Cultured fish cells metabolize octadecapentaenoic acid (all-cis δ3,6,9,12,15–18∶5) to octadecatetraenoic acid (all-cis δ6,9,12,15–18∶4) via its 2-trans intermediate (trans δ2, all-cis δ6,9,12,15–18∶5)

Octadecapentaenoic acid (all-cis δ3,6,9,12,15–18∶5; 18∶5n−3) is an unusual fatty acid found in marine dinophytes, haptophytes, and prasinophytes. It is not present at higher trophic levels in the marine food web, but its metabolism by animals ingesting algae is unknown. Here we studied the metabolism of 18∶5n−3 in cell lines derived from turbot (Scophthalmus maximus), gilthead sea bream (Sparus aurata), and Atlantic salmon (Salmo salar). Cells were incubated in the presence of approximately 1 μM [U-14C] 18∶5n−3 methyl ester or [U-14C]18∶4n−3 (octadecatetraenoic acid; all-cis δ6,9,12,15–18∶4) methyl ester, both derived from the alga Isochrysis galbana grown in H14CO3 −, and also with 25 μM unlabeled 18∶5n−3 or 18∶4n−3. Cells were also incubated with 25 μM trans δ2, all-cis δ6,9,12,15–18∶5 (2-trans 18∶5n−3) produced by alkaline isomerization of 18∶5n−3 chemically synthesized from docosahexaenoic acid (all-cis δ4,7,10,13,16,19–22∶6). Radioisotope and mass analyses of total fatty acids extracted from cells incubated with 18∶5n−3 were consistent with this fatty acid being rapidly metabolized to 18∶4n−3 which was then elongated and further desaturated to eicosatetraenoic acid (all-cis δ8,11,14,17,19–20∶4) and eicosapentaenoic acid (all-cis δ5,8,11,14,17–20∶5). Similar mass increases of 18∶4n−3 and its elongation and further desaturation products occurred in cells incubated with 18∶5n−3 or 2-trans 18∶5n−3. We conclude that 18∶5n−3 is readily converted biochemically to 18∶4n−3 via a 2-trans 18∶5n−3 intermediate generated by a Δ3, Δ2-enoyl-CoA-iso-merase acting on 18∶5n−3. Thus, 2-trans 18∶5n−3 is implicated as a common intermediate in the β-oxidation of both 18∶5n−3 and 18∶4n−3.

Octadecapentaenoic acid (all-cis 18:5n-3) is a fatty acid characteristically present in certain algal groups in marine phytoplankton (1), including dinoflagellates (2), haptophytes (3,4) and prasinophytes (5), all of which have important roles in the marine ecosystem.18:5n-3 is usually co-associated in these organisms with 22:6n-3.Given that biosynthesis of 22:6n-3 involves peroxisomal chain shortening of its precursor 24:6n-3, it is possible that 18:5n-3 is biosynthesized by chain shortening of 20:5n-3 (see 6).Marine zooplankton and fish ingesting phytoplankton contain little or no 18:5n-3 demonstrating that this fatty acid is readily metabolized by marine animals.Clearly it could be completely catabolized by marine animals by β-oxidation but it may also be directly chain elongated to 20:5n-3.We decided to test the latter possibility in fish cell cultures because marine fish in general have a very limited ability to convert 18:3n-3 to 20:5n-3 and thence to 22:6n-3 (7,8).In some species of marine fish, e.g.turbot, this appears to be due to a deficiency of C 18 to C 20 fatty acid elongase (9), whereas in others, e.g.sea bream, it appears to be due to a deficiency of Δ5 fatty acid desaturase (10).The availability of 18:5n-3 can help distinguish between these two possibilities and, in the event of it being a substrate for C 18 to C 20 fatty acid elongase, algae containing this fatty acid could be useful dietary supplements in marine fish larval culture.
In this study we prepared 18:5n-3 from the haptophycean alga Isochrysis galbana and also by chemical synthesis from 22:6n-3, and studied its metabolism in cultured cells from turbot, seabream and Atlantic salmon that differ in their abilities to perform C 18 to C 20 elongation and Δ5 fatty acid desaturation reactions.The results show that 18:5n-3 is very readily converted by cells from all three species to 18:4n-3 via a 2-trans 18:5n-3 intermediate.grown in H 14 CO 3 as described by Ghioni et al. (9).In brief, total lipid was extracted from the radioactive algal cells, transmethylated by incubation with 1% sulphuric acid in methanol at 50 o C for 16 hours to generate fatty acid methyl esters, and the methyl esters of 18:4n-3 and 18:5n-3 isolated by silver nitrate thin-layer chromatography (AgNO 3 -TLC) (11).A total of 5 µCi of methyl esters of both [U-14 C]18:4n-3 and [U-14 C]18:5n-3 were obtained, with a specific activity of approximately 12 and 19 mCi/mmol, respectively.The identity of the 18:4n-3 and 18:5n-3 methyl esters was confirmed and their purity (>99%) and specific activity determined by radio-gas chromatography as (GC) as described by Buzzi et al. (12).

Fatty
Unlabeled methyl esters of 18:5n-3 and 18:4n-3 were prepared by extracting total lipid from I. galbana cultures that were not incubated with labeled bicarbonate, transmethylating as described above and recovering the fatty acid methyl ester fraction enriched in polyunsaturated fatty acids (PUFA) by TLC in hexane/diethyl ether/acetic acid (90:10:1, by vol.).Methyl esters of 18:5n-3 and 18:4n-3 were then separated from the fatty acid methyl ester fraction on an ODS C18 HPLC column (diameter 5 mm) by eluting with acetonitrile at 1.5 ml/min, using UV detection at 215 nm.Under these conditions, 18:5n-3 was the first fatty acid eluted.The purity of the methyl esters of both 18:5n-3 and 18:4n-3 was > 99% as determined by GC and GCmass spectrometry as detailed previously (9,13).
Unlabeled 18:5n-3 was also prepared in greater quantities as the free acid by chemical synthesis according to the method of Kuklev et al. (14), which involves a γ-iodo-lactonization of 22:6n-3.The 22:6n-3 used in the synthesis was a concentrate ( > 95%) kindly supplied by Croda Universal Ltd., Hull, UK.
Preliminary experiments incubating AS cells with 25µM unlabeled 18:4n-3, added either as the fatty acid salt complexed to bovine serum albumin, or as the methyl ester complexed to bovine serum albumin, or as the methyl ester in ethanol, showed no differences in the metabolism of these substrates via desaturation and elongation to 20:4n-3 and 20:5n-3 (see 9).
No methyl esters were detectable in total lipid extracted from cells incubated with methyl esters.Therefore, the methyl esters of both 18:4n-3 and 18:5n-3 (and 2-trans 18:5n-3) were used in order to avoid having to saponify the methyl ester of 18:5n-3 to its free fatty acid (see Results).
Lipid extraction and analysis.Methods for the extraction of total lipids from cells, preparing fatty acid methyl esters from total lipid, determining radioactivity in fatty acid methyl esters separated on AgNO 3 , and analysing fatty acids by GC and radio -GC were as described in detail previously (9,10,12).The identities of individual radioactive fatty acid methyl esters separated by AgNO 3 for radioassay were confirmed by direct radio -GC analyses (12).
Statistical Analysis.All results are presented as means ± S.D. of three experiments.The statistical significance of differences between mean values in Table 1 obtained for each cell line were analysed by the Student's t-test with differences reported as significant if P < 0.05 (18).

RESULTS
The initial experiments in this study were performed with radiolabeled 18:4n-3 and 18:5n-3 isolated from the alga Isochysis galbana grown in the presence of radioactive bicarbonate.The fatty acids were prepared as methyl esters following transmethylation of total lipid extracted from the alga.In preparing the free acid by saponifying the methyl ester of 18:5n-3 we noted by TLC analyses of the reaction products that all-cis 18:5n-3 was present in low yield and accompanied by other unknown compounds.We compared the incorporation of 18:5n-3 free acid and its methyl ester into lipids of the three cultured cell lines chosen for study, adding the substrates to the cells in ethanol.No differences were found in the incorporation patterns for the free acid and its methyl ester.Therefore, to maximise the use of the limited amounts of 18:5n-3 methyl ester prepared from I. galbana, we routinely used the methyl ester of 18:5n-3 in subsequent experiments.
The results of the incorporation of the methyl esters of [U- 14 C] 18:4n-3 and [U- 14 C] 18:5n-3 into the three cell lines are shown in Table 1.In these experiments fatty acid methyl esters were prepared from total lipid isolated from the cell cultures and then separated by AgNO 3 TLC for radioassay, with the results being confirmed by radio-GC.In no case was radioactive 18:5n-3 recovered from the cells.Rather, 18:4n-3, 20:4n-3 and 20:5n-3 were the major fatty acids labeled after incubation with either [U- 14   2).However, as with the marine fish cell lines, the percentage distribution patterns in the salmon cell line were the same for cells incubated with [U- 14 C] 18:4n-3 and cells incubated with [U- 14 C] 18:5n-3.Thus, the two radioactive substrates yielded the same result in a given cell line irrespective of the pattern of desaturation/elongation expressed by the cell line.Table 1 also shows that of the 0.25 µCi of radioactive fatty acid substrate added, averaged values of 32% and 23% of radioactivity were recovered as fatty acids from cells incubated with [U- 14 C] 18:4n-3 and [U- 14 C] 18:5n-3, respectively, from the three cell lines.However, the difference between the two substrates was significant only for the Atlantic salmon cell line.An obvious explanation for the findings in Table 1 is that 18:5n-3 is rapidly converted to 18:4n-3 in the cells.This possibility, together with the apparent lability of 18:5n-3 during alkaline saponification of its methyl ester prompted us to examine the stability of 18:5n-3 in more detail.Therefore, we synthesised 18:5n-3 as the free acid from 22:6n-3 using the γ-iodolactonization of 22:6n-3 method as described by Kuklev et al. (14).Treating the 18:5n-3 product with aqueous alkaline generated four compounds, two major and two minor, as shown by HPLC analysis (Materials and Methods).The two major products were identified by 1 HNMR and GC as all-cis 18:5n-3 (25% of the total products) and 2-trans 18:5n-3 (62% of the total products).
We then studied the metabolism of the methyl esters of the two isomers of 18:5n-3 (and 18:4n-3) in the Atlantic salmon cell line.As there was insufficient isotopically labeled 18:5n-3 available to produce labeled 2-trans 18:5n-3 using the method above, the experiment was performed with unlabeled fatty acid substrates at higher concentrations (25 µM) to enable analysis by conventional GC methods.The results showed that cells incubated with 25 µM of all three fatty acid substrates showed increased proportions of 18:4n-3, 20:4n-3 and, to a lesser extent, 22:4n-3 and 20:5n-3 in their lipids over time up to 24 h after supplementation (Table 3).
There was no change in the proportions of 22:6n-3 or 22:5n-3 in cell cultures incubated with any of the three fatty acid substrates.The increased concentration of 18:4n-3 in cellular lipids was somewhat less with 2-trans 18:5n-3 than all-cis 18:5n-3, but the patterns were similar for all three substrates and broadly the same as those observed with the methyl esters of [U- 14 C] 18:4n-3 and [U- 14 C] 18:5n-3 in Table 1.The results in Table 3 also show that no 18:5n-3 was detected in lipids recovered from the cells incubated with any of the fatty acid substrates.

DISCUSSION
Octadecapentaenoic acid (all-cis 18:5n-3; Fig. 1) is present in classes of algae that have major roles in the marine environment (6).Thus, dinoflagellates can form large toxic blooms; the haptophycean Phaeocystis pouchetti is a major source of scum polluting beaches and inshore waters of mainland European coasts; the haptophycean coccolithophore Emiliania huxleyi is a major source of marine CaCO 3 deposits; the latter two organisms are major sources of dimethyl sulphide implicated as a source of "acid rain".18:5n-3 can account for more than 15% of the total fatty acids in these organisms, being present in glycolipids where it can account for 66% of the total fatty acids (6).However, it is seldom if ever detected in significant amounts in phytoplankton -consuming animals (1), i.e. in zooplankton and some fish, consistent with its being rapidly metabolized by marine animals.
18:5n-3 coexists in the aforementioned organisms with 22:6n-3 and it is unusual in sharing with 22:6n-3 the property of having the maximum number of double bonds possible in a carbon chain.However, unlike 22:6n-3 whose terminal double bond is 4 carbons from the carboxyl terminus, the terminal double bond in 18:5n-3 is 3 carbons from the carboxyl terminus.The insertion of the last (Δ4) double bond in 22:6n-3 occurs by a mechanism whereby 24:5n-3 is Δ6 desaturated to 24:6n-3 which is then chain shortened in peroxisomes to 22:6n-3 (19).Similarly, the biosynthesis of 18:5n-3 is not thought to be possible solely through the action of desaturases and elongases with the conventional pathway from 18:3n-3 to 20:5n-3 proceeding via 18:4n-3 then 20:4n-3.However, by analogy with the biosynthesis of 22:6n-3, the formation of 18:5n-3 is probably straightforward, at least in principle, in that it can be formed directly from 20:5n-3 by chain shortening as postulated previously (6).The precise subcellular location(s) of these putative chain shortening steps in the biosynthesis of 22:6n-3 and 18:5n-3 in these marine phytoplankton is not known (20).
As its biosynthesis requires a special mechanism, so the catabolism of 22:6n-3 requires a special mechanism in that the 2-trans, 4-cis intermediate formed by the initial direct dehydrogenation of 22:6n-3 is converted by a 2,4 -dienoyl reductase to a 3-trans intermediate, which is then converted by a Δ 3 , Δ 2 -enoyl isomerase to a 2-trans intermediate (21,22).The latter two enzymes are auxiliary enzymes to the multifunctional enzymes of β-oxidation required for the β-oxidation of polyunsaturated fatty acids in both mitochondria and peroxisomes (23,24).The 2-trans 22:6n-3 intermediate can then continue in the conventional β-oxidation pathway through hydration across the 2,3 trans double bond to yield the 3-hydroxy and then the 3-keto intermediate.Similarly, catabolism of 18:5n-3 requires, in principle, the operation of a Δ 3 , Δ 2enoyl isomerase to generate the 2-trans intermediate required for entry into the β-oxidation pathway though, to our knowledge, this reaction has never been studied with 18:5n-3 (Fig. 1).
The results here establish the ease with which all-cis 18:5n-3 can be converted to its 2trans isomer.Thus, all-cis 18:5n-3, chemically synthesised from 22:6n-3, was readily converted in high yield to its 2-trans isomer by treatment with alkali in aqueous ethanol.The protons at C-2 of all-cis 18:5n-3 are both allylic to the cis-3,4-double bond and α-to the terminal carboxyl group, and hence should experience a reduction in pK a (relative to, for example, the corresponding protons in 18:4n-3).These combined effects would be expected to result in an increased tendency of the 18:5n-3 to undergo enolization, particularly in protic solvents.
Enolization results in the conjugation of the 3,4-double bond with the enol and hence the configurational stability of the 3,4-double bond may be compromised.Furthermore, when reprotonation of the intermediate enol occurs, it can do so either at C-2 (regenerating 18:5n-3 with either cis-or trans-stereochemistry at C-3), or at C-4, which would result in the conjugation of the double bond with the carboxylic acid.The latter process is expected to be thermodynamically preferred and would be expected to result in the preferential formation of the trans Δ2, all-cis Δ6,9,12,15-18:5n-3 acid.The chemical isomerization of all-cis 18:5n-3 to trans Δ2, all-cis Δ6,9,12,15-18:5n-3 has not, to our knowledge, been reported before.However, it may have been observed but misinterpreted in that, in reducing all-cis 18:5n-3 with hydrazine to determine double bonds, Napolitano et al. (25) noted a loss of 18:5n-3 and mentioned the occurrence of an extra peak of 18:4n-3 in gas chromatograms of the reaction products.It is possible that the extra peak was a 2-trans 18:5n-3 isomer whose formation was prompted by availability of protons in the reaction.
Irrespective of how the relevant anabolic and catabolic pathways are controlled, the ease of conversion of all-cis 18:5n-3 to 2-trans 18:5n-3 and thence to 18:4n-3, demonstrated here in fish cells, readily accounts for the absence of 18:5n-3 from marine trophic levels higher than the algae.
C] 18:4n-3 or [U-14 C] 18:5n-3 in all three cell lines.The percentage distribution of radioactivity between 18:4n-3, 20:4n-3 and 20:5n-3 differed in the different cell lines depending upon the relative activities of C 18 -C 20 elongase and Δ5 desaturase.In the turbot cell line (TF) the percentages of radioactivity recovered in 18:4n-3, 20:4n-3 and 20:5n-3 were the same in cells incubated with [U-14 C] 18:4n-3 and [U-14 C] 18:5n-3, with 18:4n-3 containing a high percentage of radioactivity, 20:5n-3 a modest percentage and 20:4n-3 a minor percentage.A similar result was obtained for the seabream cell line (SAF-1) except that the percentages of radioactivity in 20:4n-3 and 20:5n-3 were essentially reversed compared to the turbot cell line.The Atlantic salmon (AS) cell line gave a different result in that the highest percentage of radioactivity was recovered in 20:5n-3, with both 18:4n-3 and 20:4n-3 having moderate percentages of radioactivity.The species (cell line) differences were emphasised by presenting the data as the summed products for each enzymic step in the pathway, with the marine fish cells clearly showing considerably lower C 18 -C 20 elongase activity and, in the case of sea bream cells, lower Δ5 desaturase activity compared to the salmon cells (Table