QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kew, S. Articles by Calder, P. C. Search for Related Content PubMed PubMed Citation Articles by Kew, S. Articles by Calder, P. C.

---

Nutritional Immunology

The Effect of Eicosapentaenoic Acid on Rat Lymphocyte Proliferation Depends Upon Its Position in Dietary Triacylglycerols¹

S. Kew, S. Wells, F. Thies², G. P. McNeill^,3, P. T. Quinlan^,4, G. T. Clark^*,5, H. Dombrowsky^*, A. D. Postle^* and P. C. Calder⁶

Institute of Human Nutrition and ^* Division of Infection, Inflammation and Repair, University of Southampton, Southampton, UK and Unilever Research Colworth Laboratory, Sharnbrook, UK

⁶To whom correspondence should be addressed. E-mail: pcc{at}soton.ac.uk.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Animal and human studies have shown that greatly increasing the amount of fish oil [rich in long-chain (n-3) PUFA] in the diet can decrease lymphocyte functions. The effects of a more modest provision of long-chain (n-3) PUFA and whether eicosapentaenoic acid (20:5) and docosahexaenoic acid (22:6) have the same effects as one another are unclear. Whether the position of 20:5 or 22:6 in dietary triacylglycerols (TAG) influences their incorporation into immune cells and their subsequent functional effects is not known. In this study, male weanling rats were fed for 6 wk one of 9 diets that contained 178 g lipid/kg and that differed in the type of (n-3) PUFA and in the position of these in dietary TAG. The control diet contained 4.4 g -linolenic acid (18:3)/100 g total fatty acids. In the other diets, 20:5 or 22:6 replaced a portion (50 or 100%) of 18:3, and were in the sn-2 or the sn-1(3) position of dietary TAG. There were significant dose-dependent increases in the proportion of 20:5 or 22:6 in spleen mononuclear cell phospholipids when 20:5 or 22:6 was fed. These increases were at the expense of arachidonic acid and were largely independent of the position of 20:5 or 22:6 in dietary TAG. Spleen lymphocyte proliferation increased dose dependently when 20:5 was fed in the sn-1(3) position of dietary TAG. There were no significant differences in interleukin-2, interferon- or interleukin-10 production among spleen cells from rats fed the different diets. Prostaglandin E₂ production by spleen mononuclear cells was decreased by inclusion of either 20:5 or 22:6 in the diet in the sn-1(3) position. Thus, incorporation of 20:5 or 22:6 into spleen mononuclear cell phospholipids is not influenced by the position in dietary TAG. However, the pattern of incorporation may be influenced, and there are some differential functional effects of the position of long-chain (n-3) PUFA in dietary TAG. A moderate increase in the intake of 20:5 at the sn-1(3) position of dietary TAG increases lymphocyte proliferation.

KEY WORDS: • eicosapentaenoic acid • docosahexaenoic acid • structured triacylglycerol • lymphocyte • rats

In recent years, there has been increasing interest in the effects of different types of dietary fatty acids on the immune system [see (1) for a review]. Much of the research has focused on the effects of the long-chain (n-3) PUFA found in fish oil [eicosapentaenoic acid (20:5; EPA)⁶ and docosahexaenoic acid (22:6; DHA)]. Feeding laboratory animals diets containing fish oil decreased ex vivo lymphocyte proliferation (2–6) and production of the immunoregulatory cytokines interleukin (IL)-2 (6–8) and interferon (IFN)- (8–10). Typically these studies have fed diets in which 20:5 and 22:6 contributed between 20 and 30 g/100 g of dietary fatty acids and between 6 and 12% of dietary energy. This makes the application of their findings to the human situation difficult. However, lower amounts of long-chain (n-3) PUFA in the rat diet (4.4 or 6.6 g/100 g of dietary fatty acids equivalent to 1.7 or 2.5% of dietary energy) have the same effects as the larger amounts, but to a lesser degree (11). Human studies providing 2.4–5 g 20:5 + 22:6/d, in the form of fish oil, report decreased blood lymphocyte proliferation and IL-2 and IFN- production (12–15). Studies employing fish oil do not allow the immunologic effects of 20:5 and 22:6 to be distinguished. However, a few animal studies have compared the effects of 20:5 and 22:6, and these indicate that both of these (n-3) PUFA have similar suppressive effects upon rodent spleen lymphocyte proliferation (6,16) and IL-2 (6) and IFN- (17) production. Despite these studies, the effects of lower doses of 20:5 and 22:6, which are more comparable to achievable human intakes, are unclear.

Recent studies have established that the position of fatty acids in dietary triacylglycerols (TAG) may be important in terms of the subsequent physiologic and biochemical effects of those fatty acids. These studies have focused on the positional effects of palmitic, stearic and oleic acids, largely in relation to lipid metabolism and blood lipid concentrations (18–26). Little is known about the effects of positional isomerism of fatty acids in dietary TAG on immune function. Arachidonic acid [20:4(n-6); 20:4] is most often found esterified at the sn-2 position of membrane phospholipids (PL). The mechanism by which (n-3) PUFA affect inflammation and immune function relates to their antagonism of 20:4 metabolism [see (1) for a review]. If the fatty acid originally at the sn-2 position of dietary TAG is retained in that position through digestion and subsequent metabolism, as appears to be the case in some studies (22,27), then feeding (n-3) PUFA in the sn-2 position of dietary TAG may facilitate their incorporation into membrane PL at the expense of 20:4. This might result in greater immunologic and anti-inflammatory effects than if (n-3) PUFA are fed in the sn-1(3) position of dietary TAG. Thus, the objectives of this study were to determine the effects of moderate amounts of 20:5 or 22:6 in the diet on rat spleen mononuclear cell (MNC) functions and to identify whether there are differential effects of the position of 20:5 or 22:6 in dietary TAG on these functions.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Materials.

Histopaque, HEPES-buffered RPMI medium (glutamine-free), fetal calf serum (FCS), glutamine, penicillin, streptomycin, heparin, concanavalin A (Con A), bovine serum albumin (BSA), formaldehyde, boron trifluoride (140 g/L methanol), all solvents and all standard chemicals were purchased from Sigma Chemical (Poole, UK). [6-³H] Thymidine (2 Ci/mmol) was obtained from Amersham International (Amersham, UK). Fluorescein isothiocyanate-labeled goat anti-mouse IgG (GAM-FITC) was obtained from Serotec (Kidlington, UK). Monoclonal antibodies to rat CD3, a T cell marker (R73), CD4 (W3/25), CD8 (OX8), CD19, a B cell marker (OX12), and CD14, a monocyte marker (OX42), were gifts from the Sir William Dunn School of Pathology, University of Oxford. Cytokine concentrations were determined using Cytoscreen ELISA kits from Biosource (Camarillo, CA). Prostaglandin E₂ concentrations were determined using ELISA kits from Neogen (Lexington, KY).

Production of structured triacylglycerols.

Four types of structured TAG⁷ were prepared; these were enriched with either 20:5 or 22:6 (from fish oil) attached predominantly to either carbon 2 of the glycerol backbone of the TAG (referred to as the sn-2 position) or to carbons 1 and 3 of the glycerol backbone of the TAG [referred to as the sn-1(3) position]. These structured TAG were subsequently blended with vegetable oils to provide fat for the animal diets used in this study (see below). The structured TAG were named as follows according to the predominant long-chain PUFA and its position on the TAG: 20:5 sn-2; 20:5 sn-1(3); 22:6 sn-2; 22:6 sn-1(3).

Synthesis of 22:6 sn-1(3).

Fish oil was selectively hydrolyzed using a lipase from Candida rugosa according to a procedure described elsewhere (28,29). The free fatty acids (FFA) were removed from the unhydrolyzed glycerides by short-path distillation and the glyceride fraction chemically hydrolyzed using ethanolic KOH. After neutralization with HCl, the resultant FFA fraction contained 40% 22:6. This FFA fraction was enzymatically esterified to glycerol (3:1 ratio of FFA to glycerol) under vacuum at 55°C using an immobilized sn-1(3) selective lipase from Rhizomucor miehei (Novozyme, Bagsvaerd, Denmark) for 2 d. Under the conditions used, the major product was TAG due to acyl migration. However, because the lipase is also selective against 22:6, the 22:6 was found to be predominantly in the sn-1(3) position.

Synthesis of 22:6 sn-2.

Fish oil was selectively hydrolyzed as described above. After inactivation of the C. rugosa lipase (heating at 60°C for 30 min), the oil was further hydrolyzed by a mono-, diacylglycerol selective lipase (Lipase G from Penicillium camembertii; Amano Pharmaceutical, Nagoya, Japan) to remove the partial glycerides. The FFA fraction was removed from the mixture by short-path distillation. The resultant glyceride fraction was subjected to a sn-1(3) selective acidolysis with oleic acid using lipase from R. miehei to reduce the 22:6 content in the sn-1(3) position [see Synthesis of 22:6 sn-1(3) above]. The FFA fraction was removed from the product by short-path distillation and the TAG product (rich in 22:6 sn-2) was collected.

Synthesis of 20:5 sn-1(3).

The FFA fraction resulting from the C. rugosa hydrolysis of fish oil described above was collected and found to be enriched in EPA (30%). This FFA fraction was used in an sn-1(3) selective acidolysis of high oleic-sunflower oil using lipase from R. miehei as described above. The resultant mixture was treated with short-path distillation and the resultant TAG fraction (rich in 20:5 sn-1(3)) was collected.

Synthesis of 20:5 sn-2.

The FFA fraction enriched in 20:5 used for "Synthesis of 20:5 sn-1(3)" was converted enzymatically into TAG as described in "Synthesis of 22:6 sn-2" above. Because the lipase is not selective against 20:5, this acid was randomly distributed in the TAG. The resultant TAG were subjected to selective sn-1(3) acidolysis with oleic acid and the product TAG (rich in 20:5 sn-2) was collected by short-path distillation.

Analysis of structured triacylglycerols.

The fatty acid compositions of the structured TAG synthesized above were determined by GC of the methyl esters as described elsewhere (28) (Table 1). The sn positional distribution of 20:5 and 22:6 in TAG was measured by ¹³C NMR at the carbonyl carbons (30) and by a Brucker AMX400 spectrometer. The distributions of 20:5 and 22:6 at the sn-2 and sn-1(3) positions in the structured TAG are indicated in Table 1. For the sn-1(3) structured TAG, 80% of the fatty acid of interest was found at the sn-1(3) position (Table 1). In comparison, 41 and 53% of EPA and DHA, respectively, was found in the sn-2 position in the sn-2 structured TAG (Table 1).

All studies were in accordance with the Home Office Animals (Scientific Procedures) Act of 1986. Weanling male Lewis rats were purchased from Harlan Olac, Bicester, UK. They were housed individually for a period of 6 wk before killing. During this time they had free access to water and to one of the 9 experimental diets (provided by Unilever Research Colworth Laboratory, Sharnbrook, UK) (n = 8 per diet). Each diet contained (g/kg): 182 high nitrogen casein, 520 starch, 60 fiber (Solkafloc), 42 AIN-76 mineral mix (31), 12 AIN-76 vitamin mix (31), 4 DL-methionine, 2 choline bitartrate, 178 lipid and 0.17 -tocopherol. The -tocopherol content of the oil blends was measured and normalized by the addition of commercial -tocopherol (Sigma Type V; Sigma Chemical) to give an -tocopherol content equivalent to 80 mg/kg of the final diet; the diets also contained 90 mg/kg -tocopherol as a component of the AIN-76 vitamin mix. The blends of oils used for the preparation of each diet are shown in Table 2, and the fatty acid composition of each diet is shown in Table 3. The diets contained similar proportions of palmitic (16:0), stearic (18:0), oleic [18:1(n-9)] and linoleic [18:2(n-6)] acids (Table 3). It was intended to maintain the total proportion of (n-3) PUFA at 4.4 g/100 g total fatty acids, thus maintaining the ratio of (n-3) to (n-6) PUFA at 7 in all diets. However, once the oils were blended and the fatty acid compositions determined, it was apparent that there was some variation from this (Table 3). Nevertheless, the key differences among the diets were in the proportions of the different (n-3) PUFA that they contained and in the position [sn-2 or sn-1(3)] of 20:5 and 22:6 within the dietary triacylglycerols (Table 3). The control diet contained 18:3 as the (n-3) PUFA. In the 4.4 g/100 g diets, 20:5 or 22:6 replaced 18:3 (Table 3). In the 2.2 g/100 g diets, 20:5 or 22:6 replaced approximately half of the 18:3. The diets were powdered, stored at -20°C and provided fresh to the rats every 2 d. The rats were killed in the fed state by an overdose of CO₂. Blood was collected into heparinized tubes by cardiac puncture and kept at room temperature. Plasma was then collected by centrifugation at 800 x g for 10 min and stored at -20°C until analysis. The spleen and thymus were removed and weighed.

Spleen MNC (a mixture of lymphocytes and monocytes) were prepared as described elsewhere (8). They were counted using a Coulter cell counter (Model Z1; Coulter Electronics, Luton, UK) and the cell concentration adjusted as required.

Analysis of spleen MNC subpopulations.

Purified MNC (1 x 10⁷) were washed three times in PBS supplemented with BSA (1 g/L) and sodium azide (0.65 g/L) (mPBS). They were then resuspended in mPBS and incubated for 20 min at 4°C with monoclonal antibodies (MAb) to the rat cell surface markers CD3 (MAb R73), CD4 (MAb W3/25), CD8 (MAb MRC OX8), light chain (CD19; MAb MRC OX12) and CD14 (MAb MRC OX42). Incubation with a MAb to the human C3b inactivator protein (MRC OX21) was used as a negative control. After the incubation, the cells were washed twice with mPBS, resuspended in mPBS and incubated with GAM-FITC for 20 min at 4°C. The cells were then washed twice with mPBS, suspended in FACS-Fix (20 mL formaldehyde/L PBS) and examined for fluorescence using a Becton Dickinson FACScalibur fluorescence-activated cell sorter (Becton Dickinson, Oxford, UK). Fluorescence data were collected on 2 x 10⁴ viable cells. Both the percentage of marker-positive cells and median fluorescence intensity, a measure of the expression of a surface marker, were determined.

Fatty acid composition analysis.

Total lipid was extracted from spleen MNC using chloroform/methanol/water (2:1:0.5 v/v/v). Total PL was isolated by TLC using a mixture of hexane/diethyl ether/acetic acid (90:30:1 v/v/v) as the elution phase. FAME were prepared by incubation with 140 g/L boron trifluoride in methanol at 80°C for 60 min. FAME were isolated by extraction into hexane, dried and separated and identified by GC as described elsewhere (8).

MS analysis of spleen MNC phospholipid species.

Spleen MNC PL were fractionated into phosphatidylcholine (PtdCho) and an acidic phospholipid fraction containing phosphatidylinositol (PtdIns) and phosphatidylserine using aminopropyl BondElut sample preparation cartridges (32). Electrospray ionization MS of PtdCho and PtdIns was performed on a Micromass Quattro Ultima triple quadrupole mass spectrometer (Micromass, Wythenshaw, UK), equipped with an electrospray ionization interface. Samples were dissolved in methanol/chloroform/water (7:2:1, v/v/v) and introduced into the MS by nanoflow infusion. PtdCho species were preferentially detected using positive ionization, and PtdIns species by negative ionization. After fragmentation with argon gas, sodium adducts of PtdCho molecules produced a fragment with m/z = +147 (33); parent scans of the m/z 184 moiety provided diagnostic determination of PtdCho as their sodium adducts ([M + 22]⁺). Collision gas-induced fragmentation of PtdIns species generated a common dehydrated inositol phosphate fragment with m/z = -241, and parent scans of the m/z 241 moiety provided diagnostic identification of PtdIns (33). Data were acquired and processed using MassLynx NT software (Micromass, Wythenshaw, UK). After conversion to centroid format according to area, correction for ¹³C isotope effects and for reduced response with increasing m/z values, the PtdCho and PtdIns species were expressed as percentages of their respective totals present in the sample. The predominant molecular species present for each ion peak resolved was determined by analysis of fatty acyl ion fragments generated by collision gas–induced fragmentation under negative ionization (33).

Lymphocyte proliferation.

Spleen MNC were cultured in triplicate as described elsewhere (8), except that 100 mL/L FCS and a range of concentrations of Con A were used. Proliferation was determined as incorporation of [6-³H] thymidine over the final 18 h of a 66-h culture period (8). Thymidine incorporation values for the triplicate cultures were averaged (CV was always <10% and usually <5%). Data are expressed as thymidine incorporation (Bq/well).

Cytokine and prostaglandin (PG)E₂ production by spleen MNC.

Spleen MNC were cultured as described elsewhere (8), except that 100 mL/L FCS was used. The Con A concentration used was 2.5 mg/L. After 24 h of culture, the plates were centrifuged and the medium was collected and frozen for later analysis. Cytokine and PGE₂ concentration were determined using ELISA kits according to the manufacturer’s instructions. The CV was <10% for all assays. Limits of detection were 5 ng/L (IL-2), 13 ng/L (IFN-), 5 ng/L (IL-4, IL-10) and 0.5 µg/L (PGE₂). Cross-reactivity of the PGE₂ ELISA assay with PGE₃ is 17.7% (data supplied by the manufacturer of the kits).

Statistical analysis.

Except for the PtdCho molecular species data, all data shown are means ± SEM for 8 rats. Data were tested for normality using the Kolmogorov-Smirnov test; except for PGE₂ production and the PtdCho molecular species data, all data were normally distributed. The effect of diet was determined by one-factor ANOVA; when this was significant, differences among dietary groups were determined using the Bonferroni-corrected Student’s t test. PGE₂ production data were log-transformed before these analyses. PtdCho molecular species data are shown as medians and 25th and 75th percentile values for 3 rats. The effect of diet on these data was determined by Kruskal-Wallis ANOVA; when this was significant, differences among dietary groups were determined using the Mann-Whitney U-test. Linear relationships between the amount of EPA or DHA in the diet and the proportions of certain fatty acids in spleen MNC PL were determined as Spearman’s linear rank correlation coefficients (). All analyses were performed using SPSS Version 11.0 (SPSS, Chicago, IL); in all cases, P < 0.05 was taken to indicate statistical significance.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Body and lymphoid tissue weights of rats fed structured TAG diets.

Rats weighed 225 g at the start of the study and gained 185 g during the 6 wk of the study. There were no differences among the groups at the start or the end of the feeding period (data not shown). There also were no differences in the relative weights of the thymus (0.3 g/100 g body weight) or spleen (0.17 g/100 g body weight) (data not shown).

Fatty acid composition of spleen MNC phospholipids.

The diets did not affect the proportions of 16:0, 18:0, 18:1(n-9) or 18:2(n-6) in spleen MNC PL (all P > 0.05; Table 4). However, they did affect the proportions of 20:4 (P = 0.004), 20:5 (P < 0.001), docosapentaenoic acid (22:5) (P = 0.009) and 22:6 (P = 0.028) and the 20:4 to 20:5 ratio in spleen MNC PL (P < 0.001) (one-factor ANOVA). The proportion of 20:4 in spleen MNC PL was lower when either 20:5 or 22:6 was fed (Table 4). The feeding of 20:5 dose-dependently increased the proportion of 20:5 and 22:5 in spleen MNC PL (Table 4). The increase in the proportion of 20:5 or 22:5 was independent of the position of 20:5 in dietary TAG (Table 4). There were positive linear correlations between the amount of 20:5 in the diet and the proportion of 20:5 in spleen MNC PL ( = 0.933, P < 0.001 for diets with 20:5 in the sn-2 position; = 0.937, P < 0.001 for diets with 20:5 in the sn-1(3) position; = 0.902, P < 0.001 for all 20:5-containing diets). There were negative linear correlations between the amount of 20:5 in the diet and the proportion of 20:4 in spleen MNC PL ( = -0.500, P = 0.035 for diets with 20:5 in the sn-2 position; = -0.764, P < 0.001 for diets with 20:5 in the sn-1(3) position; = -0.475, P = 0.009 for all 20:5-containing diets). Feeding 20:5 did not affect the 22:6 content of spleen MNC PL (Table 4). Feeding 22:6 did not alter the proportion of 20:5 or 22:5 in spleen MNC PL compared with the control group (Table 4). However, there was a dose-dependent increase in the proportion of 22:6 in spleen MNC PL when 22:6 was fed (Table 4). There were positive linear correlations between the amount of 22:6 in the diet and the proportion of 22:6 in spleen MNC PL ( = 0.628, P = 0.007 for diets with 22:6 in the sn-2 position; = 0.679, P = 0.011 for diets with 22:6 in the sn-1(3) position; = 0.629, P = 0.001 for all 22:6-containing diets).

The phospholipid present in the greatest amount in spleen MNC was PtdCho. There was an effect of diet on the proportions of 18:0/20:4, 18:1alk/20:4, 16:0/20:5, 16:0/18:2 and 18:0/18:2 and on the proportions of total 20:4-containing and total 20:5-containing PtdCho species (P < 0.05; Kruskal-Wallis ANOVA) (Tables 5, and 6). Both 20:5 and 22:6 reduced the proportion of 20:4-containing PtdCho species compared with the control group. For example, 18:0/20:4 PtdCho, one of the two main 20:4-containing PtdCho species, was up to 60% lower with 20:5 feeding and up to 50% lower with 22:6 feeding (Table 5). At the highest intakes from the diet, 20:5 and 22:6 also reduced the proportion of 18:1alk/20:4 PtdCho compared with the control group (Table 5). Overall, the proportion of PtdCho species containing 20:4 was up to 40% lower after feeding diets containing 20:5 or 22:6. This effect of the long-chain (n-3) PUFA was not associated with the position of the fatty acid in dietary TAG. The 20:5-containing PC species were rare, contributing only 0.5% of total PtdCho species in the control rats. When 20:5 was fed, there was a significant increase (up to sevenfold) in the proportion of 16:0/20:5 PtdCho (Table 5). The total proportion of 20:5-containing PtdCho species was higher when 20:5 was fed. This increase was greater when 20:5 was in the sn-2 position of dietary TAG than when it was in the sn-1(3) position. There was no effect of feeding 22:6 on the proportion of any 22:6- or 20:5-containing PtdCho species.

A total of 16 PtdIns species were identified in rat spleen MNC. However, there were no effects of diet on the proportions of these different species (data not shown). The most predominant species measured was 18:0/20:4PtdIns (66.9 ± 4.0% of total PtdIns), followed by 18:1/20:4PtdIns (8.9 ± 2.6%), 16:0/20:4PtdIns (4.7 ± 1.5) and 18:1/18:1PtdIns (4.4 ± 1.9%). None of the other 12 species individually contributed >2% to total PtdIns and no species containing 20:5 was identified.

Spleen MNC subpopulations.

There was no effect of diet on the percentage of CD3+ cells (T cells; 45–50% of spleen MNC), CD4+ cells (T helper cells; 35–40% of spleen MNC), CD8+ cells (cytotoxic T cells; 15% of spleen MNC), CD19+ cells (B cells; 25–30% of spleen MNC) or CD14+ cells (monocytes; 10% of spleen MNC) (P > 0.05 in all cases; data not shown).

Proliferative response of lymphocytes to Con A.

Thymidine incorporation into lymphocytes from all rats was maximal at a Con A concentration of 2.5 mg/L. There was an effect of diet on thymidine incorporation at this concentration of Con A (P = 0.025; one-factor ANOVA), but not at any other. DHA did not affect lymphocyte proliferation in response to any Con A concentration (Fig. 1 shows the response at 2.5 mg/L Con A). However, 20:5 in the sn-1(3) position of dietary TAG, but not at the sn-2 position, dose-dependently increased lymphocyte proliferation (P = 0.048 for 4.4 20:5 sn-1(3) vs. control and P = 0.035 vs. 4.4 20:5 sn-2) (Fig. 1). The mean increase in proliferation was 20% for 2.2 20:5 sn-1(3) and 37% for 4.4 20:5 sn-1(3).

View larger version (18K): [in this window] [in a new window]   FIGURE 1 Thymidine incorporation into concanavalin A-stimulated spleen mononuclear cells from rats fed structured triacylglycerols (TAG) enriched with 20:5 or 22:6. Rats were fed diets containing 4.4 g (n-3) PUFA/100 g total fatty acids in the form of -linolenic acid (Control), eicosapentaenoic (EPA; 20:5) or docosahexaenoic (DHA; 22:6). The fatty acids 20:5 and 22:6 were fed at two levels (2.2 or 4.4 g/100 g total fatty acids) and predominantly in either the sn-1(3) or sn-2 position of dietary triacylglycerols (TAG). Spleen mononuclear cells were prepared and lymphocyte proliferation in response to 2.5 mg/L concanavalin A (Con A) determined as [3H]thymidine incorporation over the final 18 h of a 66-h culture period. Values are means ± SEM, n = 8. Means labeled "a" differ from that labeled "b," P < 0.05 (one-factor ANOVA and the Bonferroni-corrected Student’s t test).

There was no effect of diet on the production of IL-2 (concentration 25 U/L), IFN- (concentration 3 µg/L) or IL-10 (concentration 150 ng/L) by Con A-stimulated MNC (P > 0.05 in all cases; data not shown). IL-4 concentrations were below the detectable limit in all cultures (< 5 ng/L).

Production of PGE₂ by cultured spleen MNC.

There was an effect of diet on the production of PGE₂ by Con A-stimulated spleen MNC (P = 0.027; one-factor ANOVA). PGE₂ production by MNC in the control group was 6.7 ± 2.4 µg/L. PGE₂ production was lower in the 2.2 20:5 sn-1(3) (4.2 ± 0.5 µg/L; P = 0.041), 4.4 20:5 sn-1(3) (3.0 ± 0.6 µg/L; P = 0.029) and 2.2 22:6 sn-1(3) (2.9 ± 0.4 µg/L; P = 0.024) groups. There were no other differences in PGE₂ production among the dietary groups (data not shown).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Most animal studies of the effects of (n-3) PUFA on immune cell composition and/or function have used diets in which the (n-3) PUFA make a relatively large contribution to total fatty acid intake. For example, if fish oil is fed as the major fat source in a diet in which fat provides 21 g/100 g [see, for example, 4,5,8)] 20:5 and 22:6 together provide 20–30 g/100 g dietary fatty acids and 8–12% of dietary energy. The results of such studies are difficult to apply to human settings, where 20:5 + 22:6 normally provide <0.5 g/100 g dietary fatty acids and <1% of dietary energy. Relatively few animal studies have combined high fat feeding, characteristic of the human situation, with modest provision of (n-3) PUFA, which might be analogous to the human setting of dietary supplementation with fish oil or regular oily fish consumption. This was the approach used in the current study in rats. The diets used contained 17.8 g/100 g fat, which is 35% of energy as fat, similar to the contribution of fat to dietary energy in many Western countries (34). The diets used provided 4.4 g/100 g fatty acids as (n-3) PUFA. These were provided in a highly controlled way, such that the control diet contained 4.4 g of the precursor (n-3) PUFA 18:3/100 g total fatty acids and the other diets replaced a portion of this (50 or 100%) with either 20:5 or 22:6. Thus, this study investigated the effect of replacing 18:3 with its long-chain derivatives. The reason for using this approach was to keep the ratio of (n-6) to (n-3) PUFA as constant as possible for all diets. The amounts of all fatty acids other than individual (n-3) PUFA were kept constant in all of the diets. The position of EPA and DHA in dietary TAG was varied because this may affect metabolic handling of the fatty acids (27). It is possible that this might influence the incorporation of these fatty acids into cell membrane phospholipids, thus altering membrane characteristics and cell functions.

The lack of appearance of 18:3 in spleen MNC PL in rats fed the control diet, which contained a large amount of 18:3, is in agreement with observations made elsewhere (11). Furthermore, human blood MNC PL rarely contains 18:3 (35,36), even though it contributes 1 g/100 g fatty acids in the typical Western diet (34). Indeed, increasing 18:3 intake to 9.5 g/d did not lead to the appearance of 18:3 in human blood MNC (36). In the current study, spleen MNC PL from rats in the control group contained large proportions of 20:5, 22:5 and 22:6 despite the absence of these fatty acids in the diet. This indicates a substantial ability to metabolize 18:3 to its long-chain derivatives. When long-chain (n-3) PUFA were provided in the diet, significant changes in spleen MNC PL fatty acid composition were observed. There were dose-dependent increases in the proportions of 20:5 or 22:6 in spleen MNC PL when these were fed, which were accompanied by a decrease in the proportion of 20:4. These changes are consistent with an earlier study in rodents using moderate doses of 20:5 or 22:6 (11). Feeding diets containing 20:5 significantly enriched spleen PL in 22:5, confirming the high capacity for elongation of 20:5 in rodents.

The changes observed in fatty acid composition of MNC PL were independent of the position of 20:5 or 22:6 in dietary TAG, suggesting that position does not affect the ability of these fatty acids to be incorporated into immune cell PL. Furthermore, the pattern of incorporation of long-chain (n-3) PUFA into PtdCho molecular species and the accompanying decrease in 20:4-containing PtdCho species appeared to be little influenced by the position of (n-3) PUFA in dietary TAG. However, these analyses demonstrate that there is considerable molecular selectivity to the patterns of change of PtdCho molecular species after consumption of long-chain (n-3) PUFA, which cannot be predicted from total fatty acid composition measurements. The precise nature of this selective response is highlighted by the results of the analysis of PtdIns species, which showed no incorporation of 20:5 in any dietary group.

Although the proportion of 22:6 was increased in the bulk spleen MNC PL of rats fed diets containing 22:6, there was no increase in 22:6-containing PtdCho or PtdIns molecular species. This may be because 22:6 is more commonly associated with phosphatidylethanolamine (PtdEtn) than with PtdCho (37,38) and because the fatty acid composition of lymphocyte PtdIns is relatively resistant to dietary manipulation, compared with other PL classes (38). In the current study, PtdEtn molecular species composition was not analyzed. However, it is possible that the proportion of PtdEtn species containing 22:6 was increased after 22:6 feeding (39), just as the proportion of PtdCho species containing 20:5 was increased after 20:5 feeding. Changes in function could result from alterations in the composition of PtdCho and PtdEtn molecular species because these are sources of signaling molecules. For example, activation of T lymphocytes is coupled to hydrolysis of PtdCho by phospholipase D to generate phosphatidic acid (40). Although phosphatidic acid is a second messenger in its own right, it can be dephosphorylated by a phosphohydrolase to yield diacylglycerol, an activator of protein kinase C. Phospholipase D could give rise to the sustained phase of diacylglycerol generation that follows T cell activation [see (41) for a review]. A PtdCho-specific phospholipase C was also described in lymphocytes. This enzyme can generate diacylglycerol directly from PtdCho and thus may play an important signaling role in activated lymphocytes (42) and other immune cells (43,44). The molecular species composition of the diacylglycerol generated by either of these pathways is similar to that of the parent PtdCho [see (41,45)]. Diacylglycerols with different fatty acid compositions have been shown to activate protein kinase C to different extents (46,47). Thus, altering the molecular species of PtdCho, as observed in the current study, could lead to generation of diacylglycerol with an altered composition and potentially different potency to activate protein kinase C. The current study did not determine the diacylglycerol molecular species composition in stimulated MNC. It will be important to perform such analyses in future studies of this type.

PtdIns is also important as a source of second messengers, including diacylglycerols. The current study did not detect any differences in PtdIns molecular species composition. Although this might suggest that any of the functional consequences of feeding (n-3) fatty acids were not mediated by signaling mechanisms involving PtdIns metabolism, it is possible that the total concentration of PtdIns or of signaling molecules formed from PtdIns is influenced by (n-3) fatty acids (48,49).

In contrast to several studies in laboratory animals using fish oil (2–5,8), EPA or DHA (6,11) in which lymphocyte proliferation decreased, proliferation increased in the current study when 20:5 was fed in the sn-1(3) position of dietary TAG. There was no effect of 20:5 or 22:6 when fed in the sn-2 position. The limited effect of feeding 20:5 or 22:6 on the proportions of different MNC in the spleen is in agreement with other studies investigating rodent lymphoid tissues and blood (4,11,50) and human blood (35,36,51). In particular, feeding 20:5 did not alter the proportions of the various lymphocyte populations in the spleen. Thus, the changes in lymphocyte proliferation after feeding some 20:5-rich diets are unlikely to be due to changes in the types of lymphocyte present.

Previous animal studies investigating the effect of fish oil or long-chain (n-3) fatty acids on lymphocyte responses have used much larger amounts of the fatty acids than used here (2–5,7–10,17). This alone may explain why marked decreases in lymphocyte responses were observed in these studies, which was not the case in the current study. A second possible factor is the nature of the comparison being made (i.e., the nature of the control diet). In the current study, the control diet contained the same amount of fat as the diets containing 20:5 or 22:6 and also contained the same amount of (n-3) PUFA, but in a different form (i.e., 18:3), and had a similar ratio of (n-6) to (n-3) PUFA. In the other studies referred to, the control diet was either a low fat diet or a high fat diet that was (n-3) PUFA-poor, and rich in either saturated fatty acids or 18:2(n-6). Thus, a different comparison is being made in the current study and these earlier ones. One earlier study used 20:5 and 22:6 as major components of a low fat diet (30 g/100 g total fatty acids); the control was a low fat diet rich in 18:2(n-6) (6). Thus, the current study differs from this earlier one in several respects, and these might explain the different observations made.

Human studies examining the effect of 22:6, in the absence of 20:5, on lymphocyte functions have provided 0.7 or 6 g 22:6/d (35,52). These studies found no effect on lymphocyte proliferation (35,52), on the number of IL-2–producing lymphocytes (52) or on the production of IL-2 or IFN- (35). The lack of effect of 22:6 at a level in the diet of up to 4.4 g/100 g fatty acids in the current study agrees with the findings of these human studies. Taken together, these studies indicate that even substantial increases in intake of 22:6 will not influence lymphocyte function, in either a beneficial or a detrimental way.

Human studies of the effect of fish oil on lymphocyte functions have provided 1–5 g 20:5 + 22:6/d (12–15,35,36,51,53,54). The findings of those studies are inconsistent, with some studies reporting decreased proliferation (12–14,35,53) and production of IL-2 (12,14,15) and IFN- (15), and others reporting no effect on proliferation (36,51,54) or production of IL-2 (22,36,51,53,54) or IFN- (35,36,51,54). There are several possible reasons for these inconsistencies. The dose of long-chain (n-3) PUFA provided may have been too low to achieve an effect in some studies. The characteristics of the subjects differed among these studies, and some of these (e.g., age, gender, genotype) might be important in determining sensitivity to long-chain (n-3) PUFA. The amount of antioxidants, especially vitamin E, provided differed among the studies, and this might be an important factor in determining the precise effect of long-chain (n-3) PUFA. Finally, the current study indicates that the position of 20:5 in dietary TAG might be an important factor, with significant differences between the effects of 20:5 in the sn-2 and sn-1(3) position of dietary TAG. Most fish oils have a random positional distribution of 20:5 in the constituent TAG (30). It will be important to compare the effects of preparations with EPA in different positions of dietary TAG on lymphocyte functions in humans.

There are two possible mechanisms to explain the observed effect on lymphocyte proliferation. The first relates to the alteration in the generation of second messengers from membrane PL, as described above. The second mechanism involves a decrease in PGE₂ production, as a result of changes in the availability of 20:4, the substrate, and of 20:5 and 22:6, which inhibit 20:4 metabolism (55,56). PGE₂ inhibits lymphocyte proliferation and the production of IL-2 and IFN- (57–59). Thus, a decrease in production of PGE₂ might be expected to result in an increase in the production of IL-2 and IFN- and in lymphocyte proliferation. Therefore, the decrease in PGE₂ production by spleen MNC from rats fed diets containing 20:5 in the sn-1(3) position of dietary TAG might explain the increased proliferation observed in those cells. However, IFN- production is very sensitive to the presence of PGE₂ (60), and there was no effect of these diets on IFN- production. Furthermore, PGE₂ production was decreased by one of the 22:6-rich diets, and this diet had no effect on lymphocyte proliferation or cytokine production. Thus, it seems unlikely that a decrease in the production of PGE₂ can explain the observations. There was some cross-reactivity of the PGE₂ assay with PGE₃, which is formed from 20:5. Therefore, a small proportion of what was measured as PGE₂ may in fact have been PGE₃. However, PGE₃ is not generated in large amounts even when large decreases in the production of PGE₂ occur after administration of (n-3) PUFA (61). Furthermore, PGE₃ is equipotent to PGE₂ in terms of its ability to inhibit lymphocyte proliferation (62) and production of IL-2 and IFN- (60).

In summary, modest levels of 20:5 or 22:6 in the diet significantly affected the fatty acid composition of spleen MNC PL and PtdCho molecular species. The position of 20:5 or 22:6 in dietary TAG has limited effect on the gross fatty acid composition changes observed in PL and on the composition of PtdCho molecular species. At the levels used here, 20:5, but not 22:6, significantly influenced lymphocyte proliferation. The effect was dose-dependent and was related to the position of 20:5 in dietary TAG. The current study indicates that 20:5, rather than 22:6, is the long-chain (n-3) PUFA with the strongest modulating effects upon lymphocyte proliferation. The precise nature of the effects of 20:5 depend upon its level in the diet and its position in dietary TAG.

	FOOTNOTES

 
¹ Supported by a grant to P.C.C. from the Ministry of Agriculture, Fisheries and Food and Unilever Research Colworth Laboratory under the Agri-Food LINK Programme (AFQ51). S.K. was supported by a Postgraduate Studentship from the Ministry of Agriculture, Fisheries and Food. Flow cytometry facilities were provided by a grant to P.C.C. from the Medical Research Council under the JREI Scheme (G9717948). Mass spectrometry facilities were provided by a grant to A.D.P. and P.C.C. from The Wellcome Trust (057405).

² Present address: Department of Medicine and Therapeutics, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK.

³ Present address: Loders Croklaan NA, Channahon, IL 60410.

⁴ Present address: Unilever Health Institute, Unilever Research Vlaardingen, 3130 AC Vlaaardingen, The Netherlands.

⁵ Present address: Ferring Pharmaceuticals, Chilworth, Southampton SO16 7NP, UK.

⁷ Abbreviations used: 18:3, -linolenic acid; 20:4, arachidonic acid; BSA, bovine serum albumin; Con A, concanavalin A; 22:6 (DHA), docosahexaenoic acid; 22:5, docosapentaenoic acid; 20:5 (EPA), eicosapentaenoic acid; FCS, fetal calf serum; FFA, free fatty acid; FO, fish oil; GAM-FITC, fluorescein isothiocyanate-labeled goat anti-mouse IgG; IFN, interferon; IL, interleukin; 18:2, linoleic acid; MAb, monoclonal antibody; MNC, mononuclear cell; 18:1, oleic acid; 16:0, palmitic acid; PG, prostaglandin; PL, phospholipid; PtdCho, phosphatidylcholine; PtdEtn, phosphatidylethanolamine; PtdIns, phosphatidylinositol; 18:0, stearic acid; TAG, triacylglycerol.

⁸ Phospholipid molecular species are designated by fatty acyl moieties at the sn-1 and sn-2 positions followed by the headgroup; for example, 16:0/22:6PtdCho is sn-1 palmitoyl sn-2 docosahexaenoyl phosphatidylcholine, whereas 18:0/20:4PtdIns is sn-1 stearoyl sn-2 arachidonoyl phosphatidylinositol. Inclusion of alk at the sn-1 position denotes this is an alkyl ether–linked fatty acid. All other species are diacyl.

Manuscript received 17 April 2003. Initial review completed 16 June 2003. Revision accepted 25 September 2003.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

1. Calder, P. C. (2001) Polyunsaturated fatty acids, inflammation and immunity. Lipids 36:1007-1024.[Medline]

2. Alexander, N. J. & Smythe, N. L. (1988) Dietary modulation of in vitro lymphocyte function. Ann. Nutr. Metab. 32:192-199.[Medline]

3. Kelley, D. S., Nelson, G. J., Serrato, C. M., Schmidt, P. C. & Branch, L. B. (1988) Effects of type of dietary fat on indices of immune status in rabbits. J. Nutr. 118:1376-84.

4. Yaqoob, P., Newsholme, E. A. & Calder, P. C. (1994) The effect of dietary lipid manipulation on rat lymphocyte subsets and proliferation. Immunology. 82:603-610.[Medline]

5. Yaqoob, P. & Calder, P. C. (1995) The effects of dietary lipid manipulation on the production of murine T-cell-derived cytokines. Cytokine 7:548-553.[Medline]

6. Jolly, C. A., Jiang, Y.-H., Chapkin, R. S. & McMurray, D. N. (1997) Dietary (n-3) polyunsaturated fatty acids suppress murine lympho-proliferation, interleukin-2 secretion and the formation of diacylglycerol and ceramide. J. Nutr. 127:37-43.[Abstract/Free Full Text]

7. Turek, J. J., Schoenlein, I. A., Clark, L. K. & van Alstine, W. G. (1994) Dietary polyunsaturated fatty acids effects on immune cells of the porcine lung. J. Leuk. Biol. 56:599-604.[Abstract]

8. Wallace, F. A., Miles, E. A., Evans, C., Stock, T. E., Yaqoob, P. & Calder, P. C. (2001) Dietary fatty acids influence the production of Th1, but not Th2, type cytokines. J. Leukoc. Biol. 69:449-457.[Abstract/Free Full Text]

9. Fritsche, K. L., Byrge, M. & Feng, C. (1999) Dietary omega-3 polyunsaturated fatty acids from fish oil reduce interleukin-12 and interferon-gamma production in mice. Immunol. Lett. 65:167-173.[Medline]

10. Byleveld, P. M., Pang, G. T., Clancy, R. L. & Roberts, D. C. (1999) Fish oil feeding delays influenza virus clearance and impairs production of interferon-gamma and virus-specific immunoglobulin A in the lungs of mice. J. Nutr. 129:328-335.[Abstract/Free Full Text]

11. Peterson, L. D., Thies, F., Sanderson, P., Newsholme, E. A. & Calder, P. C. (1998) Low levels of eicosapentaenoic and docosahexanoic acids mimic the effects of fish oil upon rat lymphocytes. Life Sci. 62:2209-2217.[Medline]

12. Meydani, S. N., Endres, S., Woods, M. M., Goldin, B. R., Soo, C., Morril-Labrode, A., Dinarello, C. & Gorbach, S. L. (1991) Oral (n-3) fatty acid supplementation suppresses cytokine and lymphocyte proliferation: comparison between young and older women. J. Nutr. 121:547-555.

13. Molvig, J., Pociot, F., Worsaae, H., Wogensen, L. D., Baek, L., Christensen, P., Mandruppoulsen, T., Andersen, K., Madsen, P., Dyerberg, J. & Nerup, J. (1991) Dietary supplementation with omega 3 polyunsaturated fatty acids decreases mononuclear cell proliferation and interleukin 1 beta content but not monokine secretion in healthy and insulin dependent diabetic individuals. Scand. J. Immunol. 34:399-410.[Medline]

14. Endres, S., Meydani, S. N., Ghorbani, R., Schindler, R. & Dinarello, C. A. (1993) Dietary supplementation with n-3 fatty acids suppresses interleukin-2 production and mononuclear cell proliferation. J. Leukoc. Biol. 54:599-603.[Abstract]

15. Gallai, V., Sarchielli, P., Trequattrini, A., Franceschin, M., Floidi, A., Firenze, C., Alberti, A., Di Benedetto, D. & Stragliotto, E. (1993) Cytokine secretion and eicosanoid production in the peripheral blood mononuclear cells of MS patients undergoing dietary supplementation with n-3 polyunsaturated fatty acids. J. Neuroimmunol. 56:143-153.

16. Peterson, L. D., Jeffrey, N. M., Thies, F., Sanderson, P., Newsholme, E. A. & Calder, P. C. (1998) Eicosapentaenoic and docosahexaenoic acids alter rat spleen leukocyte fatty acid composition and prostaglandin E₂ production but have different effects on lymphocyte functions and cell-mediated immunity. Lipids 33:171-180.[Medline]

17. Fritsche, K. L., Anderson, M. & Feng, C. (2000) Consumption of eicosapentaenoic acid and docosahexaenoic acid impair murine interleukin-12 and interferon-gamma production in vivo. J. Infect. Dis. 182:S54-S61.

18. Innis, S. M., Dyer, R., Quinlan, P. & Diersen-Schade, D. (1995) Palmitic acid is absorbed as sn-2 monopalmitin from milk and formula with rearranged triacylglycerols and results in increased plasma triglyceride sn-2 and cholesteryl ester palmitate in piglets. J. Nutr. 125:73-81.

19. Innis, S. M., Quinlan, P. & Diersen-Schade, D. (1996) Saturated fatty acid chain length and positional distribution in infant formula: effects on growth and plasma lipids and ketones in piglets. Am. J. Clin. Nutr. 57:382-390.

20. Zampelas, A., Williams, C. M., Morgan, L. M., Wright, J. & Quinlan, P. T. (1994) The effect of triacylglycerol fatty acid positional distribution on postprandial plasma metabolite and hormone responses in normal adult men. Br. J. Nutr. 71:401-410.[Medline]

21. Carnielli, V. P., Luijendijk, I.H.T., van Beek, R.H.T., Boerma, G.J.M., Degenhert, H. J. & Sauer, P.J.J. (1995) Effect of dietary triacylglycerol fatty acid positional distribution on plasma lipid classes and their fatty acid composition in preterm infants. Am. J. Clin. Nutr. 62:776-781.[Abstract/Free Full Text]

22. Pufal, D. A., Quinlan, P. T. & Salter, A. M. (1995) Effect of dietary triacylglycerol structure on lipoprotein metabolism: a comparison of the effects of dioleoylpalmitoylglycerol in which palmitate is esterified to the 2- or 1 (3)-position of the glycerol. Biochim. Biophys. Acta 1258:41-48.[Medline]

23. Aoyama, T., Fukui, K., Taniguichi, K., Nagaoka, S., Yamaoto, T. & Hashimoto, Y. (1996) Absorption and metabolism of lipids in rats depend on fatty acid isomeric position. J. Nutr. 126:225-231.

24. Jeffery, N. M., Sanderson, P., Newsholme, E. A. & Calder, P. C. (1997) Effects of varying the type of saturated fatty acid in the rat diet upon serum lipid levels and spleen lymphocyte functions. Biochim. Biophys. Acta 1345:223-236.[Medline]

25. Summers, L. K., Fielding, B. A., Ilic, V., Quinlan, P. T. & Frayn, K. N. (1998) The effect of triacyl-fatty acid positional distribution on postprandial metabolism in subcutaneous adipose tissue. Br. J. Nutr. 79:141-147.[Medline]

26. Summers, L. K., Fielding, B. A., Herd, S. L., Ilic, V., Clark, M. L., Quinlan, P. T. & Frayn, K. N. (1999) Use of structured triacylglycerols containing predominantly stearic and oleic acids to probe early events in metabolic processing of dietary fat. J. Lipid Res. 40:1890-1898.[Abstract/Free Full Text]

27. Small, D. M. (1991) The effects of glyceride structure on absorption and metabolism. Annu. Rev. Nutr. 11:413-434.[Medline]

28. McNeill, G. P., Ackman, R. G. & Moore, S. R. (1996) Lipase-catalyzed enrichment of long-chain polyunsaturated fatty acids. J. Am. Oil Chem. Soc. 73:1403-1407.

29. Moore, S. R. & McNeill, G. P. (1996) Production of triglycerides enriched in long-chain n-3 polyunsaturated fatty acids from fish oil. J. Am. Oil Chem. Soc. 73:1409-1414.

30. Gunstone, F. D. & Seth, S. (1994) A study of the distribution of eicosapentaenoic acid and docosahexaenoic acid between the alpha and beta glycerol chains in fish oils by C-13-NMR spectroscopy. Chem. Phys. Lipids 72:119-126.

31. Bieri, J. G., Stoewsand, G. S., Briggs, G. M., Phillips, R. W., Woodard, J. C. & Knapka, J. J. (1977) Report of the American Institute of Nutrition ad hoc committee on standards for nutritional studies. J. Nutr. 107:1340-1348.

32. Hunt, A. N., Clark, G. T., Attard, G. S. & Postle, A. D. (2001) Highly saturated endonuclear phosphatidylcholine is synthesized in situ and co-located with CDP-choline pathway enzymes. J. Biol. Chem. 276:8492-8499.[Abstract/Free Full Text]

33. Han, X. L. & Gross, R. W. (1995) Structural determination of picomole amounts of phospholipids via electrospray ionization tandem mass spectrometry. J. Am. Soc. Mass Spectrom. 6:1202-1210.

34. British Nutrition Foundation (1992) Report of the Task Force on Unsaturated Fatty Acids: Nutritional and Physiological Significance 1992 Chapman & Hall London, UK.

35. Thies, F., Nebe-von-Caron, G., Powell, J. R., Yaqoob, P., Newsholme, E. A. & Calder, P. C. (2001) Dietary supplementation with -linolenic acid or fish oil decreases T lymphocyte proliferation in healthy older humans. J. Nutr. 131:1918-1927.[Abstract/Free Full Text]

36. Kew, S., Banerjee, T., Minihane, A. M., Finnegan, Y. E., Muggli, R., Albers, R., Williams, C. M. & Calder, P. C. (2003) Lack of effect of foods enriched with plant- or marine-derived n-3 fatty acids on human immune function. Am. J. Clin. Nutr. 77:1287-1295.[Abstract/Free Full Text]

37. Akoh, C. C. & Chapkin, R. S. (1990) Composition of mouse peritoneal macrophage phospholipid molecular species. Lipids 25:613-617.[Medline]

38. Brouard, C. & Pascaud, M. (1990) Effects of moderate dietary supplements with n-3 fatty acids on macrophage and lymphocyte phospholipids and macrophage eicosanoid synthesis in the rat. Biochim. Biophys. Acta 1047:19-28.[Medline]

39. Chapkin, R. S. & Carmichael, S. L. (1990) Effects of dietary n-3 and n-6 polyunsaturated fatty acids on macrophage phospholipid classes and subclasses. Lipids 25:827-834.[Medline]

40. Reid, P. A., Gardner, S. D., Williams, D. M. & Harnett, M. M. (1997) The antigen receptors on mature and immature T lymphocytes are coupled to phosphatidylcholine-specific phospholipase D activation. Immunology 90:250-256.[Medline]

41. Wakelam, M. O. & Harnett, M. M. (1998) Phospholipase A₂ (EC 3.1.1.4) and D (EC 3.1.4.4) signalling in lymphocytes. Proc. Nutr. Soc. 57:551-554.[Medline]

42. Nofer, J. R., Junker, R., Seedorf, U., Assmann, G., Zidek, W. & Tepel, M. (2000) D609-phosphatidylcholine-specific phospholipase C inhibitor attenuates thapsigargin-induced sodium influx in human lymphocytes. Cell. Signalling 12:289-296.[Medline]

43. Marignani, P. A. & Sebaldt, R. J. (1995) Formation of second messenger diradylglycerol in murine peritoneal macrophages is altered after in vivo (n-3) polyunsaturated fatty acid supplementation. J. Nutr. 125:3030-3040.

44. Marignani, P. A. & Sebaldt, R. J. (1996) The formation of diradylglycerol molecular species in murine peritoneal macrophages varies dose-dependently with dietary purified eicosapentaenoic and docosahexaenoic ethyl esters. J. Nutr. 126:2738-2745.

45. Heung, Y.M.M. & Postle, A. D. (1995) The molecular selectivity of phospholipase D in HL60 granulocytes. FEBS Lett. 364:250-254.[Medline]

46. Marignani, P. A., Epand, R. M. & Sebaldt, R. J. (1996) Acyl chain dependence of diacylglycerol activation of protein kinase C activity in vitro. Biochem. Biophys. Res. Commun. 225:469-473.[Medline]

47. Bell, M. V. & Sargent, J. R. (1987) Effects of the fatty acid composition of phosphatidylserine and diacylglycerol on the in vitro activity of protein kinase C from rat spleen: influences of (n-3) and (n-6) polyunsaturated fatty acids. Comp. Biochem. Physiol. 86B:227-232.

48. Fowler, K. H., McMurray, D. N., Fan, Y. Y., Aukema, H. M. & Chapkin, R. S. (1993) Purified dietary n-3 polyunsaturated fatty acids alter diacylglycerol mass and molecular species composition in concanavalin A-stimulated murine splenocytes. Biochim. Biophys. Acta 1210:89-96.[Medline]

49. Sanderson, P. & Calder, P. C. (1998) Dietary fish oil appears to prevent the activation of phospholipase C- in lymphocytes. Biochim. Biophys. Acta 1392:300-308.[Medline]

50. Sanderson, P., Yaqoob, P. & Calder, P. C. (1995) Effects of dietary manipulation upon rat spleen lymphocyte functions and the expression of lymphocyte surface molecules. J. Nutr. Environ. Med. 5:119-132.

51. Yaqoob, P., Pala, H. S., Cortina-Borja, M., Newsholme, E. A. & Calder, P. C. (2000) Encapsulated fish oil enriched in -tocopherol alters plasma phospholipid and mononuclear cell fatty acid compositions but not mononuclear cell functions. Eur. J. Clin. Investig. 30:260-274.[Medline]

52. Kelley, D. S., Taylor, P. C., Nelson, G. J. & Mackey, B. E. (1998) Dietary docosahexaenoic acid and immunocompetence in young healthy men. Lipids 33:559-566.[Medline]

53. Meydani, S. N., Lichtenstein, A. H., Cornwall, S., Meydani, M., Goldin, B. R., Rasmussen, H., Dinarello, C. A. & Schaffer, E. J. (1993) Immunologic effects of national cholesterol education step-2 diets with and without fish-derived n-3 fatty acid enrichment. J. Clin. Investig. 92:105-113.

54. Wallace, F. A., Miles, E. A. & Calder, P. C. (2003) Comparison of the effects of linseed oil and different doses of fish oil on mononuclear cell function in healthy human subjects. Br. J. Nutr. 89:679-689.[Medline]

55. Corey, E. J., Shih, C. & Cashman, J. R. (1983) Docosahexaenoic acid is a strong inhibitor of prostaglandin but not leukotriene biosynthesis. Proc. Natl. Acad. Sci. U.S.A. 80:3581-3584.[Abstract/Free Full Text]

56. Obata, T., Nagakura, T., Masaki, T., Maekawa, K. & Yamashita, K. (1999) Eicosapentaenoic acid inhibits prostaglandin D₂ generation by inhibiting cyclo-oxygenase-2 in cultured human mast cells. Clin. Exp. Allergy 29:1129-1135.[Medline]

57. Chouaib, S., Welte, K., Mertelsmann, R. & Dupont, B. (1985) Prostaglandin E₂ acts at 2 distinct pathways of T lymphocyte activation: inhibition of interleukin 2 production and down-regulation of transferrin receptor expression. J. Immunol. 135:1172-1179.[Abstract]

58. Betz, M. & Fox, B. S. (1991) Prostaglandin E₂ inhibits production of Th1 but not Th2 lymphokines. J. Immunol. 146:108-11344.[Abstract]

59. Snijdewint, F.G.M., Kalinski, P., Wierenga, E. A., Bos, J. D. & Kapsenberg, M. L. (1993) Prostaglandin E₂ differentially modulates cytokine secretion profiles of human T helper lymphocytes. J. Immunol. 150:5321-5329.[Abstract]

60. Miles, E. A., Aston, L. & Calder, P. C. (2003) In vitro effects of eicosanoids derived from different 20-carbon fatty acids on Th1- and Th2-cytokine production in human whole blood cultures. Clin. Exp. Allergy 33:624-632.[Medline]

61. Hawkes, J. S., Cleland, L. G. & James, M. J. (1991) Separation of PGE3 following derivatization with panacyl bromide by high pressure liquid chromatography with fluorometric detection. Prostaglandins 42:355-368.[Medline]

62. Dooper, M.M.B.W., Wassink, L., M’Rabet, L. & Graus, Y.M.F. (2002) The modulatory effects of prostaglandin-E on cytokine production by human peripheral blood mononuclear cells are independent of the prostaglandin subtype. Immunology 107:152-159.[Medline]

This article has been cited by other articles:

				R. J Deckelbaum, T. S Worgall, and T. Seo n-3 Fatty acids and gene expression Am. J. Clinical Nutrition, June 1, 2006; 83(6): S1520 - 1525S. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kew, S. Articles by Calder, P. C. Search for Related Content PubMed PubMed Citation Articles by Kew, S. Articles by Calder, P. C.