. 3 Plasma antioxidant status in eight patients  4 Fig. 4 Markers of antioxidant capacity and oxidative stress  Discussion This pilot study was conducted to obtain some first indications for potential beneficial effects of an antioxidant intervention on clinically relevant parameters for rheumatoid arthritis. In our present study, a significant reduction in Disease Activity Score (DAS) was observed following antioxidant intervention of 10 weeks. This was accompanied by significant increases in blood levels of antioxidants administered. The reduction of DAS is remarkable as all patients had an active (high initial mean DAS of 5.8) longstanding disease and had tried all kinds of disease-modifying anti-rheumatic drugs, including combination therapy. The unchanged level of vitamin A, not present in the spread, reinforces the significance of the increased levels of the blood antioxidant status. As NSAIDs can influence the absorption of vitamin C, this could explain the non-significant changes of the serum concentration of this antioxidant. 6 7 8 9 In our study, not only the effect of vitamin E on the inflammatory response and clinical symptoms was evaluated but also the interactions with bioavailable natural antioxidants such as carotenoids, vitamin C and others. In view of the limited number of controlled studies, the supporting evidence for beneficial effects of antioxidants on clinical characteristics of rheumatoid arthritis may be considered limited but promising. Another point is that the potential mechanism of action of antioxidants in rheumatoid arthritis needs further attention. The aspect of damage by RS and loss of critical functions can be analysed by use of ‘molecular markers’. These markers may include antioxidant status, products which arise as a consequence of oxidative damage to lipid, protein and DNA and tissue damage (as assessed by lactic dehydrogenase release) or cytokine levels (cq anti-TNFα). Although markers are very informative from a mechanistic point of view, a drawback is that none of them has been validated against clinical conditions of patients with RA. In this study, a trend for reduced levels of measures of antioxidant capacity and of ‘footprints’ of oxidative stress was observed, e.g. statistically non-significant increase in FRAP and reduction in F2-isoprostane levels. Furthermore, these markers showed a trend for correlation with clinical conditions. The data indicate that statistically significant effects may be observed when the number of patients enrolled is increased. This open pilot study therefore should be viewed as a first step in assessing potential beneficial effects of the antioxidants tested on rheumatoid arthritis. The data indicate there may be potential in using mixes of antioxidants in the treatment of RA alongside drug treatment so that drug dosage may be reduced while hopefully retaining efficacy. Further, multi-drug/antioxidant therapy could be used to reduce drug dose and thus side effects of treatment. In conclusion, our present pilot study indicated that intervention with antioxidant-enriched margarine in RA results in consistent and significant relief of clinical symptoms. Furthermore, increases in blood antioxidant status and indications for effects on oxidative stress markers were observed. These data are promising and indicate need for a double-blind, placebo-controlled randomised human trial to establish effect and demonstrate causality.