Peripheral blood mononuclear cell fatty acid ... - Clinical Nutrition

ARTICLE IN PRESS Clinical Nutrition (2004) 23, 647–655

www.elsevier.com/locate/clnu

ORIGINAL ARTICLE

Peripheral blood mononuclear cell fatty acid composition and inflammatory mediator production in adult Crohn’s disease Timothy M. Trebblea,*,1, Nigel K. Ardenb, Stephen A. Woottona, Mark A. Mulleec, Philip C. Caldera, Graham C. Burdgea, David R. Finec, Mike A. Strouda,d a

School of Medicine, Institute of Human Nutrition, University of Southampton, Tremona Road, Southampton SO16 6YD, UK b MRC Environmental Epidemiology Unit, University of Southampton, Tremona Road, Southampton SO16 6YD, UK c Medical Statistics, Health Care Research Unit, University of Southampton, Tremona Road, Southampton SO16 6YD, UK d Department of Gastroenterology, Southampton University Hospitals Trust, Tremona Road, Southampton SO16 6YD, UK Received 4 July 2003; accepted 31 October 2003

KEYWORDS Mononuclear cell; Cytokines; Polyunsaturated fatty acids; Crohn’s disease

Summary Background & aims: Crohn’s disease (CD) is associated with nutritional deficiencies, altered plasma concentrations of polyunsaturated fatty acids (PUFA) and an anti-inflammatory response to fish oil that contains n-3 PUFA. This suggests that, in CD, immune cells may have altered n-3 PUFA composition with functional consequences. The aim of this study is to investigate n-3 and n-6 PUFA composition and synthetic function of peripheral blood mononuclear cells (PBMC) in the basal state. Methods: A case control study of 52 adult CD patients and healthy, age- and sexmatched controls. Composition of PBMC and plasma phospholipids were measured by gas chromatography and production of tumour necrosis factor-a, prostaglandin E2 (PGE2) and interferon-g (IFN-g) by PBMC were measured by ELISA. Results: CD was associated with higher concentrations of eicosapentaenoic acid and other n-3 PUFA, and lower arachidonic acid (AA) (n-6 PUFA) in PBMC. This was not explained by differences in dietary fat intake. Lower rates of production of PGE2 and IFN-g by PBMC were noted in quiescent and active CD, respectively, compared to controls. Conclusions: CD is associated with a greater availability, and not a deficiency, of n3 PUFA in PBMC, but lower concentrations of AA, and lower rates of production of PGE2 and IFN-g, compared to healthy controls. & 2003 Elsevier Ltd. All rights reserved.

*Corresponding author. 1 Supported by grants to TT from The Southampton Rheumatology Trust, South and East NHS Executive Research & Development; Nutricia Clinical Care. S0261-5614/$ - see front matter & 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.clnu.2003.10.017

ARTICLE IN PRESS 648

T.M. Trebble et al.

increased production of pro-inflammatory mediators, (TNF-a, PGE2 and interferon-g (IFN-g)).

Introduction Crohn’s disease (CD) is a chronic inflammatory condition of the gastrointestinal tract. CD is associated with osteoporosis and other extraintestinal manifestations by an unconfirmed mechanism that may involve systemic exposure to soluble inflammatory mediators including tumour necrosis factor-a (TNF-a) and prostaglandin E2 (PGE2).1–4 Peripheral blood mononuclear cells (PBMC), isolated from the systemic circulation, demonstrate a capacity to produce such mediators that may be altered in CD.5–9 CD is associated with malnutrition,10 dietary restrictions,11 a reduced body fat mass,10,12 and nutritional deficiencies.12,13 Eicosapentaenoic acid (EPA) is an n-3 polyunsaturated fatty acid (PUFA) acquired directly from the diet or by metabolism from the essential fatty acid a-linolenic acid (aLNA) (Fig. 1). EPA is incorporated within mononuclear cells and competes with the n-6 PUFA arachidonic acid (AA) as substrates in the production of eicosanoids that result in contrasting effects on the inflammatory response.14 In healthy subjects dietary fish oil supplementation increases EPA and decreases AA concentration in PBMC14 and inhibits production of PGE2 and TNF-a.15,16 In CD, dietary fish oil supplementation results in a therapeutic anti-inflammatory response and a reduction in systemic markers of inflammation.17 There is evidence that differences in plasma concentrations of EPA and AA occur in CD in the basal state compared to healthy subjects.18–22 The relative availabilities of EPA, AA and other PUFA in PBMC in CD are uncertain but, if modulated, may have functional consequences with respect to production of eicosanoids and cytokines. We hypothesised that CD is associated with a reduced concentration of EPA within PBMC and compared to healthy controls and, as a consequence, an DPA via δ6, E,δ5

αLNA (EFA)

LA (EFA)

Prostaglandin synthesis

δ5

δ6, E

DGLA

DHA

EPA

AA

Figure 1 Schematic representation of synthetic pathways of n-3 and n-6 long chain polyunsaturated fatty acids (PUFA) from dietary essential fatty acids (EFA) n-3 PUFA: g-linolenic acid (aLNA); eicosapentaenoic acid (EPA); docosapentaenoic acid (DPA); docosahexaenoic acid (DHA); n-6 PUFA: linoleic acid (LA); dihomo-glinolenic acid (DGLA); arachidonic acid (AA). d5-desaturase (d5); d6-desaturase (d6); elongase (E).

Methods Subjects This was a case control study of CD patients and age- and sex-matched healthy controls. Patients were identified through a database of CD outpatients under active and recent follow-up at a large university hospital. The diagnosis of CD was based on endoscopic, histological or radiological findings. All recruited patients underwent a questionnaire-based assessment of previous gastroenterological and medical history, and medical notes were reviewed for further information. The CD cohort was stratified into active and quiescent disease groups by laboratory markers of disease activity. Active disease was indicated by a CRP of greater than 10 mg/l23 and/or an erythrocyte sedimentation rate (ESR) of greater than 20 mm/h. A secondary analysis was performed by stratifying the CD cohort by the Crohn’s Disease Activity Index (CDAI),24 a composite clinical disease score assessing symptoms (stool frequency, abdominal pain and general well being), extra-intestinal manifestations of CD, use of anti-diarrhoeals, presence of an abdominal mass, weight differential from predicted weight and haematocrit. A score of greater than 150 indicated active disease.24 Age- and sex-matched control subjects were recruited from amongst academic, clinical and general staff. All subjects underwent assessment of body height, using a CMS Stadiometer, weight, using electronic scales (Solude), and body mass index (weight (kg)/height(m)2). A validated semiquantitative self-administered Food Frequency Questionnaire25 was completed at the time of sample collection to estimate habitual dietary nutrient intake. Subjects were excluded if they required nutritional support in any form, had undergone major small bowel intestinal resection, total or sub-total colectomy; exhibited any features of short bowel or evidence of inflammatory disease unrelated to CD, had received oral or intravenous corticosteroid medication within the previous 4 weeks; had a history of hyperlipidaemia or diabetes mellitus; or had recently consumed n-3 PUFA dietary supplements. The study was approved by the Southampton and South West Hampshire Joint Research Ethics Committee. All subjects gave informed consent.

ARTICLE IN PRESS PBMC composition and function in Crohn’s disease

Cell and plasma preparation For assessment of fatty acid composition and synthetic function of PBMC, peripheral venous blood samples (30 ml) were taken into heparinised bottles from the ante-cubital fossa following an overnight fast. Blood was layered over 20 ml of Histopaque (density 1.077 g/ml; Sigma Chemical Co., Poole, United Kingdom) and centrifuged (720g) for 15 min at 201C. The plasma layer was removed and stored at 701C. The PBMC layer was collected from the interface, washed and resuspended in medium (RPMI containing 1.875 mmol/l glutamine and antibiotics). A second cycle was performed to reduce erythrocyte contamination. Cells were resuspended in 1 ml of medium, counted and removed for culture.

Fatty acid composition analysis Internal standards (phosphatidylcholine (PC) 15:0/ 15:0 and phosphatidylethanolamine (PE) 17:0/17:0) were added to all samples prior to analysis. Total plasma (1 ml) and PBMC lipids were isolated by extraction with chloroform/methanol (2:1 vol/ vol)26 containing 50 mg/l butylated hydroxytoluene. Plasma PC was purified by solid phase extraction on aminopropylsilica cartridges (Varian, Surrey, UK). Plasma PC and total PBMC fatty acids were converted to methyl esters by incubation with methanol containing 2% (vol/vol) sulphuric acid at 501C for 18 h. Fatty acid methyl esters were separated, re-dissolved in hexane and analysed by capillary gas chromatography using a HewlettPackard 5890 GC (Hewlett-Packard, Stockport, Ches., UK) equipped with an HP7686 GC autosampler using a BPX-70 fused silica capillary column (50 m 0.25 mm 0.32 mm) with flame ionisation detection. Peaks were identified by retention times relative to standards. Fatty acids are reported as proportionate values (g/100g total fatty acids). Coefficient of variation was less than 5% for determination of fatty acid composition.

Analysis of cytokine and eicosanoid production by PBMC Purified PBMC at a concentration of 1 106 cells/ml were incubated in 2 ml medium containing 5% autologous plasma, with and without the monocyte/macrophage stimulant lipopolysaccharide (LPS) at a concentration of 15 mg/l (for TNF-a and PGE2) or the T-cell stimulant concanavalin A (Con A) at a concentration of 25 mg/l (IFN-g), both for 24 h.

649

After this, culture plates were centrifuged (180g) for 10 min at 201C and supernatant removed and frozen at 301C. TNF-a, and IFN-g concentrations were determined using EASIATM ELISA kits (Biosource Europe S.A., Nivelles, Belgium). PGE2 concentrations were determined using NEOGENt ELISA kits (Neogen Corporation, Lexington, KY). Kits were used according to the manufacturer’s instructions. Co-efficient of variation was less than 10% for both cytokine and prostaglandin assays, and the limits of detection were to 3 ng/l for TNF-a and 0.03 kIU/l for IFN-g.

Statistical analysis The primary outcome variables, the relative concentrations of n-3 and n-6 PUFA in PBMC and plasma PC and TNF-a, IFN-g and PGE2 production by PBMC, were compared between the CD cohort, and following stratification into active and quiescent disease groups, with healthy matched control groups. Statistical comparisons between groups were performed using paired and two-sample ttests for parametric variables or Wilcoxon signedrank and Mann–Whitney U tests for non-parametric variables. Correlations of non-parametric variables were performed using Spearman’s rank correlations. Probabilities o0.01 were considered significant to adjust for multiple testing. Analyses were performed using SPSS for Windows (SPSS Inc., Chicago, IL).

Results Subjects Fifty-two CD patients were recruited with age- and sex-matched healthy controls. The CD cohort was stratified into active (n ¼ 28) and quiescent (n ¼ 24) disease groups based on laboratory markers of inflammation (CRP and ESR). Baseline characteristics of CD and control groups are shown in Table 1. Dietary fat intake, quantitatively and qualitatively, were similar between active and quiescent CD, and compared to controls (Table 2). Values for CDAI, CRP and ESR in the active CD group were consistent with mild to moderate disease.

Fatty acid composition in PBMC Proportionate concentrations of aLNA, EPA and docosapentaenoic acid (DPA) in PBMC were significantly higher in the CD cohort (n ¼ 52) compared to controls, and AA was significantly lower (Table 3).

ARTICLE IN PRESS 650

T.M. Trebble et al.

Table 1 Characteristics of active and quiescent Crohn’s disease patients (stratified by CRP and ESR) and matched healthy controls. Crohn’s disease

Controls

Quiescent (n ¼ 24)

Active (n ¼ 28)

(n ¼ 52)

Sex Male Female

12 12

9 19

21 31

Mean age7SD (yr) Mean disease duration7SD (yr)

41.5711.9 11.178.6

41.5712.7 11.478.3

41.0712.4 F

Disease site Small bowel Large bowel Small and large bowel Perianal

7 9 7 1

4 11 13

Previous intestinal resection

6

5

Current drug history Azathioprine 5-ASA

9 2

1 3

Markers of disease activity Mean CDAI7SD Median CRP (IQR) (mg/l) Mean ESR7SD (mm/h) Nutritional status Mean weight7SD (kg) Mean BMI7SD (kg/m2)

138772 3.7 (2.1,6.5) 10.975.2

172784 14.7 (10.6,21.3) 22.5713.0

73.5710.5 25.573.7

Current smoking

72.7713.7 28.176.5

9

10

0.1 (0.0,0.9)

70.0713.3 24.573.8 9

Table 2 Dietary intake of active and quiescent Crohn’s Disease patients (stratified by CRP and ESR) and matched healthy controls.

Energy (kcal) Saturated fat (g/d) Monounsaturated fat (g/d) n-6 polyunsaturated fat (g/d) n-3 polyunsaturated fat (g/d) Vitamin E (mg/d)

Control (n ¼ 24)

Quiescent Crohn’s disease (n ¼ 24) Mean7SD

Mean7SD

26147753 30.7713.4 27.6710.7 12.879.7 1.971.0 9.675.8

282671410 27.8715.9 28.9717.2 12.278.3 1.870.9 10.376.9

Pn

0.714 0.850 0.883 0.579 0.456 0.941

Active Crohn’s disease (n ¼ 28) Mean7SD

Control (n ¼ 28)

278971626 30.0716.0 26.3712.4 11.076.3 1.770.8 8.775.6

275971028 28.6713.5 27.4712.3 12.376.4 1.870.9 10.875.0

Pn

Mean7SD 0.918 0.364 0.353 0.731 0.890 0.928

n

Paired t-test.

Differences between CD patients and controls remained for AA and EPA when CD was stratified as either active or quiescent disease, but aLNA was not significantly different in active (P ¼ 0:019) or quiescent (P ¼ 0:155) CD independently, and DPA

was significantly higher in quiescent (P ¼ 0:007) but not active (P ¼ 0:175) CD. There were no significant differences between active with quiescent CD in PUFA composition.


651

Table 3 Fatty acid composition (g/100g total fatty acids) of peripheral blood mononuclear cells in Crohn’s disease patients and matched healthy controls.

LA aLNA DGLA AA EPA DPA DHA

Crohn’s disease (n ¼ 52) Mean7SD

Control (n ¼ 52) Mean7SD

Mean difference (95% CI)

Pn

8.2271.48 0.4670.32 2.3770.51 20.1872.44 0.7570.57 3.4071.36 2.6870.72

8.0470.95 0.3170.24 2.2770.49 23.9271.49 0.3170.28 2.7570.85 2.8570.72

0.18 (0.35, 0.70) 0.15 (0.04, 0.25) 0.09 (0.10, 0.28) 3.74 (4.60, 2.88) 0.44 (0.27, 0.62) 0.64 (0.23, 1.05) 0.17 (0.42, 0.07)

0.502 0.007 0.326 o0.001 o0.001 0.003 0.158

n-3 PUFA: a-linolenic acid (LNA); eicosapentaenoic acid (EPA); docosapentaenoic acid (DPA); docosahexaenoic acid (DHA). n-6 PUFA: linoleic acid (LA); dihomo-g-linolenic acid (DGLA); arachidonic acid (AA). n Paired t-test.

Fatty acid composition in plasma phosphatidylcholine The CD cohort (n ¼ 52) was associated with significantly lower docosahexaenoic acid (DHA) (P ¼ 0:005) compared to controls, but there were no other significant differences (data not shown). Active CD was associated with significantly lower DPA and DHA, and higher dihomo-g-linolenic acid (DGLA) compared to controls (Table 4). There were no significant differences between the quiescent CD group and controls (Table 4). DPA was significantly lower in active compared to quiescent CD (P ¼ 0:001), but there were no other significant differences.

Production of inflammatory mediators by PBMC The CD cohort (n ¼ 52) was associated with significantly lower production of IFN-g by Con Astimulated PBMC (P ¼ 0:003) compared to controls (data not shown), but no other differences were noted. Quiescent CD was associated with significantly lower production of PGE2 by unstimulated PBMC and active CD was associated with significantly lower production of IFN-g by Con A-stimulated PBMC, compared to controls (Table 5). There were no differences in cytokine production between active and quiescent CD.

Effect of disease stratification by clinical disease score (CDAI) CDAI did not correlate with CRP (Spearman’s r ¼ 0:087; P ¼ 0:540) or ESR (Spearman’s r ¼ 0:155; P ¼ 0:303). Stratification of the CD cohort into

active and quiescent groups by CDAI and laboratory markers of inflammation was congruous in only half of patients (Table 6). Concentrations of AA and EPA within PBMC in active and quiescent CD were similar to the cohort as whole, and similarly different from control values (data not shown). There were no significant differences in PUFA profiles of plasma PC in active and quiescent CD compared to controls, but a trend was noted towards a lower DHA in quiescent disease (P ¼ 0:033). Active CD was associated with a significantly lower production of IFN-g by Con A-stimulated PBMC compared to controls. No other differences in PBMC synthetic function were noted in active or quiescent disease compared to controls. On comparison of active and quiescent CD, there was a trend towards higher production of TNF-a by LPSstimulated PBMC in active compared to quiescent CD (P ¼ 0:022), but no other differences.

Discussion To our knowledge the current study is the first to demonstrate alterations in the proportions of n-3 and n-6 PUFA in PBMC in CD, compared to healthy matched controls. CD was associated with higher aLNA, the principle source of n-3 PUFA in the UK diet, and higher EPA, the principle source in fish oil. By comparison, AA, the main substrate in the production of prostaglandins and other eicosanoids, was lower in CD. The differences in EPA and AA were noted in both active and quiescent CD compared to respective controls and there were no significant differences associated with greater inflammatory disease activity. There is insufficient

LA aLNA DGLA AA EPA DPA DHA


Control (n ¼ 24)

22.273.2 0.3670.12 3.470.8 8.571.6 1.5270.85 1.270.3 3.871.5

22.772.2 0.3970.14 3.170.8 9.171.9 1.3070.54 1.270.3 4.471.4


Pn

Mean7SD 0.47 (2.29, 1.35) 0.03 (0.09, 0.36) 0.30 (0.18, 0.78) 0.58 (1.29, 0.13) 0.22 (0.19, 0.62) 0.05 (0.10, 0.21) 0.56 (1.39, 0.28)

0.598 0.366 0.209 0.102 0.287 0.471 0.184


Control (n ¼ 28)

20.973.3 0.3570.13 3.870.8 9.271.8 1.2170.70 1.070.3 3.771.4

23.073.2 0.3370.09 3.270.7 9.571.9 1.4170.53 1.270.2 4.671.3


Pn

2.16 (3.94, 0.38) 0.02 (0.05, 0.08) 0.62 (0.23, 1.00) 0.26 (1.38, 0.87) 0.21 (0.55, 0.13) 0.20 (0.34, 0.05) 0.84 (1.43, 0.25)

0.019 0.592 0.003 0.646 0.215 0.009 0.007

652

Table 4 Fatty acid composition of plasma phosphatidylcholine (g/100g fatty acids) active and quiescent Crohn’s disease patients (stratified by CRP and ESR) and matched healthy controls.

Mean7SD

ARTICLE IN PRESS

n-3 PUFA: a-linolenic acid (LNA); eicosapentaenoic acid (EPA); docosapentaenoic acid (DPA); docosahexaenoic acid (DHA); n-6 PUFA: linoleic acid (LA); dihomo-g-linolenic acid (DGLA); arachidonic acid (AA). n Paired t-test.

Table 5 Synthesis of tumour necrosis factor-a (TNF-a), prostaglandin E2 (PGE2), and interferon-g (IFN-g) by unstimulated or stimulated PBMC in active and quiescent Crohn’s disease patients (stratified by CRP and ESR) and matched healthy controls.

n




Pn




Pn

None LPS None LPS None Con A

9430720896 860875929 13.7723.6 18.1730.7 3.0177.09 118.17101.6

12397739718 843375203 40.0737.7 37.4735.2 3.0475.87 144.97109.5

2967 (21775, 15840) 174 (3194, 3542) 26.2 (44.8, 7.6) 19.2 (41.7, 3.2) 0.04 (4.08, 4.01) 26.8 (80.0, 26.5)

0.747 0.916 0.008 0.089 0.983 0.309

465378488 591875961 37.8752.1 23.9740.3 4.94710.18 86.8789.4

5589714811 532573063 22.3725.3 23.9742.1 3.9677.85 163.7793.8

936 (5140, 3268) 592 (1525, 2710) 15.5 (7.0, 38.0) 0.0 (24.0, 24.0) 0.99 (4.30, 6.27) 77.0 (124.6, 29.3)

0.650 0.571 0.168 0.999 0.705 0.003

Paired t-test.

T.M. Trebble et al.

TNF-a (ng/l) PGE2 (ng/ml) IFN-g (kIU/l)

Stimulus


653

Table 6 Frequency of active and quiescent CD patients stratified by clinical (CDAI) and laboratory markers of inflammation (CRP and ESR). Disease activity by CDAI

Disease activity by CRP and ESR

Active (n) Quiescent (n) Total

data in the published literature to compare aLNA, EPA and AA profiles of PBMC from CD cohorts by other authors. In plasma PC, DHA, an n-3 PUFA also found in fish oil, was significantly lower in the CD cohort (and in active CD) compared to healthy matched controls. this is consistent with the results of Geerling et al.20,21 and Kuroki et al.22 but differs with those from Esteve-Comas18,19 that noted high DHA and low AA. The possible reasons for these conflicting findings include, firstly, that different studies have reported the fatty acid composition of different plasma lipid pools and, secondly, that the severity of CD in different studies may have been different. To our knowledge, the current study is the first to demonstrate lower levels of production of PGE2 by a cohort of CD patients compared to healthy matched controls. Watkins et al.27 propose that the synthesis of PGE2 may reflect both substrate availability and enzyme activity of the prostaglandin synthetic pathway. The results of the current study are consistent with this, in that a lower content of AA in PBMC was associated with lower rates of synthesis of PGE2. The profile of higher EPA and lower AA content in PBMC from CD patients, in the current study, was unexpected in view of the results of dietary fish oil intervention studies described in the published literature. Belluzzi et al.17 demonstrated that dietary fish oil in active CD results in a therapeutic anti-inflammatory response that might suggest a nutritional deficiency of EPA in the basal state; however in the current study a paradoxically greater availability of EPA was noted in PBMC in CD patients including those with active inflammatory disease. Furthermore, the profile of high EPA and low AA noted in CD patients in the current study, including those with active inflammatory disease, mimics the profile noted in healthy subjects following increased dietary intake of EPA which is associated with down-regulation of PBMC immune function (see Calder for a review.14) The differences in n-3 and n-6 PUFA content in PBMC between CD patients and controls, in the current study, cannot be simply explained by differences in

Active (n)

Quiescent (n)

Total

12 14 26

12 14 26

24 28 56

dietary fat intake or nutritional status. Consistent with reports by other authors,20 there were no significant differences in dietary fat intakes between CD and controls, and total n-6 and n-3 PUFA intakes in CD were comparable to average adult values in the UK (10.2 g/d and 1.8 g/d, respectively).28 Furthermore, body mass index, which crudely reflects body fat mass, was similar between quiescent and active CD groups and compared to controls. These differences, therefore, may reflect alterations in the respective rates of utilisation of EPA and AA for the synthesis of inflammatory mediators and other products (Fig. 1). The results of this study may have implications for the use of dietary fish oil in CD. In order to synthesise prostaglandins and other eicosanoids, the PBMC utilises AA from both endogenous and plasma sources.29 In healthy subjects, dietary fish oil supplementation increases the content of EPA and decreases the content of AA in PBMC30 and plasma.31 Similarly, in CD, dietary fish oil supplementation increases EPA, DHA and aLNA content and decreases AA content of erythrocyte membranes and plasma phospholipid.32 The response to dietary fish oil supplementation in CD, therefore, may include a further increase in the apparent imbalance between the availability of EPA and the availability of AA in peripherally circulating immune cells. This may further lower the rates of production of prostaglandins and other eicosanoids by PBMC within the systemic circulation, with possible consequences for a number of physiological systems that include bone turnover.33 CD is associated with an increased prevalence of reduced bone mass leading to osteoporosis34 and it may be speculated that alterations in the availability of AA and production of PGE2 following fish oil dietary supplementation may lead to therapeutic benefits with respect to bone loss. The primary method of stratification for inflammatory disease activity involved values for laboratory markers of inflammation (CRP and ESR). Both CRP and ESR are predictive of disease relapse,17,35,36 and, it is postulated, are a more sensitive indicator of low grade or sub-clinical

ARTICLE IN PRESS 654 inflammation.36,37 The value of a clinical disease score in assessing inflammatory activity in CD is uncertain. In conclusion, the population in the current study represent quiescent to mild/moderate disease activity, were not underweight (by BMI) and are typical of patients routinely assessed in a gastroenterology outpatient’s clinic in the UK The results of the current study demonstrate that there was significantly lower availability of AA in PBMC with possible functional consequences regarding production of PGE2. There was no evidence of lower availability of EPA in plasma or PBMC in CD despite the therapeutic benefits of dietary fish oil supplementation described in the published literature.17 An intervention study to assess the response of PBMC function and systemic manifestations of CD (including bone turnover) to PUFA dietary supplementation is indicated.

References 1. MacDonald BR, Gowen M. Cytokines and bone. Br J Rheumatol 1992;31:149–55. 2. Warren RS, Starnes Jr. HF, Gabrilove JL, Oettgen HF, Brennan MF. The acute metabolic effects of tumor necrosis factor administration in humans. Arch Surg 1987;122:1396–400. 3. Baumann H, Gauldie J. The acute phase response. Immunol Today 1994;15:74–80. 4. Beisel WR. Herman award lecture 1995: infection-induced malnutritionFfrom cholera to cytokines. Am J Clin Nutr 1995;62:813–9. 5. Bouma G, Oudkerk PM, Scharenberg JG, et al. Differences in the intrinsic capacity of peripheral blood mononuclear cells to produce tumor necrosis factor alpha and beta in patients with inflammatory bowel disease and healthy controls. Scand J Gastroenterol 1995;30:1095–100. 6. Nakamura M, Saito H, Kasanuki J, Tamura Y, Yoshida S. Cytokine production in patients with inflammatory bowel disease. Gut 1992;33:933–7. 7. Mazlam MZ, Hodgson HJ. Peripheral blood monocyte cytokine production and acute phase response in inflammatory bowel disease. Gut 1992;33:773–8. 8. Bernstein CN, Sargent M, Rawsthorne P, Rector E. Peripheral blood lymphocyte beta 2 integrin and ICAM expression in inflammatory bowel disease. Dig Dis Sci 1997;42:2338–49. 9. Pallone F, Fais S, Squarcia O, Biancone L, Pozzilli P, Boirivant M. Activation of peripheral blood and intestinal lamina propria lymphocytes in Crohn’s disease. In vivo state of activation and in vitro response to stimulation as defined by the expression of early activation antigens. Gut 1987;28: 745–53. 10. Capristo E, Mingrone G, Addolorato G, Greco AV, Gasbarrini G. Metabolic features of inflammatory bowel disease in a remission phase of the disease activity. J Intern Med 1998; 243:339–47. 11. Rigaud D, Angel LA, Cerf M, et al. Mechanisms of decreased food intake during weight loss in adult Crohn’s disease patients without obvious malabsorption. Am J Clin Nutr 1994;60:775–81.

T.M. Trebble et al.

12. Geerling BJ, Badart-Smook A, Stockbrugger RW, Brummer RJ. Comprehensive nutritional status in patients with longstanding Crohn’s disease currently in remission. Am J Clin Nutr 1998;67:919–26. 13. Harries AD, Heatley RV. Nutritional disturbances in Crohn’s disease. Postgrad Med J 1983;59:690–7. 14. Calder PC. n-3 polyunsaturated fatty acids, inflammation and immunity: pouring oil on troubled waters or another fishy tale? Nutr Res 2003;21:309–41. 15. Endres S, Ghorbani R, Kelley VE, et al. The effect of dietary supplementation with n-3 polyunsaturated fatty acids on the synthesis of interleukin-1 and tumor necrosis factor by mononuclear cells. N Engl J Med 1989;320:265–71. 16. Meydani SN, Endres S, Woods MM, et al. Oral (n-3) fatty acid supplementation suppresses cytokine production and lymphocyte proliferation: comparison between young and older women. J Nutr 1991;121:547–55. 17. Belluzzi A, Brignola C, Campieri M, Pera A, Boschi S, Miglioli M. Effect of an enteric-coated fish-oil preparation on relapses in Crohn’s disease. N Engl J Med 1996;334: 1557–60. 18. Esteve-Comas M, Nunez MC, Fernandez-Banares F, et al. Abnormal plasma polyunsaturated fatty acid pattern in non-active inflammatory bowel disease. Gut 1993;34: 1370–3. 19. Esteve-Comas M, Ramirez M, Fernandez-Banares F, et al. Plasma polyunsaturated fatty acid pattern in active inflammatory bowel disease. Gut 1992;33:1365–9. 20. Geerling BJ, Houwelingen AC, Badart-Smook A, Stockbrugger RW, Brummer RJ. Fat intake and fatty acid profile in plasma phospholipids and adipose tissue in patients with Crohn’s disease, compared with controls. Am J Gastroenterol 1999;94:410–7. 21. Geerling BJ, Houwelingen AC, Badart-Smook A, Stockbrugger RW, Brummer RJ. The relation between antioxidant status and alterations in fatty acid profile in patients with Crohn’s disease and controls. Scand J Gastroenterol 1999; 34:1108–16. 22. Kuroki F, Iida M, Matsumoto T, Aoyagi K, Kanamoto K, Fujishima M. Serum n-3 polyunsaturated fatty acids are depleted in Crohn’s disease. Dig Dis Sci 1997;42:1137–41. 23. Leiper K, Woolner J, Mullan MM, et al. A randomised controlled trial of high versus low long chain triglyceride whole protein feed in active Crohn’s disease. Gut 2001;49: 790–4. 24. Best WR, Becktel JM, Singleton JW, Kern Jr. F. Development of a Crohn’s disease activity index. National cooperative Crohn’s disease study. Gastroenterology 1976;70:439–44. 25. Shaheen SO, Sterne JA, Thompson RL, Songhurst CE, Margetts BM, Burney PG. Dietary antioxidants and asthma in adults: population-based case-control study. Am J Respir Crit Care Med 2001;164:1823–8. 26. Burdge GC, Wright P, Jones AE, Wootton SA. A method for separation of phosphatidylcholine, triacylglycerol, nonesterified fatty acids and cholesterol esters from plasma by solid-phase extraction. Br J Nutr 2000;84:781–7. 27. Watkins BA, Lippman HE, Le Bouteiller L, Li Y, Seifert MF. Bioactive fatty acids: role in bone biology and bone cell function. Prog Lipid Res. 2001;40:125–48. 28. British Nutrition Foundation. n-3 Fatty acids and health. 1999. 29. Brash AR. Arachidonic acid as a bioactive molecule. J Clin Invest 2001;107:1339–45. 30. Yaqoob P, Pala HS, Cortina-Borja M, Newsholme EA, Calder PC. Encapsulated fish oil enriched in alpha-tocopherol alters plasma phospholipid and mononuclear cell fatty acid


compositions but not mononuclear cell functions. Eur J Clin Invest 2000;30:260–74. 31. Gibney MJ, Hunter B. The effects of short- and long-term supplementation with fish oil on the incorporation of n-3 polyunsaturated fatty acids into cells of the immune system in healthy volunteers. Eur J Clin Nutr 1993;47:255–9. 32. Belluzzi A, Brignola C, Campieri M, et al. Effects of new fish oil derivative on fatty acid phospholipid-membrane pattern in a group of Crohn’s disease patients. Dig Dis Sci 1994; 39:2589–94. 33. Watkins BA, Li Y, Seifert MF. Lipids as modulators of bone remodelling. Curr Opin Clin Nutr Metab Care 2001; 4:105–10.

655

34. Andreassen H, Rungby J, Dahlerup JF, Mosekilde L. Inflammatory bowel disease and osteoporosis. Scand J Gastroenterol 1997;32:1247–55. 35. Brignola C, Campieri M, Bazzocchi G, Farruggia P, Tragnone A, Lanfranchi GA. A laboratory index for predicting relapse in asymptomatic patients with Crohn’s disease. Gastroenterology 1986;91:1490–4. 36. Schreiber S, Nikolaus S, Hampe J, et al. Tumour necrosis factor alpha and interleukin 1beta in relapse of Crohn’s disease. Lancet 1999;353:459–61. 37. Brignola C, Lanfranchi GA, Campieri M, et al. Importance of laboratory parameters in the evaluation of Crohn’s disease activity. J Clin Gastroenterol 1986;8:245–8.