Lipids (2009) 44:217–224 DOI 10.1007/s11745-008-3276-0

ORIGINAL ARTICLE

Uptake and Incorporation of Pinolenic Acid Reduces n-6 Polyunsaturated and Downstream Prostaglandin Formation in Murine Macrophage

Lu-Te Chuang Æ Po-Jung Tsai Æ Chia-Long Lee Æ Yung-Sheng Huang

Received: 10 July 2008 / Accepted: 19 November 2008 / Published online: 8 January 2009 Ó AOCS 2008

Abstract Many reports have shown the beneficial effects from dihomo-c-linolenic acid (DGLA, D8,11,14–20:4) and of consumption of pine seeds and pine seed oil. However, PGE2 from (ARA, D5,8,11,14–20:4) were few studies have examined the biological effect of pino- also suppressed by the presence of PNA and its metabolite. lenic acid (PNA; D5,9,12–18:3), the main fatty acid in As the expression of COX-2 was not suppressed, the pine seed oil. In this study, using murine macrophage inhibitory effect of PNA on PG activity was attributed in RAW264.7 cells as a model, we examined the effect of part to substrate competition between the PNA metabolite PNA on polyunsaturated fatty acid (PUFA) metabolism, (i.e., D7,11,14–20:3) and DGLA (or ARA). prostaglandin (PG) biosynthesis and -2 (COX-2) expression. Results showed that PNA was readily Keywords Non-methylene interrupted fatty acids Á taken up, incorporated and elongated to form eicosatrienoic Sciadonic acid Á Gamma-linolenic acid Á Cyclooxygenase 2 acid (ETrA, D7,11,14–20:3) in macrophage cells. A small portion of this elongated metabolite was further elongated Abbreviations to form D9,13,16–22:3. The degree of incorporation of ADA Adrenic acid (D7, 10, 13, 16–22:4) PNA and its metabolites into cellular phospholipids varied ARA Arachidonic acid (D5, 8, 11, 14–20:4) with the length of incubation time and the concentration of COX-2 Cyclooxygenase 2 PNA in the medium. Incubation of PNA also modified the DGLA Dihomo-gamma-linolenic acid (D8, 11, fatty acid profile of phospholipids: the levels of 18- and 14–20:3) 20-carbon PUFA were significantly decreased, whereas DHA (22:6) those of 22-carbon fatty acids increased. This finding DMEM Dulbecco’s modified Eagles medium suggests that PNA enhances the elongation of 20-carbon DMSO Dimethyl sulfoxide fatty acids to 22-carbon fatty acids. The syntheses of PGE1 DPA (22:5) DTrA Docosatrienoic acid (22:3) EDA Eicosadienoic acid (20:2) EPA (D5, 8, 11, 14, 17–20:5) L.-T. Chuang Á C.-L. Lee Department of Biotechnology, Yuanpei University, ETA Eicosatetraenoic acid (D8, 11, 14, 17–20:4) Hsinchu, Taiwan ETrA Eicosatrienoic acid (20:3) FACES Fatty acid chain elongation system P.-J. Tsai GC Gas chromatography Department of Human Development and Family Studies, National Taiwan Normal University, Taipei, Taiwan GC-MS Gas chromatograph mass spectrometry GLA Gamma-linolenic acid (D6, 9, 12–18:3) Y.-S. Huang (&) NMIFA Non-methylene-interrupted fatty acids Department of Food Science and Biotechnology, PGE Prostaglandin E National Chung Hsing University, 250 Kuo-Kuang Rd, Taichung 402, Taiwan PNA Pinolenic acid (D5, 9, 12–18:3) e-mail: [email protected] PSO Pine seed oil 123 218 (2009) 44:217–224

SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel PUFA metabolism and eicosanoid synthesis (Scheme 1, electrophoresis [12]). Similar to GLA, PNA can also be elongated by the fatty acid chain elongation system (FACES) in rat liver microsomes and human hepatoma HepG2 cells to form eicosatrienoic acid (ETrA, D7,11,14–20:3) [10, 13]. It has Introduction also been shown that ETrA, similar to its isomer DGLA (D8,11,14–20:3), could substitute ARA for incorporation Pine seed and pine seed oil (PSO) have been consumed for into cellular phospholipid fractions [13]. Therefore, the years by the general public in many parts of the world. present study, using murine macrophage-like RAW264.7 Several animal studies have shown that PSO supplemen- cells as a model, was carried out to examine whether PNA tation lowers blood lipids, reduces blood pressure and and/or its metabolites affect the polyunsaturated fatty acid affects lipoprotein metabolism [1–4]. However, the mech- (PUFA) metabolism and prostaglandin production. anism as to how PSO exerts these beneficial effects is still not fully understood. Pinolenic acid (PNA, D5,9,12–18:3) is the main fatty acid present in PSO. It is a rare n-6 Experimental Procedures octadecatrienoic acid, belonging to a group of non-meth- ylene-interrupted polyunsaturated fatty acids (NMIFAs). Chemicals Its chemical structure is very similar to its positional isomer, gamma-linolenic acid (GLA; D6,9,12–18:3). Pre- Authentic fatty acids, i.e., PNA, ARA and n-3 eicosatet- viously, GLA supplementation has been demonstrated to raenoic acid (n-3 ETA, D8,11,14,17–20:4) and PGE assay exert beneficial effects, such as lowering inflammation- 2 kit were obtained from Cayman Chemicals (Ann Arbor, related diseases [5, 6], hypertension and other chronic MI). GLC standard RL-461, triheptadecanoin, and DGLA diseases [7, 8]. In mammals, dietary GLA is rapidly elon- were purchased from Nu-Chek-Prep, Inc. (Elysian, MN). gated to form dihomo-c-linolenic acid (DGLA; D8,11,14– Lipopolysaccharide (LPS, from Escherichia coli 026:B6), 20:3) [9, 10], a precursor of anti-inflammatory eicosanoid, dimethyl sulfoxide (DMSO), cell proliferation kit, Tween- prostaglandin E (PGE ). DGLA competes with arachi- 1 1 20, protease inhibitors were supplied by Sigma Chemical donic acid (ARA; D5,8,11,14–20:4) as substrate for (St. Louis, MO). Dulbecco’s modified Eagles medium cyclooxygenase-2 (COX-2), and subsequently reduces the (DMEM), fetal bovine serum (FBS) and phosphate-buf- synthesis of pro-inflammatory prostaglandins [11]. Thus, fered saline (PBS) were obtained from Gibco (Carlsbad, the effect of GLA was exerted through modulation of n-6 CA). PGE1 assay kit was purchased from Assay Designs, Inc. (Ann Arbor, MI). All reagent-grade organic solvents were from Burdick & Jackson (Muskegon, MI).

Cell Culture and Culture Conditions

The murine macrophage RAW 264.7 cell line was obtained from the Bioresource Collection and Research Center (Hsinchu, Taiwan). Cells were maintained in DMEM medium supplemented with 10% heated-inactivated FBS,

and incubated at 37 °C and 5% CO2 in a humidity-con- trolled atmosphere. Macrophage cells (5 9 105) were seeded in a 100-mm tissue culture plate, and allowed to adhere for 4 h at 37 °C. After cell adhesion, the culture medium was replaced by DMEM medium with different levels of PNA. The first study was carried out to examine the uptake and metabolism of PNA, cells were incubated in the DMEM serum medium supplemented with 50 lMof PNA for 24 h. The second study was performed to examine whether the uptake of PNA was time- or dose-dependent, the same number of macrophage cells (5 9 105) were cultured in the same DMEM medium supplemented with Scheme 1 n-6 and n-3 polyunsaturated fatty acid metabolism in 50 lM of PNA for 12, 24, 48 or 72 h, or with different mammalian cells [19] levels of PNA (0, 10, 25, 50 or 100 lM) for 24 h. The cell 123 Lipids (2009) 44:217–224 219 viability was monitored by the methods of trypan blue dye sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), exclusion [14] and MTT assay [15]. and transferred on a polyvinylidene difluoride membrane. After nonspecific blocking, membranes were probed with a Extraction and Fatty Acid Analysis 1:500 dilution of anti-mouse COX-2 antibody (BD Bio- sciences, Franklin Lakes, NJ), washed with PBS-T (10% After incubation, cells in plates were collected and harvested Tween 20), and then reacted with a 1:5,000 dilution of anti- by centrifugation. The pellet was washed twice with sterile mouse immunoglobulin-alkaline phosphatase (Sigma, St. PBS. Total cellular lipids were then extracted following with Louis, MO, USA). Following further washing in PBS-T minor modification the Folch method [16]. Briefly, cell three times, the immuno-complexes were visualized by pellets were extracted with 15 ml chloroform–methanol colorimetric blotting substrates BCIP/NBT (Sigma, St. (2:1,v/v) at 4 °C for overnight. After adding 3 ml of 0.9% Louis, MO, USA). (w/v) NaCl solution, the mixture was vortexed and centri- fuged. The chloroform phase containing the total lipid Statistical Analyses extract was then removed from the aqueous phase, and evaporated at 40°C using a stream of nitrogen. To separate Data were analyzed by analysis of variance (ANOVA) and the total phospholipid fraction, the lipid extracts were frac- Fisher’s protected least significant difference (LSD) to tionated by thin-layer chromatography using hexane/diethyl determine differences between means of the uptake rates ether/ (70:30:1, v/v/v) as the developing system. and between means of the conversion rates. Means differ- Fatty acids of the phospholipid fraction were methylated, ences were considered significant at P B 0.05. and fatty acid methyl esters analyzed by an Agilent 6890 gas chromatography (GC) equipped with a flame-ionization detector and a fused-silica capillary column (Omegawax; Results 30 m 9 0.32 mm, i.d., film thickness 0.25 lm, Supelco, Bellefonte, PA, USA) [17]. To separate the isomer of Uptake and Metabolism of PNA in Macrophages eicosatrienoic acid (ETrA), an HP-88 capillary column (100 m 9 0.25 mm, i.d., film thickness 0.2 lm, Hewlett- To examine the uptake and metabolism of PNA by mac- Packard, Sunyvale, CA, USA) was utilized. For quantitation rophage, RAW264.7 cells were incubated in the culture of fatty acids, a known amount of triheptadecanoin was medium containing 50 lM of PNA for 24 h. Results from added to each lipid sample as the internal standard. The the gas chromatographic analysis (GC analysis) show that a identity of PNA elongated metabolite was determined by gas significant level of PNA (14% by weight of cellular phos- chromatograph mass spectrometry (GC-MS), using a Hew- pholipids) and eicosatrienoic acid (ETrA) (19% by weight lett Packard mass selective detector operating at an of cellular phospholipids) were detected in cells incubated ionization voltage of 70 eV with a scan range of 20–500 Da. with PNA as compared to the control where neither PNA nor ETrA was detected (Fig. 1). Further GC analysis using

In-Vitro Study of PGE1 and PGE2 Production the HP-88 capillary column (100 m 9 0.25 mm) revealed that the ETrA peak contained two isomers (Fig. 1, inset A). RAW264.7 cells were seeded in 24-well plates at 5 9 105 Gas chromatography-mass spectrometric analysis shows cells/well with complete DMEM medium containing 10% that the major ETrA peak was the PNA elongation metab- FBS. The cells were allowed to adhere for 4 h at 37 °C. olite (D7,11,14–20:3), whereas the minor peak was DGLA After 24 h of treatment of PNA and/or other fatty acids, (\3% of ETrA) (data not shown). A low level (2% of cel- cells were stimulated with 0.1 lg/ml LPS for 16 h. Cell- lular phospholipids) of docosatrienoic acid (DTrA) was also free supernatants were collected and concentrations of both observed. The DTrA peak has an identical retention time to prostaglandins (PGE1 and PGE2) were analyzed with n-6 docosatrienoic acid (D10,13,16–22:3), an elongation respective enzyme immunoassay kits. metabolite of DGLA (Fig. 1, inset B). As this peak could only be observed in cells incubated with PNA but not in the Detection of COX-2 Expression by Western Blotting controls, it was tentatively identified as D9,13,16–22:3, a possible elongation product of D7,11,14–20:3. Confluent macrophage cells (2.5 9 107) were pre-incu- bated with 50 lM of PNA and/or other fatty acids for 24 h Effect of Incubation Time and Dose Response on PNA and then stimulated with LPS for 16 h. Total protein was Metabolism in Macrophages extracted from cells, and its concentration determined using the BCA Protein Assay Reagent Kit (Pierce, Rockford, IL). To examine the effect of the length of the incubation time Denatured proteins were separated by 10% sodium dodecyl on PNA uptake and metabolism, cells were incubated with 123 220 Lipids (2009) 44:217–224

Fig. 1 Gas chromatogram of fatty acids of total lipids in the shows the gas chromatogram of cellular phospholipids fatty acids in RAW264.7 cells cultured in the medium containing 50 lM of PNA. an HP-88 capillary column. Inset B compares gas chromatograms of Solid arrows show the presence of pinolenic acid (PNA; D5,9,12– fatty acids in cells incubated in medium containing either PNA or 18:3), eicosatrienoic acid (ETrA; D7,11,14–20:3 and D8,11,14–20:3) GLA and docosatrienoic acid (DTrA; D9,13,16–22:3), respectively. Inset A

Fig. 2 Effects of different incubation times (a) and PNA concentration (b) on the levels of D5,9,12–18:3 (PNA) (j), D7,11,14–20:3 (m) and D9,13,16–22:3 (d) in cellular phospholipids. RAW264.7 cells were incubated with medium containing 50 lM of PNA for 48 h, or different levels (0, 10, 25, 50, or 100 lM) of PNA for 24 h

50 lM PNA for 12, 24, 48 or 72 h. The phospholipids fatty incorporated into cellular phospholipids. The proportion of acid composition in total cellular lipids was then analyzed. PNA elevated rapidly, reached the maximum at 12 h and The results in Fig. 2a show that PNA was readily fell thereafter. A significant portion of PNA was elongated 123 Lipids (2009) 44:217–224 221 to D7,11,14–20:3. A small portion of D7,11,14–20:3 was (LA; D9,12–18:2), GLA, eicosadienoic acid further elongated to D9,13,16–22:3. The levels of these (EDA; D11,14–20:2), DGLA and ARA. In contrast, PNA two PNA metabolites increased as the incubation time incubation raised the levels of the 22-carbon fatty acids, increased and reached a plateau after 24 h of incubation. i.e., adrenic acid (ADA; D7,10,13,16–22:4) and n-6 doco- To examine the effect of varying PNA concentration in sapentaenoic acid (n-6 DPA; D4,7,10,13,16–22:5). The the medium on phospholipid fatty acid composition, the increase reached a plateau when the concentration excee- cells were incubated with 10, 25, 50 or 100 lM of PNA for ded 50 lM. 24 h. Results in Fig. 2b show that the level of PNA and its To examine the effect of PNA incubation on n-3 fatty metabolites increased as the concentration of PNA in the acid metabolism, macrophage cells were incubated with a medium increased. medium containing 25 lM of n-3 eicosatetraenoic acid (n-3 ETA; D8,11,14,17–20:4) alone or in combination with Effect of PNA Incubation on Phospholipid Fatty Acid 25 lM of PNA for 24 h. Results in Fig. 4a show that levels Composition of the 20-carbon n-3 fatty acids (n-3 ETA and EPA) were decreased, whereas those of 22-carbon n-3 fatty acids, i.e., We also examined the composition of other fatty acids in n-3 docosapentaenoic acid (n-3 DPA, D7,10,13,16,19– cells incubated with different levels of PNA. The results in 22:5) and docosahexaenoic acid (DHA; D4,7,10,13,16,19– Fig. 3a show that when the levels of PNA and its metab- 22:6) were increased. However, the levels of total n-3 olites in cellular phospholipids increased, those of total PUFA were decreased in cells incubated with PNA monounsaturated fatty acids and n-6 PUFA decreased. The (Fig. 4b). effects were dose-dependent. With respect to levels of saturated fatty acids and n-3 PUFA, a significant lowering Effect of PNA on Synthesis of Prostaglandin E1 and E2 effect was observed only when the concentration of PNA in the medium exceeded 25 lM. The results shown in Fig. 3b demonstrate that PNA incu- Among n-6 PUFA, incubation with PNA significantly bation lowered the level of 20-carbon n-6 PUFA, i.e., lowered the levels of 18- and 20-carbon fatty acids, e.g., DGLA and ARA in macrophage cellular phospholipids. It

Fig. 3 Effects of PNA on the level of total saturated fatty acids (SFA), total monounsaturated fatty acids (MUFA), total n-6 or n-3 polyunsaturated fatty acids (n-6 or n-3 PUFA) (a), and individual n-6 polyunsaturated fatty acids (b). RAW264.7 cells were incubated with the medium containing 0 lM(h), 10 lM( ), 25 lM( ), 50 lM ( ) or 100 lM(j) of PNA for 24 h. Each value represented the mean ± SE of three independent experiments. In each fatty acid or group of fatty acids, values with different letters are significantly different from each other at P \ 0.05

123 222 Lipids (2009) 44:217–224

Fig. 4 Effects of PNA incubation on levels of total n-3 polyunsaturated fatty acids (n-3 PUFA) (a), and individual n-3 polyunsaturated fatty acids (b). Cells were incubated with 25 lM of n-3 ETA alone (h)or a combination of 25 lM of n-3 ETA and 25 lM of PNA (j) for 24 h. Each value represents the mean ± SE of three independent experiments. In each category, symbol (*) denotes the difference between 2 values is significant at P \ 0.05 is possible that reduction of DGLA and ARA, could lead to a decrease in synthesis of PGE1 and PGE2, respectively. To examine this possibility, macrophage cells were incubated with varying levels of PNA (0, 12, 25, 50, and 100 lM) for 24 h and then treated with LPS for 16 h to stimulate the prostaglandin synthesis. Results in Fig. 5 (upper panel) show that LPS stimulation increased significantly the synthesis of both PGE1 and PGE2. Addition of PNA in the medium decreased the levels of both PGE1 and PGE2. The effect was dose-dependent. In a separated study, we examined the effect of PNA

(50 lM) on synthesis of PGE1 or PGE2 in macrophage cells pre-incubated with 50 lM of DGLA or ARA. The results in Fig. 5 (lower panel) show that cells pre-treated with DGLA or ARA significantly increased the production of PGE1 or PGE2, respectively. However, addition of PNA into medium significantly suppressed the level of PGE1 in cells pre-incubated with DGLA by 53%, and PGE2 in cells pre-incubated with ARA by 22%.

Effect of PNA on Expression of COX-2 Protein

To understand the mechanism of the inhibitory effect of Fig. 5 Effect of PNA on formation of prostaglandin E (h) and PNA on PGE1 and PGE2 activity, we examined the effect 1 prostaglandin E2 (j) in the LPS-treated RAW264.7 macrophage of PNA on the expression of COX-2 enzyme. Results in cells. Cells were incubated in the medium containing different levels Fig. 6 show that expression of COX-2 in RAW264.7 cells (0, 10, 25, 50, or 100 lM) of PNA (upper panel) for 24 h, or in the was highly induced upon LPS simulation (Fig. 6, lane 2 vs. medium containing either 50 lM of DGLA (or AA) alone or in a lane 1). When cells were treated with ARA or PNA alone, combination of 50 lM of PNA and 50 lM of DGLA (or AA) for 24 h (lower panel), followed by LPS treatment for 16 h. Each value the levels of COX-2 protein in comparison with the control represents the mean ± SE of three independent experiments. In PGE1 were increased by 18%, and 12%, respectively (lanes 3 and (h) or PGE2 (j), values with different letters or numbers are 5). When cells were treated with a combination of ARA significantly different from each other at P \ 0.05 and PNA, the levels of COX-2 protein were increased synergistically (26%, P \ 0.01). In contrast, incubation Discussion with DGLA, the cellular level of COX-2 protein was decreased by 11% (lane 7). Addition of PNA in the med- In this study, we observed that PNA was readily taken up, ium eradicated the lowering effect of DGLA: the level of incorporated and metabolized in murine RAW264.7 mac- COX-2 protein was raised to between those in cells incu- rophage cells. In cells, PNA was rapidly elongated to bated with PNA and those with DGLA (lane 6). form D7,11,14–20:3. A similar finding has previously been 123 Lipids (2009) 44:217–224 223

fatty acids (conversion of ARA to n-6 DPA or EPA to DHA) was enhanced in cells incubated with PNA (Scheme 1). In this study, we observed the inhibitory effect of PNA on eicosanoid production (Fig. 5). This effect could be exerted via the competitive incorporation of PNA and D7,11,14-20:3 into phospholipids which reduced the level of DGLA and ARA available for formation of eicosanoids (Fig. 3b and 5a). Previously, Li and his colleagues have shown that one of conjugated linoleic acid isomers

(10t,12c-CLA) reduced LPS-stimulated PGE2 production by decreasing the level of COX-2 protein expression in RAW264.7 cells [21]. Similarly, the inhibitory effect of PNA on PGE production could be due to its ability to modulate protein production. Results in the present study showed an increase in the level of COX-2 in macrophage cells treated with PNA (Fig. 6). This finding is comparable with the report that EPA treatment induced the expression

of COX-2, but suppressed the formation of PGE2 in cul- Fig. 6 Effects of PNA on the expression of COX-2 in RAW264.7 tured human lung cancer A549 cells [22]. Thus, the macrophage cells. Cells were incubated with only medium, or inhibitory effect of PNA on prostaglandin production was medium containing 50 lM of AA, DGLA or PNA alone, or a mixture of 50 lM AA and 50 lM PNA or 50 lM DGLA and 50 lM PNA for not exerted through suppression of COX-2 expression. 24 h, followed by LPS treatment for 16 h. Cyclooxygenase-2 It is of interest to note that sciadonic acid (D5,11,14– expression was determined by Western blot analysis. a-tubelin was 20:3), another NMIFA found in Podocarpus nagi (Podo- used as a loading control. Each value represents the mean ± SE of carpaceae) seed oil, behaves very similarly to PNA. This three independent experiments. Values with different letters are significantly different from each other at P \ 0.05 acid could be taken up, incorporated into phospholipids, and reduced both levels of ARA and PGE2 in cultured skin keratinocytes [23]. In comparison with PNA, the inhibitory reported in human hepatoma HepG2 cells [13]. We have effect of sciadonic acid on the level of ARA in membrane also observed the presence of D9,13,16–22:3 in cellular phospholipids became evident only when the concentration phospholipids (Fig. 1). The level of D9,13,16–22:3 of sciadonic acid in the culture medium exceeded 15 lM, increased as the length of incubation time and concentra- and inhibition on PGE2 production was observed only tion of PNA in medium increased. These findings suggest when the concentration of sciadonic acid reached 100 lM that a portion of D7,11,14–20:3 underwent further elon- [22]; while the inhibitory effects of PNA on the level of gation. This reaction was probably catalyzed by the fatty DGLA (or ARA) and its respective prostaglandin could be acid chain elongation system (FACES) which converts observed when the concentration of PNA was only 10 lM specifically 20-carbon fatty acids to 22-carbon fatty acids, (Fig. 2b and 5a). This finding suggests that PNA as com- such as ARA to ADA, EPA to n-3 DPA [19, 20]. Chapkin pared to sciadonic acid is more potent on suppressing the and his colleague has demonstrated in macrophages the PG production. The explanation may be attributed to the elongation of DGLA (D8,11,14–20:3) to n-6 docosatrienoic difference in chemical structures between these two acids: acid (DTrA; D10,13,16–22:3) by this enzyme system [18]. the PNA elongation product (D7,11,14–20:3) is more Incubation of PNA also modified compositions of cell similar to DGLA (D8,11,14–20:3) and ARA (D5,8,11,14– membrane fatty acids in RAW264.7 cells. In cells incu- 20:4) than is sciadonic acid (D5,11,14–20:3) in competing bated with PNA, significant portions of total n-6 PUFA, 18- for the catalytic sites of the COX-2 enzyme. This differ- carbon and 20-carbon n-6 fatty acids in phospholipids were ence in chemical structure could also explain the greater replaced by PNA and its metabolites (Fig. 3a). In spite of inhibitory effect of PNA on conversion of DGLA to PGE1 this, levels of 22-carbon ADA and n-6 DPA were increased (53%) than that on conversion of ARA to PGE2 (22%). significantly in cells incubated with PNA (Fig. 3b). Simi- In this study, we have clearly shown that PNA was larly, PNA decreased levels of total n-3 fatty acids, n-3 metabolized to form D7,11,14–20:3 and D9,13,16–22:3, ETA and EPA when n-3 ETA was supplemented into the and they significantly modulated n-6 PUFA metabolism medium, but levels of 22-carbon n-3 fatty acids (i.e., DPA and PGE synthesis. However, in rats, fed PSO failed to and DHA) were increased (Fig. 4b). These findings suggest modulate prostacyclin [1, 24]. This could be due to low that the metabolism of 20-carbon fatty acids to 22-carbon incorporation of PNA into phospholipid fractions (2–4% of 123 224 Lipids (2009) 44:217–224 total fatty acids) [1, 25], and to a lower rate of elongation is an essential structure for metabolism by the fatty acid chain of PNA to D7,11,14–20:3 as compared to that of GLA to elongation system of rat liver. Biochim Biophys Acta 1393:299– 306 DGLA [1, 10, 24]. 11. Levin G, Duffin KL, Obukowicz MG, Hummert SL, Fujiwara H, In conclusion, by using murine RAW264.7 macrophage Needleman P, Raz A (2002) Differential metabolism of dihomo- cells, we demonstrated that exogenous PNA could be taken c-linolenic acid (DGLA) and arachidonic acid (AA) by cyclo- up, incorporated and metabolized to form elongation oxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2): Implications for cellular synthesis of PGE1 and PGE2. Biochem J products. The incorporated PNA and its metabolite mod- 365:489–496 ulated fatty acid compositions of cellular phospholipids 12. Sprecher H, Luthria DL, Mohammed BS, Baykousheva SP and PUFA metabolism. Since DGLA (or ARA) was (1995) Reevaluation of the pathways for the biosynthesis of substituted by PNA and its metabolites, the synthesis of polyunsaturated fatty acids. J Lipid Res 36:2471–2477 13. Tanaka T, Takimoto T, Morishige J, Kikuta Y, Sugiura T, Sa- PGE1 (or PGE2) were also influenced. 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