Rheumatoid arthritis (RA) is an autoimmune disease characterised by synovial proliferation in joints as well as infiltration of the synovial stroma by B cells, CD4+ helper T cells, plasma cells, and macrophages [¹]. The inflammation in RA results in an overproduction of PGE2, cytokines, and nitrogen monoxide. Tumor necrosis factor (TNF) α and interleukin (IL)-1 are key pro-inflammatory molecules implicated in the cytokine cascade in RA [²]. The beneficial effects of n-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) found in fish and fish oils on clinical parameters of RA were demonstrated in at least 17 of 18 randomized controlled clinical trials [³]. Eico-sapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3), as well as n-6 PUFA, such as arachidonic acid (AA, C20:4n-6) are present in cell phoshpolipids. EPA is a precursor of prostaglandins (PGs; series-3) and leukotrienes (LTs; series-5). Supplementation with n-3 LC-PUFA results in a replacement of AA in cell membranes [⁴,⁵]. Additionally, n-3 LC-PUFA regulate inflammatory and immunological processes by competing with the enzymatic conversion to eicosanoids thereby diminishing the formation of inflammatory series-2 PGs and series-4 LTs from AA. In fact, n-3 LC-PUFA are involved in many physiological processes through their influence on membrane fluidity, eicosanoid synthesis, receptor affinity, cell signalling, gene expression, cytokine production, and the production of pro-resolving mediators [³].

γ-linolenic acid (C18:2n-6, GLA) derived from plant seed oils, e.g., borage, black current seed, and primrose oil can be metabolized to dihomo-γ-linolenic acid (C20:3n-6; DGLA), the immediate precursor of series-1 PGs. Anti-inflammatory and immunoregulatory effects of these eicosanoids have also been proposed. Several clinical trials have shown that large doses of GLA (between 1400 - 2800 mg/d) may also exhibit anti-inflammatory properties and reduce disease activity in patients with RA [^6-13].

In the present trial, patients with RA or psoriatic arthritis (PA) received capsules containing either n-3 LC-PUFA, GLA, or olive oil as a control. A synergistic effect due to the combination of n-3 LC-PUFA and GLA was also regarded in the present study. The primary outcome measure of this study was the relative variation of the eicosanoid precursor PUFA (EPA, AA, AA/EPA ratio, DGLA) in erythrocyte membranes (EM) indicative of change in the production of eicosanoids. The secondary outcome measure was the disease activity (disease activity score DAS28, visual analog scale VAS).

We expect an increased incorporation of the supplemented PUFA and hypothesized that the resulting changes on the amount of eicosanoid precursor fatty acids influence disease activity.

Fifty four patients with RA (49 f, 5 m), and six patients with psoriatic arthritis (4 f, 2 m) started the trial (intent to treat, ITT), full data after three-month of intervention could be retrieved for 47 (per protocol, PP). For these patients mean age was 56.2 ± 13.6 years (mean BMI 26.4 ± 6.1 kg/m²). Thirteen patients, representing a drop out rate of 22% did not complete the study because of the following reasons: they claimed to experience smell and odor, felt an insufficient relief of their symptoms, or showed other reasons of non-compliance (Figure 2).

Red blood cell membranes are biomarkers of medium-term FA intake as they reflect the intake ac-cumulated over the lifespan of erythrocytes (approx. 120d). Fatty acids, found in plasma lipids (e.g., CE) reflect food consumption over the last few days [^21-23]. In the present study, concentrations of supplemented PUFA increased in the analysed tissues (PL, CE, EM) in proportion to the supplemented dosages (Table 2, 3, 4), indicating a good compliance and a high bioavailability of the supplemented PUFA. An assessment of the resulting changes in PL, CE, and EM in response to the supplementation (group 1-4) indicated that PL and EM are reliable biomarkers for PUFA intake (Table 2, 4). The alterations in CE cannot be clearly interpreted due to the comparably high standard deviations within the groups (Table 3), possibly resulting from the methods used for sample preparation including a double TLC purification.

The consumption of 3 g n-3 LC-PUFA resulted in a high increase in EPA and DHA proportions, in turn, leading to a decrease of AA concentrations in EM as well as the AA/EPA ratio in PL, CE, and EM (Table 2, 3, 4). In RA, various pro-inflammatory metabolites of AA, such as LTB4 and PGE2 contribute to tissue destruction and pain [¹]. The AA concentration of the membranes is an important regulatory step in the synthesis of these prostanoids and LTs. In effect, the AA/EPA ratio in cell lipids determines the degree of competitive eicosanoid inhibition. The observed outcome on the ratio of these eicosanoid precursory FAs in EM indicated for a reduction in the production of PGs and LTs from AA. Additionally, the increase of EPA and DHA could promote the production of proresolving mediators like resolvins, protectins, and maresins. Following from this, a significant reduction of disease activity parameters (DAS28, VAS scale) was observed in this group. Previous intervention trials with fish oil in patients with RA have shown a decrease in LTB4 as the most prominent feature of lipoxygenase inhibition due to n-3 LC-PUFA [^24-26]. Recent RCT's as well as the results of the present trial suggest that daily fish oil supplementation at dosages of around 3 g n-3 LC-PUFA for approximately 12 wks are sufficient to effectuate a clinical benefit in patients with RA [^25-35]. There is also increased evidence that the restriction of AA intake enhanced the anti-inflammatory effects of n-3 LC-PUFA intervention in RA patients [⁴,²⁴,³⁴,³⁶].

Additionally, the AA/EPA ratio and the n-3 FA index which represent highly discriminative risk factors for sudden cardiac death [¹⁷,³⁷] were significantly improved due to consumption of 3 g n-3 LC-PUFA (group 1; Table 4). This is an added benefit because the CV mortality is strongly elevated in patients with RA [³⁸,³⁹].

The administration of GLA leads to a dose-dependendent increase of GLA and of its elongation metabolite DGLA in EM (Table 4). The disease activity parameters were also diminished in this group. The increase of DGLA concentration in the membrane indicates for blocking the metabolism of AA and changing the balance of lipid mediators towards the production of less potent 1-series PGs via the cyclooxygenase (COX) pathway and via the 15-lipoxygenase (15-LOX) pathway to 15(S)-hydroxyeicosatrienoic acid (15-HETrE; [⁴⁰]). Because DGLA competes with AA for COX and 5-LOX, the production of 2-series PGs and 4-series LTs from AA is reduced [⁴¹]. PGE1 has a negative feedback role in chronic inflammation, initially aiding in the development of the cardinal signs of inflammation but later suppressing inflammation. PGE1 suppressed the immune response by increasing intracellular cyclic AMP (cAMP) in polymorphonuclear leukocytes resulting in a reduction in the release of lysosomal enzymes, polymorphonuclear leukocyte chemotaxis, adherence of leukocytes in the blood vessels, and PGE1 regulates IL-6 signal transduction [^42-45].

Eskimos living on a traditional diet rarely suffer from coronary heart disease (CHD), psoriasis, asthma, and RA. It is hypothesized that not only the high dietary intake of EPA, but also the comparably low plasma concentrations of AA as well as the high plasma concentrations of DGLA are responsible for the low prevalence of these diseases [⁴⁶]. The combination of n-3 LC-PUFA and GLA tested in the present study (group 3) had no significant effect on disease activity parameters (DAS28, VAS scale; [¹⁸,¹⁹,⁴⁷]). However, because of the high initial AA concentrations due to the Western diet, it is almost impossible for us to reproduce the very low Eskimo AA concentra-tions which result from a lifetime of genetically reduced AA production.

GLA is a potential precursor of AA via its metabolism by elongase along with d5-desaturase activities [⁴⁸]. Accordingly, a marked increase in the quantity of AA in PL and CE due to supplementation of 3 g GLA/d was demonstrated in the present trial (group 2; Table 2, 3), indicating for the potential of dietary GLA to be elongated to DGLA and subsequently desaturated to AA. A similar rise was not seen in EM which is indicative of a temporary accumulation of AA (Table 4). Interestingly, there was no increase of AA in PL, CE and EM for supplementation with combined n-3-LC-PUFA + GLA/d (group 3; Table 2, 3, 4). Concluding from this, the addition of EPA due to GLA intervention attenuates the conversion of DGLA to AA by blocking d5-desaturase activity. Further, comparison of variations in DGLA concentrations in groups 2 and 3 showed a strong dose-dependent increase of DGLA in CE and EM (Table 3, 4).

Olive oil has classically been used as a placebo in studies investigating the effects of fish oil in pa-tients with RA. In the present trial, ingesting of 3 g olive oil revealed a trend towards a decrease of DAS28 [⁴⁷]. A low improvement of disease activity was also observed in some earlier studies [²⁵,³⁴,⁴⁹] because olive oil contains minor components with antiinflammatory potential, e.g., squalene, plant sterols, tocopherols, and polyphenols [^50-52]. These data suggest that olive oil itself could have beneficial effects in RA/PA.

The concentrations of the supplemented PUFA increased in the analysed tissues (PL, CE, EM) in proportion to the supplemented dosages indicating a good compliance as well as a high bioavailability of the supplemented PUFA. The intake of capsules enriched with n-3 LC-PUFA or GLA (3 g/d) resulted in an increased incorporation of the eicosanoid precursor FAs (EPA, DHA, DGLA) in plasma lipids and cell membranes as well as a subsequent improvement in clinical status of patients with chronic inflammatory diseases. These changes on FA distribution are indicative for a reduction in the production of inflammatory eicosanoids from AA. In addition, the intake of n-3 LC-PUFA improved cardiovascular risk factors, e.g., AA/EPA ratio and n-3 FA index.

AA: arachidonic acid; ACR: American College of Rheumatology; ALA: α-linolenic acid; cAMP: cyclic AMP; CE cholesteryl esters, CHD: coronary heart disease; CLA: conjugated fatty acids; COX: cyclooxygenase; CRP: C-reacitve protein, CV: cardiovascular; DAS28: disease activity scor; DGLA: dihomo-γ-linolenic acid, DHA: docosahexaenoic acid, DMARD: disease-modifying antirheumatic drugs; DPA: docosapentaenoic acid, EM: erythrocyte membranes, EMS: duration of early morning stiffness; EPA: eicosapentaenoic acid; ESR: erythrocyte sedimentation rate; ESSG: European Spondyloarthropathy Study Group; FA: fatty acid; FAME: fatty acid methyl esters; FFP: Food Frequency Protocol; GLA: γ-linolenic acid; HETrE: hydroxyeicosatrienoic acid; ICAM: intercellular adhesion molecule; IL: interleukin, ITT: intent to treat; LA: linoleic acid, LOX: lipoxygenase; LTs: leukotrienes; MUFA: monounsaturated fatty acids; n-3 LC-PUFA: n-3 long-chain polyunsaturated fatty acids; NSAID: nonsteroidal anti-inflammatory drugs; PA: psoriatic arthritis; PGs: prostaglandins; PI3K: phosphoinositide 3-kinase; PL: plasma lipids; PP: per protocol; PUFA: polyunsaturated fatty acids; RA: rheumatoid arthritis; SFA: saturated fatty acids; TLC: thin layer chromatography; TNF: Tumor necrosis factor, VAS visual analog scale.

Each author has made substantial contributions to the conception and design of the study, or aquisition of data, or analysis and interpretation of data, drafting the article or revising it critically for important intellectual content. Each author has seen and approved the contents of the submitted manuscript. There are no competing interests because none of the authors had any personal (political, religious, ideological, academic, intellectual, or commercial) or financial conflicts of interest.

CD, MS, and GJ were involved in designing the study. RS, UH, and MV were responsible for the clinical investigations and medical care of the patients. CD was accountable for the analytical and statistical analysis of the fatty acids, data interpretation, and production of manuscript. CD and GJ were responsible for the critical revision of the manuscript. MS was in charge of obtaining funding. GJ supervised this work. All authors read and approved the final manuscript.

Special thanks for support founding go to the Future Agency Brandenburg http://www.zab.eu/en/18.aspx, to the Non-Profit Organisation for Food and Environmental Research (ILU Institut für Lebensmittel- und Umweltforschung e.V. Nuthetal, http://www.ilu-ev.de/) and Goerlich pharma international, Edling http://www.goerlich-pharma.com/, for founding and allocating the capsules. The funders had no role in study design, data collection and analysis and interpretation of data, writing the manuscript, decision to publish, or preparation of the manuscript.

RS, UH, and MV obtained funding from the Future Agency Brandenburg, the Non-Profit Organisation for Food and Environmental Research (ILU Institut für Lebensmittel- und Umweltforschung e.V. Nuthetal) and Goerlich pharma international, Edling. CD and GJ obtained funding from the Non-Profit Organisation for Food and Environmental Research (ILU Institut für Lebensmittel- und Umweltforschung e.V. Nuthetal).

Previous exhibition

37. Kongress der Deutschen Gesellschaft für Rheumatologie e.V., Köln, Germany, 23.-26. September 2009

7th Euro Fed Lipid Congress "Lipids, Fats and Oils: From Knowledge to Application", Graz, Aus-tria, 18.-21. October 2009

5th International Congress on Complementary Medicine Research, Tromsø, Norwegen, 2010