eicosa-5-11-14-trienoic-acid and juniperonic-acid

The lipids of gymnosperms frequently feature unusual polyunsaturated fatty acids (PUFAs) such as sciadonic acid (20:3Δ5,11,14) and juniperonic acid (20:4Δ5,11,14,17) showing a first double bond on C-5 which is separated from the next double bond by five methylene units. Compared to "classic" fatty acids, these fatty acids are not easily commercially available and their prices are quite high. For this reason, we wished to isolate those fatty acids from the seed oil of Podocarpus falcatus by countercurrent chromatography (CCC) after conversion of the fatty acids to methyl esters (FAMEs). The contribution of sciadonic acid (20:3Δ5,11,14) and juniperonic acid (20:4Δ5,11,14,17) in the unfractionated sample was 10% and 6% respectively, while oleic acid (18:1Δ9) and linoleic acid (18:2Δ9,12) were the major fatty acids. After a first CCC run with FAMEs from Podocarpus falcatus, fractions enriched in the target compounds were chosen for subsequent isolation by means of two subsequent CCC runs. Initially, 13mg of juniperonic acid was recovered with a purity of 92% according to analysis by gas chromatography with mass spectrometry (GC/MS). Further purification of this fraction yielded 2.7mg with a purity of 99% according to GC/MS. The isolation of sciadonic acid was hampered by high amounts of linoleic acid with the same equivalent chain length in suitable fractions of the first CCC separation. After an enrichment step by CCC, the critical pair sciadonic acid and linoleic acid was finally separated as free fatty acids. After this step, 4.4mg of sciadonic acid was recovered with 99% purity. The methodology could also be applied to isolate larger amounts of those fatty acids or for the isolation of other minor fatty acids.

Topics: Arachidonic Acids; Countercurrent Distribution; Embryophyta; Fatty Acids, Unsaturated; Gas Chromatography-Mass Spectrometry; Linoleic Acid

Juniperonic acid (Delta-5c,11c,14c,17c-20:4, JA) is a polymethylene-interrupted (PMI) fatty acid that occurs in Biota orientalis. In this study, we found that JA has an antiproliferative activity. Swiss 3T3 cells were preloaded with fatty acids before stimulation with bombesin, a mitogenic neuropeptide, and proliferation of the cells was assessed by [(3)H]thymidine incorporation. Preloading of linoleic acid (Delta-9c,12c-18:2) significantly enhanced bombesin-induced proliferation. In contrast, preloading of eicosapentaenoic acid (Delta-5c,8c,11c,14c,17c-20:5, EPA) suppressed proliferation. Likewise, cells preloaded with JA showed a significantly curtailed response to bombesin. The antiproliferative potency of JA was equivalent to that of EPA. Sciadonic acid (Delta-5c,11c,14c-20:3), an omega-6 analogue of JA did not show antiproliferative activity, suggesting the importance of the omega-3 double bond rather than the PMI structure. The EPA-like activity of JA may be involved in the pharmaceutical activity of biota seeds, a psychoactive Chinese traditional medicine.

Topics: Animals; Arachidonic Acids; Bombesin; Cell Proliferation; Eicosapentaenoic Acid; Fatty Acids; Fatty Acids, Unsaturated; Lipid Metabolism; Mice; Phospholipids; Seeds; Swiss 3T3 Cells; Thuja

Sciadonic acid (20:3 Delta-5,11,14) and juniperonic acid (20:4 Delta-5,11,14,17) are polyunsaturated fatty acids (PUFAs) that lack the Delta-8 double bond of arachidonic acid (20:4 Delta-5,8,11,14) and eicosapentaenoic acid (20:5 Delta-5,8,11,14,17), respectively. Here, we demonstrate that these conifer oil-derived PUFAs are metabolized to essential fatty acids in animal cells. When Swiss 3T3 cells were cultured with sciadonic acid, linoleic acid (18:2 Delta-9,12) accumulated in the cells to an extent dependent on the concentration of sciadonic acid. At the same time, a small amount of 16:2 Delta-7,10 appeared in the cellular lipids. Both 16:2 Delta-7,10 and linoleic acid accumulated in sciadonic acid-supplemented CHO cells, but not in peroxisome-deficient CHO cells. We confirmed that 16:2 Delta-7,10 was effectively elongated to linoleic acid in rat liver microsomes. These results indicate that sciadonic acid was partially degraded to 16:2 Delta-7,10 by two cycles of beta-oxidation in peroxisomes, then elongated to linoleic acid in microsomes. Supplementation of Swiss 3T3 cells with juniperonic acid, an n-3 analogue of sciadonic acid, induced accumulation of alpha-linolenic acid (18:3 Delta-9,12,15) in cellular lipids, suggesting that juniperonic acid was metabolized in a similar manner to sciadonic acid. This PUFA remodeling is thought to be a process that converts unsuitable fatty acids into essential fatty acids required by animals.

Topics: alpha-Linolenic Acid; Animals; Arachidonic Acids; Fatty Acids, Essential; Fatty Acids, Unsaturated; Linoleic Acid; Metabolic Networks and Pathways; Mice; Microsomes, Liver; Peroxisomes; Rats; Swiss 3T3 Cells

Sciadonic acid (20:3, delta-5c,11 c,14c) is a polymethylene-interrupted PUFA (PMI-PUFA) that is present in conifer seeds and known to be incorporated into animal cells and to accumulate in membrane PI as a substitute for arachidonate. In this study, we investigated whether PI having sciadonate could serve as source of DAG that could activate protein kinase C (PKC). When Swiss 3T3 cells cultured with sciadonic acid were stimulated with 100 nM of bombesin, 1-stearoyl-2-sciadonoyl-glycerol (G) and 1-stearoyl-2-arachidonoyl-G were produced. The net increments of these two molecular species of DAG reflected the levels of the two molecular species in the PI in the cells. When cells cultured with juniperonic acid (20:4, delta-5c,11c,14c,17c) were stimulated, 1-stearoyl-2-juniperonoyl-G was produced in proportion to the level of this molecular species in PI in the cells. We also examined PKC activation by synthetic DAG using a partially purified PKC fraction from rat brain and found that both 1-stearoyl-2-sciadonoyl-G and 1-stearoyl-2-juniperonoyl-G could activate PKC comparably to 1 -stearoyl-2-arachidonoyl-G. These results indicate that 1-stearoyl-PI having these C20 PMI-PUFA residues can serve as sources of potential signaling molecules.

Topics: Animals; Arachidonic Acids; Bombesin; Brain; Diglycerides; Enzyme Activation; Fatty Acids, Unsaturated; Male; Mice; Protein Kinase C; Rats; Signal Transduction; Swiss 3T3 Cells

Sciadonic acid (20:3 Delta-5,11,14) is an n-6 series trienoic acid that lacks the Delta8 double bond of arachidonic acid. This fatty acid is not converted to arachidonic acid in higher animals. In this study, we characterized the metabolic behavior of sciadonic acid in the process of acylation to phospholipid of HepG2 cells. One of the characteristics of fatty acid compositions of phospholipids in sciadonic acid-supplemented cells is a higher proportion of sciadonic acid in phosphatidylinositol (PtdIns) (27.4%) than in phosphatidylethanolamine (PtdEtn) (23.2%), phosphatidylcholine (PtdCho) (17.3%) and phosphatidylserine (PtdSer) (20.1%). Similarly, the proportion of arachidonic acid was higher in PtdIns (35.8%) than in PtdEtn (29.1%), PtdSer (18.2%) and PtdCho (20.2%) in arachidonic-acid-supplemented cells. The extensive accumulation of sciadonic acid in PtdIns resulted in the enrichment of newly formed 1-stearoyl-2-sciadonoyl molecular species (38%) in PtdIns and caused the reduction in the level of pre-existing arachidonic-acid-containing molecular species. The kinetics of incorporation of sciadonic acid to PtdEtn, PtdSer and PtdIns of cells were similar to those of arachidonic acid. In contrast to sciadonic acid, neither eicosapentaenoic acid (20:5 Delta-5,8,11,14,17) nor juniperonic acid (20:4 Delta-5,11,14,17) accumulated in the PtdIns fraction. Rather, these n-3 series polyunsaturated fatty acids, once incorporated into PtdIns, tended to be excluded from PtdIns. In addition, the level of arachidonic-acid-containing PtdIns molecular species remained unchanged by eicosapentaenoic-acid-supplementation. These results suggest that sciadonic acid or sciadonic-acid-containing glycerides are metabolized in a similar manner to arachidonic acid or arachidonic-acid-containing glyceride in the biosynthesis of PtdIns and that sciadonic acid can effectively modify the molecular species composition of PtdIns in HepG2 cells. In this regard, sciadonic acid will be an interesting experimental tool to clarify the significance of arachidonic acid-residue of PtdIns-origin bioactive lipids.

Topics: Acyltransferases; Animals; Arachidonic Acid; Arachidonic Acids; Chromatography, High Pressure Liquid; Eicosapentaenoic Acid; Fatty Acid Desaturases; Fatty Acids, Unsaturated; Humans; Hydrogenation; Kinetics; Lysophospholipids; Microsomes, Liver; Phosphatidylinositols; Rats; Substrate Specificity; Tumor Cells, Cultured