Alexander E. A. Porter

Biosynthesis and tissue deposition of docosahexaenoic acid (22∶6n−3) in rainbow trout (Oncorhynchus mykiss)

Rainbow trout (Oncorhynchus mykiss) weighing ca. 5 g and previously acclimated for 8 wk on a diet comprising vegetable oil (11%), fish meal (5%), and casein (48%) as the major constituents were fed a pulse of diet containing deuterated (D5) (17,17,18,18,18)-18∶3n−3 ethyl ester. The synthesis and tissue distribution of D5-22∶6n−3 was determined 3,7,14, 24, and 35 d after the pulse. The whole-body accumulation of D5-22∶6n−3 was linear over the first 7 d, corresponding to a rate of 0.54±0.12 μg D5-22∶6n−3/g fish/mg D5-18∶3n−3 eaten/d. Maximal accretion of D5-22∶6n−3 was 4.3±1.2 μg/g fish/mg of D5-18∶3n−3 eaten after 14 d. The amount of D5-22∶6n−3 peaked in liver at day 7, in brain and eyes at day 24, and plateaued after day 14 in visceral and eye socket adipose tissue and in the whole fish. The majority of D5-22∶6n−3 was found in the carcass (remaining tissues minus the above tissues analyzed separately) at all times. On a per milligram lipid basis, liver and eyes had the highest concentration of D5-22∶6n−3. The experimental diet also contained 21∶4n−6 ethyl ester as a marker to estimate the amount of food eaten by individual fish. From such estimates it was calculated that the great majority of the D5-tracer was catabolized, with the combined recovery of D5-18∶3n−3 plus D5-22∶6n−3 being 2.6%. The recovery of 21∶4n−6 was 57.6%. The concentration of 22∶6n−3 in the fish decreased during the 13-wk period, and the amount of 22∶6n−3 synthesized from 18∶3n−3 was only about 5% of that obtained directly from the fish meal in the diet.

Pyloric ceca are significant sites of newly synthesized 22∶6n−3 in rainbow trout (Oncorhynchus mykiss)

In this pulse-chase study, rainbow trout fed a diet containing deuterated (D5) (17,17,18,18,18)-18∶3n−3 ethyl ester accumulated D5-22∶6n−3 in pyloric ceca to a greater extent than in liver 2 d post-dose. The ratio of newly synthesized D5-22∶6n−3 in ceca to that in liver 2 d after feeding D5-18∶3n−3 was 4.7±1.2 when expressed as per mg tissue and 5.2±2.4 when expressed as per mg protein. The amount of D5-22∶6n−3 in ceca then declined whereas that in liver and blood increased, with the ratio of ceca to liver falling to 1.7 and 1.4, respectively, by day 5 and approaching unity by day 9. A crude cecal mucosa fraction contained 123±50 ng D5-22∶6n−3/mg protein/mg D5-18∶3n−3 eaten 2 d after feeding the tracer, compared with 35±21 ng D5-22∶6n−3/mg protein/mg D5-18∶3n−3 eaten in liver. Three days later the amount in cecal mucosa had fallen by one-third and that in liver had increased threefold. Most of the D5-18∶3n−3 was catabolized very rapidly. The ratio of D5-18∶3n−3 to 21∶4n−6 (a relatively inert FA marker) in the diet was 4.0, but this fell to 0.30 in ceca and ca. 0.8 in liver, blood, and whole carcass one day after feeding. These results indicate that ceca are active in the synthesis of 22∶6n−3 and the oxidation of 18∶3n−3.

IDENTIFICATION OF THREE HYDROXYFLAVAN PHYTOALEXINS FROM DAFFODIL BULBS

Abstract Three phytoalexins were isolated from daffodil bulb scales inoculated with Botrytis cinerea and identified as 7-hydroxyflavan, 7,4′-dihydroxyflavan and 7,4′-dihydroxy-8-methylflavan. The structures of the phytoalexins were confirmed by total synthesis.

Metabolism of 18:4n-3 (stearidonic acid) and 20:4n-3 in salmonid cells in culture and inhibition of the production of prostaglandin F2α (PGF2α) from 20:4n-6 (arachidonic acid)

Arachidonic acid (AA; 20:4n-6) is the precursor of a range of highly biologically active derivatives, collectively termed eicosanoids, including prostaglandins, thromboxanes, leukotrienes and lipoxins, that act as autocrine hormones regulating many physiological processes including haemostasis, reproduction, immune and inflammatory responses. Eicosapentaenoic (EPA; 20:5n-3) and dihomo-γ-linolenic (20:3n-6) acids modulate eicosanoid metabolism by both inhibiting the conversion of AA to eicosanoids whilst simultaneously being converted to eicosanoids with different, often attenuated, properties compared to their AA homologues. Eicosatetraenoic acid (20:4n-3) is a naturally occurring C20 polyunsaturated fatty acid (PUFA), present in fish oil at levels of around 1–2%, that has been suggested to be the active metabolite responsible for the anti-inflammatory effects of plant oils containing stearidonic acid (18:4n-3). However, the biochemical properties of 20:4n-3 in terms of cellular biology have rarely been investigated, partly due to difficulties in obtaining the fatty acid in high purity. In this paper, we describe methods for the medium scale laboratory preparation of high purity 20:4n-3, and investigate its metabolism in fish cell culture systems which normally contain significant amounts of n-3 PUFA. Thus the incorporation and metabolism of 18:4n-3 and 20:4n-3, and their distribution in phospholipid classes was studied in an established cell line from Atlantic salmon (Salmo salar) (AS), and the effects of 20:4n-3 on eicosanoid production studied in freshly isolated macrophages from rainbow trout (Oncorhynchus mykiss). Both 18:4n-3 and 20:4n-3 were preferentially esterified into phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine in contrast with the accumulation of AA in phosphatidylinositol. Incorporated 18:4n-3 was readily converted to 20:4n-3, and both fatty acids were further desaturated and elongated to EPA and 22:5n-3 but not 22:6n-3. Supplementation with 20:4n-3 decreased the conversion of AA into prostaglandins, as demonstrated by the decreased levels of PGF2α produced in trout macrophages supplemented with 20:4n-3 and AA compared to cells supplemented with AA alone. In addition, 20:4n-3 was converted into eicosanoids in fish cells as indicated by the presence of Δ17,18 12-HETE, Δ17,18 PGE1 and Δ17,18 PGF1α in extracts from rainbow trout macrophages incubated with 20:4n-3.

Reductive formylation of oximes; an approach to the synthesis of vinyl isonitriles

Abstract Reduction of ketoximes using anhydrous titanium-(III)-acetate in the presence of mixed formic-acetic anhydride yields N-formyl enamines which may be dehydrated to vinyl isonitriles.

Dihydrowyerone derivatives as components of the furanoacetylenic phytoalexin response of tissues of Vicia faba.

Abstract Changes in concentrations of 7 wyerone derivatives in bean tissues undergoing resistant reactions to Botrytis cinerea or B. fabae and in cotyledons in response to mercuric chloride have been examined using high performance liquid chromatography. The proportion of derivatives occurring in their saturated (dihydro) forms varied between cotyledon, leaf and pod tissues and with time after inoculation. Unsaturated derivatives were always present in greater concentrations than their dihydro analogues.

Floral volatiles of the sweet pea Lathyrus odoratus

Abstract The volatile components of the floral fragrances from three sweet pea cultivars have been determined using polymer entrainment and solvent elution, combined with gas-chromatography–mass spectrometry. A total of 48 compounds were detected in quantifiable amounts, 41 of which were common to all three cultivars. The most abundant compounds were consistently found to be ( E )-β-ocimene and linalool.

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.

Modification of odd-chain length unsaturated fatty acids by hepatocytes of rainbow trout (Oncorhynchus mykiss) fed diets containing fish oil or olive oil

Hepatocytes isolated from rainbow trout fed on diets containing either fish oil or olive oil were incubated with individual odd-chain length unsaturated fatty acids (19∶1n−9, 19∶2n−6, 19∶3n−3, 21∶2n−6, 21∶3n−6, 21∶4n−6, 21∶3n−3, and 21∶5n−3) to examine whether these fatty acids were substrates for modification by desaturation and elongation. All odd-chain length fatty acids were readily assimilated into the lipids of hepatocytes from both dietary groups of fish, but their conversion to longer-chain, more unsaturated derivatives was more pronounced with cells from trout fed olive oil. Thus, the conversion of 19∶2n−6 and 21∶2n−6 to 21∶3n−6 and 21∶4n−6, and of 19∶3n−3 to 21∶4n−3 and 21∶5n−3, was most obvious in cells from the olive oil group, as was the conversion of 21∶3n−6 and 21∶3n−3 to 21∶4n−6 and 21∶4n−3, respectively. Elongation of 19∶1n−9 to 21∶1n−9 and 23∶1n−9 occurred in cells from both groups. No 23∶6n−3 was detectable as a product of 19∶3n−3 or 21∶3n−3. However, this fatty acid was a major product formed by cells from fish fed olive oil presented with 21∶5n−3. Cells from both groups of fish incorporated 21∶4n−6 and 21∶5n−3 into their lipids largely without modification but chain-shortened around 40, 23, and 19% of the incorporated 21∶2n−6, 21∶3n−3, and 19∶1n−9, respectively. The results demonstrate that odd-chain length unsaturated fatty acids can act as substrates for the desaturation, elongation, and chain-shortening systems of trout hepatocytes.

Tissue deposition of n-3 FA pathway intermediates in the synthesis of DHA in rainbow trout (Oncorhynchus mykiss).

The tissue distribution of newly synthesized 22∶6n-3 and intermediate PUFA was examined in rainbow trout to further our understanding of the metabolism of this EFA in fish. Rainbow trout were fed a pulse of deuterated linoleic acid (D5−17,17,18,18,18−18∶3n-3), and the tissue distribution of deuterated anabolites was determined at interval up to 35 d post-dose by GC-negative chemical ionization MS of the pentafluorobenzyl derivatives. D5−22∶6n-3 was the major deuterated FA in liver and cecal mucosa 2 and 5 d post-dose. All the n−3 FA pathway intermediates were found in liver, cecal mucosa, and blood including D5−24∶5n-3 and D5−24∶6n-3. Brain and eyes also contained the full suite of intermediate deuterated FA, but with a different profile from liver when analyzed over a longer time course up to 35 d. D5−20∶5n-3 was the major component in brain up to 7 d, after which D5−22∶6n-3 became predominant, but D5−22∶5n-3 constituted ca. 20% of FA throughout the time period. The pattern in eyes was similar but less pronounced. In visceral adipose tissue there was a much greater accumulation of the initial substrate, D5−18∶3n-3, with D5−18∶4n-3 and D5−22∶6n-3 the predominant deuterated FA at all time points. There was a similar though less pronounced trend in eye socket adipose tissue. The C24 PUFA were not detected in visceral fat and barely detected in eye socket fat. The results show that the kinetics of accumulation and depletion of the various n−3 PUFA differ between tissues. The presence of pathway intermediate FA provides evidence that liver and ceca possess the full metabolic pathway for synthesis of 22∶6n-3, whereas brain and eyes are less active, with an accumulation of pentaene intermediate FA, and adipose tissue is inactive.