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Synovial fluid and plasma n3 long chain polyunsaturated fatty acids in patients with inflammatory arthritis
Prostaglandins, Leukotrienes and Essential Fatty Acids 97 (2015) 7–12
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Synovial fluid and plasma n3 long chain polyunsaturated fatty acids in patients with inflammatory arthritis Mahin Moghaddami a,b,c, Michael James a,b,c, Susanna Proudman a,b,c, Leslie G Cleland a,b,c,n a
Arthritis Research Laboratory, Hanson Institute, SA Pathology, Adelaide, South Australia 5000, Australia Discipline of Medicine, University of Adelaide, Adelaide, South Australia 5000, Australia c Rheumatology Unit, Royal Adelaide Hospital, Adelaide, South Australia 5000, Australia b
art ic l e i nf o
a b s t r a c t
Article history: Received 11 November 2014 Received in revised form 25 February 2015 Accepted 26 February 2015
Relationships between n-3 long chain polyunsaturated fatty acids (LC-PUFA) in plasma and synovial fluid (SF) were examined in 36 patients with knee effusion within the context of a variety of rheumatic diagnoses and various stated fish oil (FO) intakes (from 0 to 30 mL of standard FO daily) of variable duration. In a sub-group of patients, correlations between PUFA in SF mononuclear cells (MNC) and cellfree supernatants of SF and between SF MNC and peripheral blood (PB) MNC were examined. Correlations were also sought between clinical data (stated FO intake, pain score) and n-3 LC-PUFA. Correlations between plasma n-3 LC-PUFA and SF n-3 LC-PUFA were very strong (r2 40.9, p o0.001). The LC-PUFA profiles of SF supernatants differed from those of MNC. PUFA profiles in PB MNC and SF MNC were similar, except for a higher proportion of DHA in the latter. Positive correlations were observed between stated intakes of FO and EPA in plasma and SF (for both r ¼0.37, p¼ 0.02) and DHA in plasma (r ¼ 0.37, p ¼0.02) and SF (r ¼ 0.36, p ¼0.03). n-3 LC-PUFA in plasma and SF correlated inversely with pain score (plasma r2 ¼0.16, po 0.02; SF r2 0.32, p¼ 0.001). In conclusion, plasma n-3 LC-PUFA is a strong indicator of SF n-3 LC-PUFA status across a broad range of rheumatic diagnoses and FO intakes. Higher n-3 LC-PUFA in plasma and SF were associated with lesser pain experience. & 2015 Elsevier Ltd. All rights reserved.
Keywords: N-3 LC-PUFA Fish oil Inflammatory arthritis Plasma Synovial fluid Mononuclear cells
1. Introduction Humans are dependent on dietary intake for n-3 PUFA and also for more abundant competitor n-6 PUFA. Humans cannot synthesise n-3 alpha-linolenic acid (ALA; C18:3,n-3) and have a limited and variable capacity to convert ALA from vegetable sources to eicospentaenoic acid (EPA; C20:5n-3), n-3 docosapentenoic acid (DPA; C22:5,n-3) and docosahexaenoic acid (DHA; C22:6n-3) [1]. Direct dietary sources of EPA and DHA include fish and fish oil (FO) and to a lesser extent red meat [2]. FO is a particularly rich source of EPA and DHA and because FO has been shown to have antiinflammatory effects, FO supplements are used medicinally to treat rheumatoid arthritis (RA) and certain other inflammatory diseases [3]. Since individuals vary in the efficiency with which they absorb ingested fat, and from time to time, in the extent to
n Corresponding author at: Rheumatology Unit, Royal Adelaide Hospital, North Terrace, Adelaide, South Australia 5000, Australia. Tel.: þ 61 8 82225190; fax: þ 61 8 82225895. E-mail addresses: [email protected] (M. Moghaddami), [email protected] (M. James), [email protected] (S. Proudman), [email protected] (L. Cleland).
http://dx.doi.org/10.1016/j.plefa.2015.02.005 0952-3278/& 2015 Elsevier Ltd. All rights reserved.
which they consume ALA and competitor n-6 PUFA [4] and in rates of conversion dietary ALA to EPA [1,5], the n-3 LC-PUFA found in blood and tissue phospholipids (PL) is not a simple reflection of dietary intakes [5]. EPA and DHA displace arachidonic acid (AA; C20:4n-6) from cell membrane PL and also inhibit synthesis of pro-inflammatory n-6 eicosanoid mediators of inflammation derived from AA [6]. EPA is also metabolised to eicosanoids, although EPA derived n-3 eicosanoids have little pro-inflammatory activity relative to their n-6 counterparts [6]. EPA and DHA are also precursors of specialized pro-resolving mediators (SPMs), which are active in the resolution of inflammation [7–9]. For these reasons, blood levels of n-3 LC-PUFA have been suggested as a potentially useful biomarker to guide medicinal use of FO [10]. However for such a biomarker to be valid, blood levels need to correlate with levels in tissues where n-3 LCPUFA are likely to have their anti-inflammatory effects. Multiple clinical trials have reported benefit in RA from medicinal use of FO in daily doses of 9 g or more of standard FO or a commensurate dose of a FO concentrate (for review see Refs. [3,11]). Meta-analysis of randomised controlled trials (RCTs) of FO in RA shows that FO reduces pain, tender joint count, morning stiffness and non-steroidal anti-inflammatory drug (NSAID) use in patients with RA [12]. Proudman and co-workers have more
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recently reported favourable effects of FO in recent onset RA in a long-term RCT comparing high dose FO (providing 5.5 g of EPA plus DHA daily) versus a comparator oil [13]. The latter contained a small dose of FO, providing 0.4 g EPA plus DHA daily diluted in a low n-6 vegetable oil for masking and to meet Heart Foundation recommendations for desirable EPA plus DHA intake [14]. All patients received treatment with combinations of disease modifying anti-inflammatory drugs (DMARDs), which were applied according to a predetermined treatment algorithm with rules for intensification of DMARD therapy in order to achieve predetermined treat-to-target criteria for remission or low disease activity [13]. The findings of the first 12 months of a three year study have been reported to date. Patients receiving the high dose FO required less intensive DMARD therapy and achieved more remissions than their counterparts taking the comparator blend. Furthermore, n-3 LC-PUFA as a continuous variable correlated positively with improved disease control when data from the treatment groups were pooled (Proudman SM, James MJ, Cleland LG submitted). A role for n-3 LC-PUFA in management of chronic pain associated with inflammation is suggested by pain modulating effects in RA [12]. The possibility of a more general effect on pain has been suggested by an open study into the use of FO as an alternative to NSAIDs in patients with discogenic spinal pain [15]. At a mechanistic level, FO has been shown inhibit synthesis of the nociceptive eicosanoid prostaglandin E2 [6,16]. Maresin-1, which is biosynthesised from DHA, has been shown to reduce pain responses in experimentally challenged mice [17]. For these reasons, possible correlations between n-3 LC-PUFA in plasma and synovial fluid (SF) and reported pain experience were examined in the present study. Correlations between stated FO dose and pain scores were also explored.
2. Materials and methods 2.1. Subjects SF and peripheral blood (PB) samples were obtained contemporaneously from 36 patients undergoing arthrocentesis of an inflammatory knee effusion as part of their care. Mononuclear cells (MNC) were isolated from PB and SF when samples became available early enough to allow separation of MNC from other cell types within the day. Patient characteristics are shown in Table 1. All patients gave informed consent and the study protocol was approved by the Human Research Ethics Committee, Royal Adelaide Hospital. 2.2. Fish oil intake
Table 1 Demographic and clinical characteristics of patients. Diagnosis
Age Sex Disease (years) duration (years)
Fish oil (mL/d)a
Duration of fish oil intake
Rheumatoid arthritis Rheumatoid arthritis Gout Psoriatic arthritis Crohn's arthropathy Rheumatoid arthritis Psoriatic arthritis Rheumatoid arthritis Monoarthritis Rheumatoid arthritis Gout Rheumatoid arthritis Gout Ulcerative colitis arthropathy Rheumatoid arthritis Monoarthritis Rheumatoid arthritis Pauci-arthritis Psoriatic arthritisb MCTDb Psoriatic arthritis Rheumatoid arthritis B27 arthropathy Rheumatoid arthritis Gout Rheumatoid arthritis Pauci-arthritisb Rheumatoid arthritis Rheumatoid arthritisb Rheumatoid arthritisb Monoarthritisb Psoriatic arthritisb CCPDb B27þ spondyloarthritisb Psoriatic arthritisb Psoriatic arthritisb
82 83 64 19 46 48 56 61 34 63 56 57 33 50
F F M M F F M F F M M F M F
30 18 30 1 30 22 7 13 26 14 12 27 4 1
15 10 30 20 11 15 13 5 0 5 30 10 0 7
6 8 8 7 1 5 5 9 0 9 4 7 0 1
60 62 62 33 58 55 60 82 62 66 54 68 35 63 42 84 57 49 67 53
F F F F F F M F M F M F F F F M M M F F
38 1 2 7 6 3 8 11 41 10 10 29 18 27 8 5 0.1 8 1 30
11 15 15 10 4 10 15 10 15 10 30 15 15 30 15 6 15 0 10 5
9 years 2 months 1 month 2 years 5 years 3 years 6 years 11 years 1 month 1 year 2 months 3 months 2 years 11 years 3 years 5 years 1 month 0 1 month 12 years
50 49
M F
1 12
15 0
1 year 0
years years months months year years years years years years years month
MCTD, mixed connective tissue disease; CCPD, calcium pyrophosphate deposition disease. a Dose of fish oil, whether taken as capsules or fluid on juice, standard strength or a concentrate, has been expressed as the equivalent dose of standard fish in mL/d. 1 mL standard fish oil weighs 0.92 g and contains 166 mg EPA and 100 mg DHA. b Provided mononuclear cell data.
2.4. Sample preparation
Most patients had been told to take FO 10–15 mL daily as a complement to their routine medical management. Subjects completed a Vital Activities and Lifestyle Index (VALI) form [18] as a routine part of clinical assessment. Our modification of the VALI form includes questions on type, dose and mode of ingestion of FO taken. Answers to these questions allowed calculation of the dose of standard FO (EPA 18% w/w þ DHA 12% w/w, 1 mL FO weighs 0.92 g) as mL/d. A small minority of patients took a concentrate of FO n-3 LC-PUFA as natural triglycerides, in which case the daily dose was expressed as the equivalent dose, with regard to EPA and DHA content, of standard FO in mL/day. Dose and duration of FO intake are detailed in Table 1.
PB and SF samples were collected into heparinised tubes and centrifuged at 10,000g for 15 min after which 2 mL aliquots of plasma and SF supernatant were removed and stored at 70 1C for later assay. MNC were isolated from SF and PB by density gradient centrifugation over Lymphoprep as described previously [19]. The harvested MNC were washed with phosphate buffered saline prior to drying under nitrogen. All subjects contributed samples for comparisons between plasma and SF fatty acids. When more than one sample was obtained, the initial sample for each subject was used for these comparisons. For analyses of MNC fatty acids, a subsequent SF sample was obtained in most cases. This sample was used for analyses of SF supernatants for paired comparisons with MNC. Comparisons between SF MNC and PB MNC were undertaken using contemporaneously obtained samples.
2.3. Pain score
2.5. Fatty acid analysis
The VALI form has an uncalibrated 100 mm line for visual analogue score (VAS) for pain (0–100), which is used by patients to document intensity of pain experience.
Plasma, SF supernatants and MNC preparations were treated with chloroform/isopropanol to extract lipids. The PL fraction was separated by thin layer chromatography and subjected to
M. Moghaddami et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 97 (2015) 7–12
methanolysis. The fatty acid methyl esters were quantified by gasliquid chromatography, as previously described [20].
2.6. Statistical analyses Pearson correlation and linear regression analyses were performed using Prism Version 5.00 and Instat 3.1 software (GraphPad Software, La Jolla, CA, USA). A p-value o0.05 was considered statistically significant. Comparisons between n-3 LC-PUFA in SF supernatants and MNC were undertaken by paired t-test.
3. Results
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Table 2 Correlation between stated daily fish oil dosea and n-3 LC-PUFA of phospholipids in plasma and synovial fluid (n¼ 36). Fatty acid
EPA DPA DHA EPA þDHA
Synovial fluid
Plasma rb
p Value
r
p Value
0.37 o 0.1 0.37 0.39
0.02 0.8 0.02 0.02
0.37 o 0.1 0.36 0.36
0.02 0.94 0.03 0.03
a Fish oil intakes are shown in Table 1 and varied from 0 to 30 mL/day of standard fish oil or equivalent. LC-PUFA, long chain polyunsaturated fatty acids; EPA; eicosapentaenoic acid; DPA, docosapentaenoic acid; DHA, docosahexaenoic acid. b Pearson correlation coefficient.
3.1. Stated FO intake Patients, who represented a variety of inflammatory arthropathies, reported consuming various amounts of FO within the range 0–30 mL/d as detailed in Table 1. The mean daily FO intake (excluding those who stated they were not taking FO) was 13.8 mL (SEM 1.29 mL) and the median 15 mL (IQR 10,15 mL). Of those taking FO, the mean duration of intake was 4.1 year (SEM 0.62 year) and median 3 year (IQR 0.67, 6 year). Eleven subjects stated they were taking FO in a dose of 15 mL daily.
3.2. Correlation between stated FO intake and n-3 LC-PUFA in phospholipids in SF and plasma There were statistically significant correlations between stated FO intake and both EPA and DHA in PL of SF (Table 2). The lack of correlation with n-3 docosapentaenoic acid (DPA) is not unexpected because FOs contains little DPA compared with EPA and DHA. Similar relationships were observed between stated dietary intake of FO and EPA and DHA in plasma PL (Table 2). Among the 11 subjects who stated they were taking FO 15 mL daily, the median EPA was 3.53 (range 2.8–8.1) and median total SF n-3 LC-PUFA was 10.4 (range 4.6–18.5) as percentage of SF PL fatty acids. Similar variability was seen in plasma PL n-3 LC-PUFA in this sub-group (EPA median 3.8; range 0.9–11.4; total n-3 LC-PUFA median 10; range 6.5–22.3 as percentage plasma PL fatty acids).
Table 3 Fatty acid composition of phospholipids in plasma and synovial fluid and correlations between them. Fatty acid
18:2n-6 (LA) 20:3n-6 (DGLA) 20:4n-6 (AA) Total n-6 LC-PUFA 18:3n-3 (ALA) 20:5n-3 (EPA) 22:5n-3 (DPA) 22:6n-3 (DHA) Total n-3 LC PUFA
Mean 7 SEM (n¼ 36) Plasma
Synovial fluid
r2a
p Value
177 0.7b 2.5 7 0.2 7.8 7 0.2 28.3 7 0.8 0.2 7 0.02 4.2 7 0.5 1.4 7 0.1 5.5 7 0.3 11.17 0.8
14.8 7 0.5 2.5 7 0.3 8.8 7 0.2 26.8 7 0.7 0.2 7 0.02 3.3 7 0.4 1.7 7 0.1 5.3 7 0.3 10.4 7 0.6
0.74 0.96 0.83 0.95 0.73 0.96 0.95 0.95 0.96
o 0.001 o 0.001 o 0.001 o 0.001 o 0.001 o 0.001 o 0.001 o 0.001 o 0.001
a
Linear regression. Individual fatty acids expressed as a percentage of total fatty acids in phospholipids. LA, linoleic acid; DGLA, dihomo-γ-linolenic acid; AA, arachidonic acid; ALA, α-linolenic acid; EPA, eicosapentaenoic acid; DPA, docosapentaenoic acid; DHA, docosahexaenoic acid. b
3.5. Comparison of fatty acids in the phospholipids of mononuclear cells from peripheral blood and synovial fluid There were no differences in any of the n-6 or n-3 fatty acids between MNC from blood and MNC from SF with the exception of DHA which was higher in the SF cells (Table 5).
3.3. Correlations between PUFA in the phospholipids of plasma and SF
3.6. Relationships between pain scores and n-3 LC-PUFA in phospholipids of plasma and synovial fluid
There were very strong correlations between plasma and SF values for the n-6 PUFA linoleic acid (LA; C18:2n-6), dihomo-γlinolenic acid (DGLA; C20:3n-6) and AA and the n-3 fatty acids ALA, EPA, DPA and DHA (Table 3).
Total n-3 PUFA in PL of plasma and SF correlated inversely with pain score (plasma r2 ¼0.16, p¼ 0.02; SF r2 ¼0.32, p¼ 0.001). Correlations were also found between EPA and DHA and pain score (Table 6). By contrast, there was not a significant correlation between stated FO intake and pain score (r2 ¼0.08, p ¼0.1) (data not shown).
3.4. Comparison between fatty acids in the phospholipids of SF supernatants and SF mononuclear cells 4. Discussion The 18-carbon n-6 fatty acid, LA, was substantially lower and its long chain 20-carbon metabolite, AA, was substantially higher in PL of SF MNC compared with PL in the supernatants of SF from which they were isolated. This pattern was not repeated for the n-3 PUFA where both 18- and 20-carbon fatty acids, ALA and EPA, were lower in SF MNC than in corresponding supernatants. The other n-3 PUFA, DPA and DHA, were similar in PL of SF MNC and SF supernatants (Table 4). Similar relationships were observed between n-3 LC-PUFA in PL of plasma and peripheral blood MNC (data not shown).
Analyses of n-3 LC-PUFA in plasma and cells within peripheral blood samples have proven useful in evaluating changes in n-3 LCPUFA status in tissues in response to experimental interventions with FO supplements or changes in amounts of n-3 LC-PUFA consumed as food [21,22]. Notwithstanding, to date fatty acid analyses have been applied systematically to few clinical settings beyond clinical trials. Support for routine use of PUFA analyses in guiding treatment and in evaluation of risk is found in the “omega-3 index” of erythrocyte EPA þDHA proposed by Harris
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Table 4 Fatty acid composition of phospholipids in synovial fluid (SF) supernatants and SF mononuclear cells (MNC) and correlations between them. Fatty acid
Mean 7 SEM (n¼ 11) SF supernatants
18:2n-6 (LA) 20:3n-6 (DGLA) 20:4n-6 (AA) Total n-6 LC-PUFA 18:3n-3 (ALA) 20:5n-3 (EPA) 22:5n-3 (DPA) 22:6n-3 (DHA) Total n-3 LC PUFA
b
17.4 7 0.5 2.8 7 0.3 8.8 7 0.3 30.0 7 0.5 0.2 7 0.03 1.7 7 0.2 2.0 7 0.5 4.0 7 0.3 7.6 7 0.4
Fatty acid SF MNC
p Valuea
8.6 7 0.7 1.7 7 0.2 12.9 7 1 25.5 7 1.6 0.017 0.01 0.9 7 0.3 2.3 7 0.4 4.0 7 0.3 7.2 7 0.7
o 0.001 o 0.001 0.001 0.02 0.002 o 0.001 0.6 0.9 0.5
a
Paired t-test. For this sub-group mean daily fish oil intake was 8.6 mL (SEM 1.80 mL), median 10 mL (IQR 4,15 mL). Comparisons are between SF MNC and the supernatants of the SF from which the MNC had been obtained. In some cases, the SF sample differed from the patient's sample which had contributed prior to plasma and SF correlations. b Individual fatty acids expressed as a percentage of total fatty acids. LA, linoleic acid; AA, arachidonic acid; DGLA, dihomo-γ-linolenic acid; ALA, α-linolenic acid; EPA, eicosapentaenoic acid; DPA, docosapentaenoic acid; DHA, docosahexaenoic acid.
Table 5 Fatty acid composition in phospholipids of mononuclear cells (MNC) in peripheral blood (PB) and synovial fluid (SF) and correlations between them. Fatty acid
18:2n-6 (LA) 20:3n-6 (DGLA) 20:4n-6 (AA) Total n-6 LC-PUFA 18:3n-3 (ALA) 20:5n-3 (EPA) 22:5n-3 (DPA) 22:6n-3 (DHA) Total n-3 LC PUFA
Table 6 Correlation between pain scores and n-3 LC-PUFA in phospholipids of plasma and synovial fluid (n¼ 29).
Mean 7 SEM (n¼ 9) PB MNC
SF MNC
p Valuea
7.4 7 0.7b 1.4 7 0.23 15.0 7 1.1 24.97 2.1 0.017 0.01 0.8 7 0.22 2.4 7 0.39 2.6 7 0.2 5.8 7 0.6
8.6 70.9 1.5 70.3 12.5 71.2 24.971 0.02 70.01 0.9 70.4 2.3 70.4 4.0 70.35 7.3 71.0
0.1 0.6 0.1 0.9 0.1 0.6 0.8 o 0.001 0.09
a
Paired t-test. Individual fatty acids expressed as a percentage of total fatty acids. LA, linoleic acid; AA, arachidonic acid; DGLA, dihomo-γ-linolenic acid; ALA, α-linolenic acid; EPA, eicosapentaenoic acid; DPA, docosapentaenoic acid; DHA, docosahexaenoic acid. b
and von Schacky as both an index of adequacy of supplementation and an estimator of cardiovascular risk [23]. The present study examines the potential utility of plasma n-3 LC PUFA profiles as a guide to SF levels of n-3 LC PUFA within a routine clinical practice setting. Navarro and co-workers studied RA and osteoarthritis patients who were not taking FO and reported reduced n-3 PUFA in SF in RA compared to osteoarthritis [24]. They did not report correlations between fatty acid profiles of plasma and SF. The present study shows that levels of n-3 LC PUFA in PL of plasma and SF are strongly correlated across a range of FO doses and different rheumatic diagnoses. By comparison, stated intake of FO was found to correlate weakly with EPA and DHA within the PL fractions of both plasma and SF supernatants. The strong correlations between PL n-3 LC-PUFA in SF and plasma indicate that, in patients with inflammatory arthropathies, n-3 LC-PUFA in plasma PL can be used as a surrogate for SF n-3 PUFA when monitoring dietary interventions aimed at increasing n-3 LC-PUFA in SF, whence they can be expected to have modulating effects on immune cells in synovium [22]. Functions potentially affected include phagocytosis, T cell signalling and antigen
EPA DHA Total n-3 LC PUFA a
Synovial fluid
Plasma r2a
p Value
r2
p Value
0.15 0.14 0.16
0.03 0.04 0.02
0.14 0.17 0.32
0.03 0.02 0.001
Linear regression.
presentation [22]. Further studies are warranted to assess the function of dendritic cells (DC), which can be isolated from SF [25], since exogenous EPA and DHA, when added in vitro to DC, which have been differentiated from human peripheral blood monocytes in vitro (MDDC), have been shown to reduce expression of HLA-DR and of co-stimulatory molecules in response to a maturation stimulus and to reduce allogeneic stimulation of T cells [26]. Interesting differences were found between LC-PUFA in SF supernatants and SF MNC. Notably AA was higher and LA and DGLA lower as a proportion of PL in SF MNC compared to SF supernatants. These differences suggest either preferential uptake of AA, or remodelling of n-6 precursors LA and DGLA to AA within SF MNC. Similar differences were seen between PUFA in PL of plasma and PB MNC. In contrast to AA, EPA was a higher proportion of PL in plasma and SF than in the respective MNC. It is notable that we have previously shown similar patterns of difference in respective abundance of LA, AA and EPA in plasma compared to PB MNC of healthy subjects whether taking FO supplements or not [4]. Accordingly, putative LC-PUFA remodelling, if it exists, is not likely to be confined to MNC activated at sites of inflammation. Based on randomised trials of efficacy and tolerance of FO in RA and other inflammatory diseases, the recommended dose of FO for anti-inflammatory effects is at least 9 g or 10 mL daily of standard FO (or a dose of a FO concentrate which delivers 2.7 g daily or more of EPA plus DHA as triglycerides) [3]. In the present study, the median stated dose of standard FO or equivalent was 15 ml/d and the mean dose 13.8 ml/d. Although some patients took higher doses, on average these doses are usual for medicinal use of FO for inflammatory diseases in trials and in practice [3,13]. Findings from clinical trials and practice experience with medicinal use of FO suggest that these doses are generally safe and are not associated with increased bleeding tendency. Four subjects in the present study took 30 mL daily. While clinical trials experience with this dose is limited, we observed no abnormal bleeding episodes in these patients. Furthermore this dose is within the range of doses (up to 5 g/daily of EPA plus DHA) deemed safe by the European Food Safety Authority [27]. Sears and co-workers used the plasma AA/EPA ratio to monitor n-6/n-3 LC-PUFA balance in the management of brain injury [28]. They chose a plasma AA/EPA ratio of unity to determine an arbitrary therapeutic ceiling for treatment with enterically administered n-3 LC-PUFA with the intent of avoiding abnormal bleeding events. These authors described two patients with brain injury who were given EPA 10.8 g and DHA 5.4 g daily in divided doses. This dose of EPA plus DHA equates with the EPA plus DHA content of 45 g or 49 mL of standard FO daily. However, EPA and DHA in their preparation were in the form of ethyl esters which are significantly less well absorbed than EPA and DHA within the natural triglycerides of FO [29]. While Sears et al. report no bleeding episode, they reduced the dose of EPA plus DHA when the plasma AA/EPA ratio was observed to fall below unity in one of their patients. We have
M. Moghaddami et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 97 (2015) 7–12
followed patients who stated they were taking FO 30 mL daily for at least one year and in some cases for several years. No significant bleeding events have been encountered. The median plasma PL AA/EPA ratio after at least 12 months of FO at the dose of 30 ml daily was 0.68 (IQR 0.48–1.07, n¼23) (unpublished data). While these findings suggest the ceiling ratio of Sears et al. is conservative, the principle of utilising LC PUFA levels as a guide for dose adjustment for efficacy and safety appears to have merit. The evidence for an increased bleeding tendency with medicinal use of FO is weak [30] as acknowledged by Sears and coauthors [28]. Concern regarding the potential for increased bleeding with FO supplements seems to have its origins in reduced platelet aggregation and increased bleeding time observed in Greenland Inuits consuming their native diet [31]. However, this predisposition is likely contributed to by the paucity of n-6 fatty acids in the Inuit diet relative to Western diets [32], in addition to the very high n-3 LC-PUFA content of the Inuit diet, the mean of which has been estimated to be as high as 13 g/d [32]. The n-3 LC-PUFA content of the Inuit diet contrasts with EPA plus AA of 4.1 g in a 15 mL daily dose of standard FO used medicinally. Furthermore, in clinical trials in which FO at or near anti-inflammatory doses was given with warfarin or aspirin plus clopridogrel, no increase in bleeding events was seen in subjects taking FO compared to their counterparts taking these anti-thrombotics without FO [33,34]. Thus, while it is not possible to give categorical reassurance regarding the potential for undesirable effects of FO supplements in situations where bleeding is a special risk, the level at which n6/n3 balance becomes hazardous in this regard remains to be determined. Four subjects stated they were not taking FO at the time of arthrocentesis. Interestingly, their levels of EPA (1.870.1, mean7SEM), DHA (4.770.2) and total n-3 PUFA (8.270.3) as a proportion of plasma PL are higher than values observed in local healthy volunteers [35]. On the other hand, their levels are lower than those found in subjects taking FO in anti-inflammatory doses in a clinical trial setting [36]. In the present study, patients, who stated they were taking FO 15 mL daily, displayed considerable variability in n-3 LC-PUFA in plasma and SF. These differences may reflect a mismatch between statements and behaviour, as well as metabolic differences between subjects, and underline the potential advantages of measurement of levels in blood in order to assess n-3 status. Meta-analysis of studies assessing musculoskeletal pain indicates that FO improves pain outcomes, particularly with respect to patient assessed pain, duration of morning stiffness and number of painful and/or tender joints [12]. We observed inverse relationships between pain score and n-3 LC-PUFA in plasma and SF. n-3 LC-PUFA could potentially reduce pain by several mechanisms. EPA and DHA are competitive inhibitors of metabolism of AA by cyclooxygenase [37], which provides a mechanism for inhibition of synthesis of the nociceptive mediator prostaglandin E2 by FO treatment [38]. Products of EPA and DHA metabolism include resolvins, protectins and maresins, which may contribute to pain relief through actions that help to resolve inflammation [17,39]. DHA has also been shown to modulate pain through an effect on opioid-receptors [39]. In conclusion, plasma PL PUFA analyses were an accurate predictor of SF values across a range of FO doses and different rheumatic diagnoses. Plasma n-3 LC-PUFA levels are a substantially more accurate predictor of SF n-3 LC-PUFA than stated dietary intakes of FO and also correlated with lesser pain experience.
Acknowledgements This work was funded in part by research Grants from Arthritis Australia and the Royal Adelaide Hospital Research Fund.
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Moghaddami, M. James, S.L. Whittle, L.G. Cleland, Synovial fluid myeloid dendritic cells display important differences compared to monocyte-derived dendritic cells prepared in vitro, Clin. Transl. Immunol. 3 (2014) e23. [26] H. Wang, Q. Hao, Q.R. Li, X.W. Yan, S. Ye, Y.S. Li, N. Li, J.S. Li, Omega-3 polyunsaturated fatty acids affect lipopolysaccharide-induced maturation of dendritic cells through mitogen-activated protein kinases p38, Nutrition 23 (2007) 474–482. [27] EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA), Scientific Opinion on the Tolerable Upper Intake Level of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA), EFSA J. 10 (2012) 2815.
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[28] B. Sears, J. Bailes, B. Asselin, Therapeutic uses of high-dose omega-3 fatty acids to treat comatose patients with severe brain injury, PharmaNutrition 1 (2013) 86–89. [29] D. Tarry, T.D. Lawson, B.G. Hughes, Human absorption of fish oil fatty acids as triacylglycerols, free acids, or ethyl esters, Biochem. Biophys. Res. Commun. 152 (1988) 328–335. [30] A.M. Villani, M. Crotty, L.G. Cleland, M.J. James, R.J. Fraser, L. Cobiac, M.D. Miller, Fish oil administration in older adults with cardiovascular disease or cardiovascular risk factors: is there potential for adverse events? a systematic review of the literature, Int. J. Cardiol. 168 (2013) 4371–4375. [31] J. Dyerberg, H.O. Bang, Haemostatic function and platelet polyunsaturated fatty acids in Eskimos, Lancet 2 (1979) 433–435. [32] H.O. Bang, J. Dyerberg, H.M. Sinclair, The composition of the Eskimo food in north western Greenland, Am. J. Clin. Nutr. 33 (1980) 2657–2661. [33] J. Eritsland, H. Arnesen, K. Gronseth, N.B. Fjeld, M. Abdelnoor, Effect of dietary supplementation with n-3 fatty acids on coronary artery bypass graft patency, Am. J. Cardiol. 77 (1996) 31–36. [34] P.D. Watson, P.S. Joy, C. Nkonde, S.E. Hessen, D.G. Karalis, Comparison of bleeding complications with omega-3 fatty acids þaspirin þ clopidogrel–versus–aspirin þ clopidogrel in patients with cardiovascular disease, Am. J. Cardiol. 104 (2009) 1052–1054.
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Contents lists available at ScienceDirect
Prostaglandins, Leukotrienes and Essential Fatty Acids journal homepage: www.elsevier.com/locate/plefa
Synovial fluid and plasma n3 long chain polyunsaturated fatty acids in patients with inflammatory arthritis Mahin Moghaddami a,b,c, Michael James a,b,c, Susanna Proudman a,b,c, Leslie G Cleland a,b,c,n a
Arthritis Research Laboratory, Hanson Institute, SA Pathology, Adelaide, South Australia 5000, Australia Discipline of Medicine, University of Adelaide, Adelaide, South Australia 5000, Australia c Rheumatology Unit, Royal Adelaide Hospital, Adelaide, South Australia 5000, Australia b
art ic l e i nf o
a b s t r a c t
Article history: Received 11 November 2014 Received in revised form 25 February 2015 Accepted 26 February 2015
Relationships between n-3 long chain polyunsaturated fatty acids (LC-PUFA) in plasma and synovial fluid (SF) were examined in 36 patients with knee effusion within the context of a variety of rheumatic diagnoses and various stated fish oil (FO) intakes (from 0 to 30 mL of standard FO daily) of variable duration. In a sub-group of patients, correlations between PUFA in SF mononuclear cells (MNC) and cellfree supernatants of SF and between SF MNC and peripheral blood (PB) MNC were examined. Correlations were also sought between clinical data (stated FO intake, pain score) and n-3 LC-PUFA. Correlations between plasma n-3 LC-PUFA and SF n-3 LC-PUFA were very strong (r2 40.9, p o0.001). The LC-PUFA profiles of SF supernatants differed from those of MNC. PUFA profiles in PB MNC and SF MNC were similar, except for a higher proportion of DHA in the latter. Positive correlations were observed between stated intakes of FO and EPA in plasma and SF (for both r ¼0.37, p¼ 0.02) and DHA in plasma (r ¼ 0.37, p ¼0.02) and SF (r ¼ 0.36, p ¼0.03). n-3 LC-PUFA in plasma and SF correlated inversely with pain score (plasma r2 ¼0.16, po 0.02; SF r2 0.32, p¼ 0.001). In conclusion, plasma n-3 LC-PUFA is a strong indicator of SF n-3 LC-PUFA status across a broad range of rheumatic diagnoses and FO intakes. Higher n-3 LC-PUFA in plasma and SF were associated with lesser pain experience. & 2015 Elsevier Ltd. All rights reserved.
Keywords: N-3 LC-PUFA Fish oil Inflammatory arthritis Plasma Synovial fluid Mononuclear cells
1. Introduction Humans are dependent on dietary intake for n-3 PUFA and also for more abundant competitor n-6 PUFA. Humans cannot synthesise n-3 alpha-linolenic acid (ALA; C18:3,n-3) and have a limited and variable capacity to convert ALA from vegetable sources to eicospentaenoic acid (EPA; C20:5n-3), n-3 docosapentenoic acid (DPA; C22:5,n-3) and docosahexaenoic acid (DHA; C22:6n-3) [1]. Direct dietary sources of EPA and DHA include fish and fish oil (FO) and to a lesser extent red meat [2]. FO is a particularly rich source of EPA and DHA and because FO has been shown to have antiinflammatory effects, FO supplements are used medicinally to treat rheumatoid arthritis (RA) and certain other inflammatory diseases [3]. Since individuals vary in the efficiency with which they absorb ingested fat, and from time to time, in the extent to
n Corresponding author at: Rheumatology Unit, Royal Adelaide Hospital, North Terrace, Adelaide, South Australia 5000, Australia. Tel.: þ 61 8 82225190; fax: þ 61 8 82225895. E-mail addresses: [email protected] (M. Moghaddami), [email protected] (M. James), [email protected] (S. Proudman), [email protected] (L. Cleland).
http://dx.doi.org/10.1016/j.plefa.2015.02.005 0952-3278/& 2015 Elsevier Ltd. All rights reserved.
which they consume ALA and competitor n-6 PUFA [4] and in rates of conversion dietary ALA to EPA [1,5], the n-3 LC-PUFA found in blood and tissue phospholipids (PL) is not a simple reflection of dietary intakes [5]. EPA and DHA displace arachidonic acid (AA; C20:4n-6) from cell membrane PL and also inhibit synthesis of pro-inflammatory n-6 eicosanoid mediators of inflammation derived from AA [6]. EPA is also metabolised to eicosanoids, although EPA derived n-3 eicosanoids have little pro-inflammatory activity relative to their n-6 counterparts [6]. EPA and DHA are also precursors of specialized pro-resolving mediators (SPMs), which are active in the resolution of inflammation [7–9]. For these reasons, blood levels of n-3 LC-PUFA have been suggested as a potentially useful biomarker to guide medicinal use of FO [10]. However for such a biomarker to be valid, blood levels need to correlate with levels in tissues where n-3 LCPUFA are likely to have their anti-inflammatory effects. Multiple clinical trials have reported benefit in RA from medicinal use of FO in daily doses of 9 g or more of standard FO or a commensurate dose of a FO concentrate (for review see Refs. [3,11]). Meta-analysis of randomised controlled trials (RCTs) of FO in RA shows that FO reduces pain, tender joint count, morning stiffness and non-steroidal anti-inflammatory drug (NSAID) use in patients with RA [12]. Proudman and co-workers have more
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recently reported favourable effects of FO in recent onset RA in a long-term RCT comparing high dose FO (providing 5.5 g of EPA plus DHA daily) versus a comparator oil [13]. The latter contained a small dose of FO, providing 0.4 g EPA plus DHA daily diluted in a low n-6 vegetable oil for masking and to meet Heart Foundation recommendations for desirable EPA plus DHA intake [14]. All patients received treatment with combinations of disease modifying anti-inflammatory drugs (DMARDs), which were applied according to a predetermined treatment algorithm with rules for intensification of DMARD therapy in order to achieve predetermined treat-to-target criteria for remission or low disease activity [13]. The findings of the first 12 months of a three year study have been reported to date. Patients receiving the high dose FO required less intensive DMARD therapy and achieved more remissions than their counterparts taking the comparator blend. Furthermore, n-3 LC-PUFA as a continuous variable correlated positively with improved disease control when data from the treatment groups were pooled (Proudman SM, James MJ, Cleland LG submitted). A role for n-3 LC-PUFA in management of chronic pain associated with inflammation is suggested by pain modulating effects in RA [12]. The possibility of a more general effect on pain has been suggested by an open study into the use of FO as an alternative to NSAIDs in patients with discogenic spinal pain [15]. At a mechanistic level, FO has been shown inhibit synthesis of the nociceptive eicosanoid prostaglandin E2 [6,16]. Maresin-1, which is biosynthesised from DHA, has been shown to reduce pain responses in experimentally challenged mice [17]. For these reasons, possible correlations between n-3 LC-PUFA in plasma and synovial fluid (SF) and reported pain experience were examined in the present study. Correlations between stated FO dose and pain scores were also explored.
2. Materials and methods 2.1. Subjects SF and peripheral blood (PB) samples were obtained contemporaneously from 36 patients undergoing arthrocentesis of an inflammatory knee effusion as part of their care. Mononuclear cells (MNC) were isolated from PB and SF when samples became available early enough to allow separation of MNC from other cell types within the day. Patient characteristics are shown in Table 1. All patients gave informed consent and the study protocol was approved by the Human Research Ethics Committee, Royal Adelaide Hospital. 2.2. Fish oil intake
Table 1 Demographic and clinical characteristics of patients. Diagnosis
Age Sex Disease (years) duration (years)
Fish oil (mL/d)a
Duration of fish oil intake
Rheumatoid arthritis Rheumatoid arthritis Gout Psoriatic arthritis Crohn's arthropathy Rheumatoid arthritis Psoriatic arthritis Rheumatoid arthritis Monoarthritis Rheumatoid arthritis Gout Rheumatoid arthritis Gout Ulcerative colitis arthropathy Rheumatoid arthritis Monoarthritis Rheumatoid arthritis Pauci-arthritis Psoriatic arthritisb MCTDb Psoriatic arthritis Rheumatoid arthritis B27 arthropathy Rheumatoid arthritis Gout Rheumatoid arthritis Pauci-arthritisb Rheumatoid arthritis Rheumatoid arthritisb Rheumatoid arthritisb Monoarthritisb Psoriatic arthritisb CCPDb B27þ spondyloarthritisb Psoriatic arthritisb Psoriatic arthritisb
82 83 64 19 46 48 56 61 34 63 56 57 33 50
F F M M F F M F F M M F M F
30 18 30 1 30 22 7 13 26 14 12 27 4 1
15 10 30 20 11 15 13 5 0 5 30 10 0 7
6 8 8 7 1 5 5 9 0 9 4 7 0 1
60 62 62 33 58 55 60 82 62 66 54 68 35 63 42 84 57 49 67 53
F F F F F F M F M F M F F F F M M M F F
38 1 2 7 6 3 8 11 41 10 10 29 18 27 8 5 0.1 8 1 30
11 15 15 10 4 10 15 10 15 10 30 15 15 30 15 6 15 0 10 5
9 years 2 months 1 month 2 years 5 years 3 years 6 years 11 years 1 month 1 year 2 months 3 months 2 years 11 years 3 years 5 years 1 month 0 1 month 12 years
50 49
M F
1 12
15 0
1 year 0
years years months months year years years years years years years month
MCTD, mixed connective tissue disease; CCPD, calcium pyrophosphate deposition disease. a Dose of fish oil, whether taken as capsules or fluid on juice, standard strength or a concentrate, has been expressed as the equivalent dose of standard fish in mL/d. 1 mL standard fish oil weighs 0.92 g and contains 166 mg EPA and 100 mg DHA. b Provided mononuclear cell data.
2.4. Sample preparation
Most patients had been told to take FO 10–15 mL daily as a complement to their routine medical management. Subjects completed a Vital Activities and Lifestyle Index (VALI) form [18] as a routine part of clinical assessment. Our modification of the VALI form includes questions on type, dose and mode of ingestion of FO taken. Answers to these questions allowed calculation of the dose of standard FO (EPA 18% w/w þ DHA 12% w/w, 1 mL FO weighs 0.92 g) as mL/d. A small minority of patients took a concentrate of FO n-3 LC-PUFA as natural triglycerides, in which case the daily dose was expressed as the equivalent dose, with regard to EPA and DHA content, of standard FO in mL/day. Dose and duration of FO intake are detailed in Table 1.
PB and SF samples were collected into heparinised tubes and centrifuged at 10,000g for 15 min after which 2 mL aliquots of plasma and SF supernatant were removed and stored at 70 1C for later assay. MNC were isolated from SF and PB by density gradient centrifugation over Lymphoprep as described previously [19]. The harvested MNC were washed with phosphate buffered saline prior to drying under nitrogen. All subjects contributed samples for comparisons between plasma and SF fatty acids. When more than one sample was obtained, the initial sample for each subject was used for these comparisons. For analyses of MNC fatty acids, a subsequent SF sample was obtained in most cases. This sample was used for analyses of SF supernatants for paired comparisons with MNC. Comparisons between SF MNC and PB MNC were undertaken using contemporaneously obtained samples.
2.3. Pain score
2.5. Fatty acid analysis
The VALI form has an uncalibrated 100 mm line for visual analogue score (VAS) for pain (0–100), which is used by patients to document intensity of pain experience.
Plasma, SF supernatants and MNC preparations were treated with chloroform/isopropanol to extract lipids. The PL fraction was separated by thin layer chromatography and subjected to
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methanolysis. The fatty acid methyl esters were quantified by gasliquid chromatography, as previously described [20].
2.6. Statistical analyses Pearson correlation and linear regression analyses were performed using Prism Version 5.00 and Instat 3.1 software (GraphPad Software, La Jolla, CA, USA). A p-value o0.05 was considered statistically significant. Comparisons between n-3 LC-PUFA in SF supernatants and MNC were undertaken by paired t-test.
3. Results
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Table 2 Correlation between stated daily fish oil dosea and n-3 LC-PUFA of phospholipids in plasma and synovial fluid (n¼ 36). Fatty acid
EPA DPA DHA EPA þDHA
Synovial fluid
Plasma rb
p Value
r
p Value
0.37 o 0.1 0.37 0.39
0.02 0.8 0.02 0.02
0.37 o 0.1 0.36 0.36
0.02 0.94 0.03 0.03
a Fish oil intakes are shown in Table 1 and varied from 0 to 30 mL/day of standard fish oil or equivalent. LC-PUFA, long chain polyunsaturated fatty acids; EPA; eicosapentaenoic acid; DPA, docosapentaenoic acid; DHA, docosahexaenoic acid. b Pearson correlation coefficient.
3.1. Stated FO intake Patients, who represented a variety of inflammatory arthropathies, reported consuming various amounts of FO within the range 0–30 mL/d as detailed in Table 1. The mean daily FO intake (excluding those who stated they were not taking FO) was 13.8 mL (SEM 1.29 mL) and the median 15 mL (IQR 10,15 mL). Of those taking FO, the mean duration of intake was 4.1 year (SEM 0.62 year) and median 3 year (IQR 0.67, 6 year). Eleven subjects stated they were taking FO in a dose of 15 mL daily.
3.2. Correlation between stated FO intake and n-3 LC-PUFA in phospholipids in SF and plasma There were statistically significant correlations between stated FO intake and both EPA and DHA in PL of SF (Table 2). The lack of correlation with n-3 docosapentaenoic acid (DPA) is not unexpected because FOs contains little DPA compared with EPA and DHA. Similar relationships were observed between stated dietary intake of FO and EPA and DHA in plasma PL (Table 2). Among the 11 subjects who stated they were taking FO 15 mL daily, the median EPA was 3.53 (range 2.8–8.1) and median total SF n-3 LC-PUFA was 10.4 (range 4.6–18.5) as percentage of SF PL fatty acids. Similar variability was seen in plasma PL n-3 LC-PUFA in this sub-group (EPA median 3.8; range 0.9–11.4; total n-3 LC-PUFA median 10; range 6.5–22.3 as percentage plasma PL fatty acids).
Table 3 Fatty acid composition of phospholipids in plasma and synovial fluid and correlations between them. Fatty acid
18:2n-6 (LA) 20:3n-6 (DGLA) 20:4n-6 (AA) Total n-6 LC-PUFA 18:3n-3 (ALA) 20:5n-3 (EPA) 22:5n-3 (DPA) 22:6n-3 (DHA) Total n-3 LC PUFA
Mean 7 SEM (n¼ 36) Plasma
Synovial fluid
r2a
p Value
177 0.7b 2.5 7 0.2 7.8 7 0.2 28.3 7 0.8 0.2 7 0.02 4.2 7 0.5 1.4 7 0.1 5.5 7 0.3 11.17 0.8
14.8 7 0.5 2.5 7 0.3 8.8 7 0.2 26.8 7 0.7 0.2 7 0.02 3.3 7 0.4 1.7 7 0.1 5.3 7 0.3 10.4 7 0.6
0.74 0.96 0.83 0.95 0.73 0.96 0.95 0.95 0.96
o 0.001 o 0.001 o 0.001 o 0.001 o 0.001 o 0.001 o 0.001 o 0.001 o 0.001
a
Linear regression. Individual fatty acids expressed as a percentage of total fatty acids in phospholipids. LA, linoleic acid; DGLA, dihomo-γ-linolenic acid; AA, arachidonic acid; ALA, α-linolenic acid; EPA, eicosapentaenoic acid; DPA, docosapentaenoic acid; DHA, docosahexaenoic acid. b
3.5. Comparison of fatty acids in the phospholipids of mononuclear cells from peripheral blood and synovial fluid There were no differences in any of the n-6 or n-3 fatty acids between MNC from blood and MNC from SF with the exception of DHA which was higher in the SF cells (Table 5).
3.3. Correlations between PUFA in the phospholipids of plasma and SF
3.6. Relationships between pain scores and n-3 LC-PUFA in phospholipids of plasma and synovial fluid
There were very strong correlations between plasma and SF values for the n-6 PUFA linoleic acid (LA; C18:2n-6), dihomo-γlinolenic acid (DGLA; C20:3n-6) and AA and the n-3 fatty acids ALA, EPA, DPA and DHA (Table 3).
Total n-3 PUFA in PL of plasma and SF correlated inversely with pain score (plasma r2 ¼0.16, p¼ 0.02; SF r2 ¼0.32, p¼ 0.001). Correlations were also found between EPA and DHA and pain score (Table 6). By contrast, there was not a significant correlation between stated FO intake and pain score (r2 ¼0.08, p ¼0.1) (data not shown).
3.4. Comparison between fatty acids in the phospholipids of SF supernatants and SF mononuclear cells 4. Discussion The 18-carbon n-6 fatty acid, LA, was substantially lower and its long chain 20-carbon metabolite, AA, was substantially higher in PL of SF MNC compared with PL in the supernatants of SF from which they were isolated. This pattern was not repeated for the n-3 PUFA where both 18- and 20-carbon fatty acids, ALA and EPA, were lower in SF MNC than in corresponding supernatants. The other n-3 PUFA, DPA and DHA, were similar in PL of SF MNC and SF supernatants (Table 4). Similar relationships were observed between n-3 LC-PUFA in PL of plasma and peripheral blood MNC (data not shown).
Analyses of n-3 LC-PUFA in plasma and cells within peripheral blood samples have proven useful in evaluating changes in n-3 LCPUFA status in tissues in response to experimental interventions with FO supplements or changes in amounts of n-3 LC-PUFA consumed as food [21,22]. Notwithstanding, to date fatty acid analyses have been applied systematically to few clinical settings beyond clinical trials. Support for routine use of PUFA analyses in guiding treatment and in evaluation of risk is found in the “omega-3 index” of erythrocyte EPA þDHA proposed by Harris
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Table 4 Fatty acid composition of phospholipids in synovial fluid (SF) supernatants and SF mononuclear cells (MNC) and correlations between them. Fatty acid
Mean 7 SEM (n¼ 11) SF supernatants
18:2n-6 (LA) 20:3n-6 (DGLA) 20:4n-6 (AA) Total n-6 LC-PUFA 18:3n-3 (ALA) 20:5n-3 (EPA) 22:5n-3 (DPA) 22:6n-3 (DHA) Total n-3 LC PUFA
b
17.4 7 0.5 2.8 7 0.3 8.8 7 0.3 30.0 7 0.5 0.2 7 0.03 1.7 7 0.2 2.0 7 0.5 4.0 7 0.3 7.6 7 0.4
Fatty acid SF MNC
p Valuea
8.6 7 0.7 1.7 7 0.2 12.9 7 1 25.5 7 1.6 0.017 0.01 0.9 7 0.3 2.3 7 0.4 4.0 7 0.3 7.2 7 0.7
o 0.001 o 0.001 0.001 0.02 0.002 o 0.001 0.6 0.9 0.5
a
Paired t-test. For this sub-group mean daily fish oil intake was 8.6 mL (SEM 1.80 mL), median 10 mL (IQR 4,15 mL). Comparisons are between SF MNC and the supernatants of the SF from which the MNC had been obtained. In some cases, the SF sample differed from the patient's sample which had contributed prior to plasma and SF correlations. b Individual fatty acids expressed as a percentage of total fatty acids. LA, linoleic acid; AA, arachidonic acid; DGLA, dihomo-γ-linolenic acid; ALA, α-linolenic acid; EPA, eicosapentaenoic acid; DPA, docosapentaenoic acid; DHA, docosahexaenoic acid.
Table 5 Fatty acid composition in phospholipids of mononuclear cells (MNC) in peripheral blood (PB) and synovial fluid (SF) and correlations between them. Fatty acid
18:2n-6 (LA) 20:3n-6 (DGLA) 20:4n-6 (AA) Total n-6 LC-PUFA 18:3n-3 (ALA) 20:5n-3 (EPA) 22:5n-3 (DPA) 22:6n-3 (DHA) Total n-3 LC PUFA
Table 6 Correlation between pain scores and n-3 LC-PUFA in phospholipids of plasma and synovial fluid (n¼ 29).
Mean 7 SEM (n¼ 9) PB MNC
SF MNC
p Valuea
7.4 7 0.7b 1.4 7 0.23 15.0 7 1.1 24.97 2.1 0.017 0.01 0.8 7 0.22 2.4 7 0.39 2.6 7 0.2 5.8 7 0.6
8.6 70.9 1.5 70.3 12.5 71.2 24.971 0.02 70.01 0.9 70.4 2.3 70.4 4.0 70.35 7.3 71.0
0.1 0.6 0.1 0.9 0.1 0.6 0.8 o 0.001 0.09
a
Paired t-test. Individual fatty acids expressed as a percentage of total fatty acids. LA, linoleic acid; AA, arachidonic acid; DGLA, dihomo-γ-linolenic acid; ALA, α-linolenic acid; EPA, eicosapentaenoic acid; DPA, docosapentaenoic acid; DHA, docosahexaenoic acid. b
and von Schacky as both an index of adequacy of supplementation and an estimator of cardiovascular risk [23]. The present study examines the potential utility of plasma n-3 LC PUFA profiles as a guide to SF levels of n-3 LC PUFA within a routine clinical practice setting. Navarro and co-workers studied RA and osteoarthritis patients who were not taking FO and reported reduced n-3 PUFA in SF in RA compared to osteoarthritis [24]. They did not report correlations between fatty acid profiles of plasma and SF. The present study shows that levels of n-3 LC PUFA in PL of plasma and SF are strongly correlated across a range of FO doses and different rheumatic diagnoses. By comparison, stated intake of FO was found to correlate weakly with EPA and DHA within the PL fractions of both plasma and SF supernatants. The strong correlations between PL n-3 LC-PUFA in SF and plasma indicate that, in patients with inflammatory arthropathies, n-3 LC-PUFA in plasma PL can be used as a surrogate for SF n-3 PUFA when monitoring dietary interventions aimed at increasing n-3 LC-PUFA in SF, whence they can be expected to have modulating effects on immune cells in synovium [22]. Functions potentially affected include phagocytosis, T cell signalling and antigen
EPA DHA Total n-3 LC PUFA a
Synovial fluid
Plasma r2a
p Value
r2
p Value
0.15 0.14 0.16
0.03 0.04 0.02
0.14 0.17 0.32
0.03 0.02 0.001
Linear regression.
presentation [22]. Further studies are warranted to assess the function of dendritic cells (DC), which can be isolated from SF [25], since exogenous EPA and DHA, when added in vitro to DC, which have been differentiated from human peripheral blood monocytes in vitro (MDDC), have been shown to reduce expression of HLA-DR and of co-stimulatory molecules in response to a maturation stimulus and to reduce allogeneic stimulation of T cells [26]. Interesting differences were found between LC-PUFA in SF supernatants and SF MNC. Notably AA was higher and LA and DGLA lower as a proportion of PL in SF MNC compared to SF supernatants. These differences suggest either preferential uptake of AA, or remodelling of n-6 precursors LA and DGLA to AA within SF MNC. Similar differences were seen between PUFA in PL of plasma and PB MNC. In contrast to AA, EPA was a higher proportion of PL in plasma and SF than in the respective MNC. It is notable that we have previously shown similar patterns of difference in respective abundance of LA, AA and EPA in plasma compared to PB MNC of healthy subjects whether taking FO supplements or not [4]. Accordingly, putative LC-PUFA remodelling, if it exists, is not likely to be confined to MNC activated at sites of inflammation. Based on randomised trials of efficacy and tolerance of FO in RA and other inflammatory diseases, the recommended dose of FO for anti-inflammatory effects is at least 9 g or 10 mL daily of standard FO (or a dose of a FO concentrate which delivers 2.7 g daily or more of EPA plus DHA as triglycerides) [3]. In the present study, the median stated dose of standard FO or equivalent was 15 ml/d and the mean dose 13.8 ml/d. Although some patients took higher doses, on average these doses are usual for medicinal use of FO for inflammatory diseases in trials and in practice [3,13]. Findings from clinical trials and practice experience with medicinal use of FO suggest that these doses are generally safe and are not associated with increased bleeding tendency. Four subjects in the present study took 30 mL daily. While clinical trials experience with this dose is limited, we observed no abnormal bleeding episodes in these patients. Furthermore this dose is within the range of doses (up to 5 g/daily of EPA plus DHA) deemed safe by the European Food Safety Authority [27]. Sears and co-workers used the plasma AA/EPA ratio to monitor n-6/n-3 LC-PUFA balance in the management of brain injury [28]. They chose a plasma AA/EPA ratio of unity to determine an arbitrary therapeutic ceiling for treatment with enterically administered n-3 LC-PUFA with the intent of avoiding abnormal bleeding events. These authors described two patients with brain injury who were given EPA 10.8 g and DHA 5.4 g daily in divided doses. This dose of EPA plus DHA equates with the EPA plus DHA content of 45 g or 49 mL of standard FO daily. However, EPA and DHA in their preparation were in the form of ethyl esters which are significantly less well absorbed than EPA and DHA within the natural triglycerides of FO [29]. While Sears et al. report no bleeding episode, they reduced the dose of EPA plus DHA when the plasma AA/EPA ratio was observed to fall below unity in one of their patients. We have
M. Moghaddami et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 97 (2015) 7–12
followed patients who stated they were taking FO 30 mL daily for at least one year and in some cases for several years. No significant bleeding events have been encountered. The median plasma PL AA/EPA ratio after at least 12 months of FO at the dose of 30 ml daily was 0.68 (IQR 0.48–1.07, n¼23) (unpublished data). While these findings suggest the ceiling ratio of Sears et al. is conservative, the principle of utilising LC PUFA levels as a guide for dose adjustment for efficacy and safety appears to have merit. The evidence for an increased bleeding tendency with medicinal use of FO is weak [30] as acknowledged by Sears and coauthors [28]. Concern regarding the potential for increased bleeding with FO supplements seems to have its origins in reduced platelet aggregation and increased bleeding time observed in Greenland Inuits consuming their native diet [31]. However, this predisposition is likely contributed to by the paucity of n-6 fatty acids in the Inuit diet relative to Western diets [32], in addition to the very high n-3 LC-PUFA content of the Inuit diet, the mean of which has been estimated to be as high as 13 g/d [32]. The n-3 LC-PUFA content of the Inuit diet contrasts with EPA plus AA of 4.1 g in a 15 mL daily dose of standard FO used medicinally. Furthermore, in clinical trials in which FO at or near anti-inflammatory doses was given with warfarin or aspirin plus clopridogrel, no increase in bleeding events was seen in subjects taking FO compared to their counterparts taking these anti-thrombotics without FO [33,34]. Thus, while it is not possible to give categorical reassurance regarding the potential for undesirable effects of FO supplements in situations where bleeding is a special risk, the level at which n6/n3 balance becomes hazardous in this regard remains to be determined. Four subjects stated they were not taking FO at the time of arthrocentesis. Interestingly, their levels of EPA (1.870.1, mean7SEM), DHA (4.770.2) and total n-3 PUFA (8.270.3) as a proportion of plasma PL are higher than values observed in local healthy volunteers [35]. On the other hand, their levels are lower than those found in subjects taking FO in anti-inflammatory doses in a clinical trial setting [36]. In the present study, patients, who stated they were taking FO 15 mL daily, displayed considerable variability in n-3 LC-PUFA in plasma and SF. These differences may reflect a mismatch between statements and behaviour, as well as metabolic differences between subjects, and underline the potential advantages of measurement of levels in blood in order to assess n-3 status. Meta-analysis of studies assessing musculoskeletal pain indicates that FO improves pain outcomes, particularly with respect to patient assessed pain, duration of morning stiffness and number of painful and/or tender joints [12]. We observed inverse relationships between pain score and n-3 LC-PUFA in plasma and SF. n-3 LC-PUFA could potentially reduce pain by several mechanisms. EPA and DHA are competitive inhibitors of metabolism of AA by cyclooxygenase [37], which provides a mechanism for inhibition of synthesis of the nociceptive mediator prostaglandin E2 by FO treatment [38]. Products of EPA and DHA metabolism include resolvins, protectins and maresins, which may contribute to pain relief through actions that help to resolve inflammation [17,39]. DHA has also been shown to modulate pain through an effect on opioid-receptors [39]. In conclusion, plasma PL PUFA analyses were an accurate predictor of SF values across a range of FO doses and different rheumatic diagnoses. Plasma n-3 LC-PUFA levels are a substantially more accurate predictor of SF n-3 LC-PUFA than stated dietary intakes of FO and also correlated with lesser pain experience.
Acknowledgements This work was funded in part by research Grants from Arthritis Australia and the Royal Adelaide Hospital Research Fund.
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