Leslie G. Cleland

Dietary polyunsaturated fatty acids and inflammatory mediator production

Many antiinflammatory pharmaceutical products inhibit the production of certain eicosanoids and cytokines and it is here that possibilities exist for therapies that incorporate n-3 and n-9 dietary fatty acids. The proinflammatory eicosanoids prostaglandin E(2) (PGE(2)) and leukotriene B(4) (LTB(4)) are derived from the n-6 fatty acid arachidonic acid (AA), which is maintained at high cellular concentrations by the high n-6 and low n-3 polyunsaturated fatty acid content of the modern Western diet. Flaxseed oil contains the 18-carbon n-3 fatty acid alpha-linolenic acid, which can be converted after ingestion to the 20-carbon n-3 fatty acid eicosapentaenoic acid (EPA). Fish oils contain both 20- and 22-carbon n-3 fatty acids, EPA and docosahexaenoic acid. EPA can act as a competitive inhibitor of AA conversion to PGE(2) and LTB(4), and decreased synthesis of one or both of these eicosanoids has been observed after inclusion of flaxseed oil or fish oil in the diet. Analogous to the effect of n-3 fatty acids, inclusion of the 20-carbon n-9 fatty acid eicosatrienoic acid in the diet also results in decreased synthesis of LTB(4). Regarding the proinflammatory ctyokines, tumor necrosis factor alpha and interleukin 1beta, studies of healthy volunteers and rheumatoid arthritis patients have shown < or = 90% inhibition of cytokine production after dietary supplementation with fish oil. Use of flaxseed oil in domestic food preparation also reduced production of these cytokines. Novel antiinflammatory therapies can be developed that take advantage of positive interactions between the dietary fats and existing or newly developed pharmaceutical products.

Dietary n-3 fatty acids and therapy for rheumatoid arthritis

OBJECTIVE To examine the potential for dietary n-3 fats to be component of therapy for rheumatoid arthritis (RA). METHODS Studies of encapsulated fish oil use in RA were reviewed and critiqued, and possible biochemical mechanisms for fish oil effects were examined. The potential for use of n-3 fats was evaluated within a dietary framework rather than a quasi-pharmaceutical framework. RESULTS There is consistent evidence from double-blind, placebo-controlled clinical trials that dietary n-3 fats, supplied as fish oil, can have beneficial effects in RA. The beneficial effects appear modest, but their size and extent may have been moderated by common trial design factors such as high n-6 polyunsaturated fat diets and concurrent antiinflammatory drug use. Mechanisms for the clinical effects of n-3 fats in RA may involve their ability to suppress production of inflammatory mediators, including n-6 eicosanoids and proinflammatory cytokines. Suppression of n-6 eicosanoid and cytokine production will be possible using foodstuffs that are rich in n-3 fats and poor in n-6 fats. CONCLUSIONS There are many overlapping biochemical effects of n-3 fatty acids and antiinflammatory pharmaceuticals that could explain the clinical actions of n-3 fats in RA. They suggest that there is the potential for complementarity between drug therapy and dietary choices that increase intake of n-3 fats and decrease intake of n-6 fats. In particular, there is the potential for drug-sparing effects. Future studies with n-3 fats in RA need to address the fat composition of the background diet and the issue of concurrent drug use.

Roles of Cyclooxygenase (COX)-1 and COX-2 in Prostanoid Production by Human Endothelial Cells: Selective Up-Regulation of Prostacyclin Synthesis by COX-2

The two cyclooxygenase (COX) isoforms, COX-1 and COX-2, both metabolize arachidonic acid to PGH2, the common substrate for thromboxane A2 (TXA2), prostacyclin (PGI2), and PGE2 synthesis. We characterized the synthesis of these prostanoids in HUVECs in relation to COX-1 and COX-2 activity. Untreated HUVEC expressed only COX-1, whereas addition of IL-1β caused induction of COX-2. TXA2 was the predominant COX-1-derived product, and TXA2 synthesis changed little with up-regulation of COX-2 by IL-1β (2-fold increase). By contrast, COX-2 up-regulation was associated with large increases in the synthesis of PGI2 and PGE2 (54- and 84-fold increases, respectively). Addition of the selective COX-2 inhibitor, NS-398, almost completely abolished PGI2 and PGE2 synthesis, but had little effect on TXA2 synthesis. The up-regulation of COX-2 by IL-1β was accompanied by specific up-regulation of PGI synthase and PGE synthase, but not TX synthase. An examination of the substrate concentration dependencies showed that the pathway of TXA2 synthesis was saturated at a 20-fold lower arachidonic acid concentration than that for PGI2 and PGE2 synthesis. In conclusion, endothelial prostanoid synthesis appears to be differentially regulated by the induction of COX-2. The apparent PGI2 and PGE2 linkage with COX-2 activity may be explained by a temporal increase in total COX activity, together with selective up-regulation of PGI synthase and PGE synthase, and different kinetic characteristics of the terminal synthases. These findings have particular importance with regard to the potential for cardiovascular consequences of COX-2 inhibition.

The Role of Fish Oils in the Treatment of Rheumatoid Arthritis

Fish oils are a rich source of omega-3 long chain polyunsaturated fatty acids (n-3 LC PUFA). The specific fatty acids, eicosapentaenoic acid and docosahexaenoic acid, are homologues of the n-6 fatty acid, arachidonic acid (AA). This chemistry provides for antagonism by n-3 LC PUFA of AA metabolism to pro-inflammatory and pro-thrombotic n-6 eicosanoids, as well as production of less active n-3 eicosanoids. In addition, n-3 LC PUFA can suppress production of pro-inflammatory cytokines and cartilage degradative enzymes.In accordance with the biochemical effects, beneficial anti-inflammatory effects of dietary fish oils have been demonstrated in randomised, double-blind, placebo-controlled trials in rheumatoid arthritis (RA). Also, fish oils have protective clinical effects in occlusive cardiovascular disease, for which patients with RA are at increased risk.Implementation of the clinical use of anti-inflammatory fish oil doses has been poor. Since fish oils do not provide industry with the opportunities for substantial profit associated with patented prescription items, they have not received the marketing inputs that underpin the adoption of usual pharmacotherapies. Accordingly, many prescribers remain ignorant of their biochemistry, therapeutic effects, formulations, principles of application and complementary dietary modifications. Evidence is presented that increased uptake of this approach can be achieved using bulk fish oils. This approach has been used with good compliance in RA patients. In addition, an index of n-3 nutrition can be used to provide helpful feedback messages to patients and to monitor the attainment of target levels.Collectively, these issues highlight the challenges in advancing the use of fish oil amid the complexities of modern management of RA, with its emphasis on combination chemotherapy applied early.

Differential Regulation of Prostaglandin E2 and Thromboxane A2 Production in Human Monocytes: Implications for the Use of Cyclooxygenase Inhibitors

There is an autocrine relationship between eicosanoid and cytokine synthesis, with the ratio of prostaglandin E2 (PGE2)/thromboxane A2 (TXA2) being one of the determinants of the level of cytokine synthesis. In monocytes, cyclooxygenase type 1 (COX-1) activity appears to favor TXA2 production and COX-2 activity appears to favor PGE2 production. This has led to speculation regarding possible linkage of COX isozymes with PGE and TXA synthase. We have studied the kinetics of PGE2 and TXA2 synthesis under conditions that rely on COX-1 or -2 activity. With small amounts of endogenously generated prostaglandin H2 (PGH2), TXA2 synthesis was greater than PGE2. With greater amounts of endogenously generated PGH2, PGE2 synthesis was greater than TXA2. Also, TXA synthase was saturated at lower substrate concentrations than PGE synthase. This pattern was observed irrespective of whether PGH2 was produced by COX-1 or COX-2 or whether it was added directly. Furthermore, the inhibition of eicosanoid production by the action of nonsteroidal anti-inflammatory drugs or by the prevention of COX-2 induction with the p38 mitogen-activated protein kinase inhibitor SKF86002 was greater for PGE2 than for TXA2. It is proposed that different kinetics of PGE synthase and TXA synthase account for the patterns of production of these eicosanoids in monocytes under a variety of experimental conditions. These properties provide an alternative explanation to notional linkage or compartmentalization of COX-1 or -2 with the respective terminal synthases and that therapeutically induced changes in eicosanoid ratios toward predominance of TXA2 may have unwanted effects in long-term anti-inflammatory and anti-arthritic therapy.

Elongase Reactions as Control Points in Long-Chain Polyunsaturated Fatty Acid Synthesis

Background Δ6-Desaturase (Fads2) is widely regarded as rate-limiting in the conversion of dietary α-linolenic acid (18:3n-3; ALA) to the long-chain omega-3 polyunsaturated fatty acid docosahexaenoic acid (22:6n-3; DHA). However, increasing dietary ALA or the direct Fads2 product, stearidonic acid (18:4n-3; SDA), increases tissue levels of eicosapentaenoic acid (20:5n-3; EPA) and docosapentaenoic acid (22:5n-3; DPA), but not DHA. These observations suggest that one or more control points must exist beyond ALA metabolism by Fads2. One possible control point is a second reaction involving Fads2 itself, since this enzyme catalyses desaturation of 24:5n-3 to 24:6n-3, as well as ALA to SDA. However, metabolism of EPA and DPA both require elongation reactions. This study examined the activities of two elongase enzymes as well as the second reaction of Fads2 in order to concentrate on the metabolism of EPA to DHA. Methodology/Principal Findings The substrate selectivities, competitive substrate interactions and dose response curves of the rat elongases, Elovl2 and Elovl5 were determined after expression of the enzymes in yeast. The competitive substrate interactions for rat Fads2 were also examined. Rat Elovl2 was active with C20 and C22 polyunsaturated fatty acids and this single enzyme catalysed the sequential elongation reactions of EPA→DPA→24:5n-3. The second reaction DPA→24:5n-3 appeared to be saturated at substrate concentrations not saturating for the first reaction EPA→DPA. ALA dose-dependently inhibited Fads2 conversion of 24:5n-3 to 24:6n-3. Conclusions The competition between ALA and 24:5n-3 for Fads2 may explain the decrease in DHA levels observed after certain intakes of dietary ALA have been exceeded. In addition, the apparent saturation of the second Elovl2 reaction, DPA→24:5n-3, provides further explanations for the accumulation of DPA when ALA, SDA or EPA is provided in the diet. This study suggests that Elovl2 will be critical in understanding if DHA synthesis can be increased by dietary means.

Interleukin-17: the missing link between T-cell accumulation and effector cell actions in rheumatoid arthritis?

The prominence of T cells and monocyte/macrophages in rheumatoid synovium suggests T cells may localize and amplify the effector functions of monocyte/macrophages in rheumatoid disease. However, while T cells are abundant in rheumatoid joints, classic T‐cell derived cytokines are scarce, especially when compared to the levels of monokines IL‐1β and TNF‐α. For this reason, it has been speculated that monocyte/macrophages may act independently of T cells in rheumatoid disease and that the role of T cells may be more or less irrelevant to core disease mechanisms. The question of T‐cell influence requires re‐evaluation in light of the characterization of IL‐17, a T‐cell derived cytokine that is abundant in rheumatoid synovium and synovial fluid. IL‐17 has a number of pro‐inflammatory effects, both directly and through amplification of the effects of IL‐1β and TNF‐α. IL‐17 is able to induce expression of pro‐inflammatory cytokines and stimulate release of eicosanoids by monocytes and synoviocytes. Furthermore, IL‐17 has been implicated in the pathogenesis of inflammatory bone and joint damage through induction of matrix metalloproteinases and osteoclasts, as well as inhibition of proteoglycan synthesis. In animal models of arthritis, intra‐articular injection of IL‐17 results in joint inflammation and damage. The recognition of IL‐17 as a pro‐inflammatory T cell derived cytokine, and its abundance within rheumatoid joints, provides the strongest candidate mechanism to date through which T cells can capture and localize macrophage effector functions in rheumatoid arthritis. As such, IL‐17 warrants consideration for its potential as a therapeutic target in rheumatoid arthritis.

A practical approach to increasing intakes of n-3 polyunsaturated fatty acids: use of novel foods enriched with n-3 fats.

Objectives: To assess the effects of providing a wide range of foodstuffs containing n-3 polyunsaturated fatty acids (PUFA), occurring naturally or from fortification, on intake and blood and tissue proportions of n-3 PUFA.Design: Before/after dietary intervention study.Setting: Adelaide, Australia.Subjects: 16 healthy males recruited from the community.Interventions: Subjects were provided with a range of foodstuffs naturally containing n-3 PUFA (fresh fish, canned fish, flaxseed meal, canola oil) and items fortified with fish oil (margarine spread, milk, sausages, luncheon meat, french onion dip). Food choices were left to the discretion of each subject. Intake was estimated by diet diary. Blood was collected at—2, 0, 2, and 4 weeks for fatty acid analysis.Main outcome measures: Dietary intakes; plasma, platelet, and mononuclear cell phospholipid fatty acids.Results: Consumption of n-3 PUFA increased significantly: α-linolenic acid (ALA) from 1.4 to 4.1 g/day (P<0.001), eicosapentaenoic acid (EPA) from 0.03 to 0.51 g/day (P<0.001), and docosahexaenoic acid (DHA) from 0.09 to 1.01 g/day (P<0.001). Linoleic acid (LA) intake decreased from 13.1 to 9.2 g/day (P<0.001). The proportions of EPA and DHA increased significantly in all phospholipid pools examined; plasma EPA from 1.13% of total fatty acids to 3.38% (P<0.001) and DHA from 3.76 to 7.23% (P<0.001); mononuclear cell EPA from 0.40 to 1.25% (P<0.001) and DHA from 2.33 to 4.08% (P<0.001); platelet EPA from 0.41 to 1.2% (P<0.001) and DHA from 1.64 to 3.07% (P<0.001).Conclusions: Incorporating fish oil into a range of novel commercial foods provides the opportunity for wider public consumption of n-3 PUFA with their associated health benefits.Sponsorship: Dawes Scholarship, Royal Adelaide Hospital.

Fish oil in recent onset rheumatoid arthritis: a randomised, double-blind controlled trial within algorithm-based drug use

Background The effects of fish oil (FO) in rheumatoid arthritis (RA) have not been examined in the context of contemporary treatment of early RA. This study examined the effects of high versus low dose FO in early RA employing a ‘treat-to-target’ protocol of combination disease-modifying anti-rheumatic drugs (DMARDs). Methods Patients with RA <12 months’ duration and who were DMARD-naïve were enrolled and randomised 2:1 to FO at a high dose or low dose (for masking). These groups, designated FO and control, were given 5.5 or 0.4 g/day, respectively, of the omega-3 fats, eicosapentaenoic acid + docosahexaenoic acid. All patients received methotrexate (MTX), sulphasalazine and hydroxychloroquine, and DMARD doses were adjusted according to an algorithm taking disease activity and toxicity into account. DAS28-erythrocyte sedimentation rate, modified Health Assessment Questionnaire (mHAQ) and remission were assessed three monthly. The primary outcome measure was failure of triple DMARD therapy. Results In the FO group, failure of triple DMARD therapy was lower (HR=0.28 (95% CI 0.12 to 0.63; p=0.002) unadjusted and 0.24 (95% CI 0.10 to 0.54; p=0.0006) following adjustment for smoking history, shared epitope and baseline anti–cyclic citrullinated peptide. The rate of first American College of Rheumatology (ACR) remission was significantly greater in the FO compared with the control group (HRs=2.17 (95% CI 1.07 to 4.42; p=0.03) unadjusted and 2.09 (95% CI 1.02 to 4.30; p=0.04) adjusted). There were no differences between groups in MTX dose, DAS28 or mHAQ scores, or adverse events. Conclusions FO was associated with benefits additional to those achieved by combination ‘treat-to-target’ DMARDs with similar MTX use. These included reduced triple DMARD failure and a higher rate of ACR remission.

Effect of Dietary n-3 Polyunsaturated Fatty Acids on the Inducibility of Ventricular Tachycardia in Patients With Ischemic Cardiomyopathy

Increased consumption of fish and/or fish oil was associated with decreased risk of sudden cardiac death (SCD). The study aim was to evaluate the antiarrhythmic effect of dietary fish oil on the inducibility of ventricular tachycardia (VT) at high risk of SCD. Patients with coronary artery disease undergoing defibrillator implantation were recruited if sustained monomorphic VT could be induced by programmed extra stimuli at 2 cycle lengths. After the initial study, 12 patients consumed 3 g/d of encapsulated fish oil for approximately 6 weeks before a repeated electrophysiologic study. To control for fluctuations in the inducibility of VT, an additional 14 patients with no dietary manipulation were also studied. Aggressiveness of stimulation required to induce VT was ranked from least aggressive to most aggressive based on cycle length and number of extra stimuli, with noninducibility ranked highest. At the repeated electrophysiologic study, in the fish-oil group, 42% had no inducible VT, 42% required more aggressive stimulation to induce VT, 8% required identical stimulation, and 8% required less stimulation compared with 7%, 36%, 36%, and 21% in the control group, respectively. Overall, there was a change to noninducible or less inducible VT in the fish-oil group, but no change in the control group (p = 0.003 and p = 0.65, respectively; Wilcoxons sign-rank test). In conclusion, dietary n-3 fatty acid supplementation decreased the inducibility of VT in patients at risk of SCD. These findings suggest that dietary fish oil can have an antiarrhythmic effect.