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(+)-Catechin and (-)-epicatechin are found in many foods and may have important effects on human health. These compounds, like many other catechols, are thought to be converted to methylated metabolites after ingestion. This paper describes the synthesis of the 3'- and 4'-methyl ethers and their unambiguous identification. These products, along with catechin, epicatechin and an internal standard, (+)-taxifolin, were separated using RP-HPLC with ultraviolet, electrochemical and fluorescence detection. The trimethylsilylated derivatives of the seven compounds were also separated by GC with mass spectrometric detection. The limits of detection and selectivity of the analytical methods were compared with respect to their application in complex matrices such as human plasma.

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The biosynthesis of 4,7,10,13,16-22:5 and 4,7,10,13,16,19-22:6 requires that when 6,9,12,15,18-24:5 and 6,9,12,15,18,21-24:6 are produced in microsomes they must move to peroxisomes for partial beta-oxidation. When the 24-carbon acids were incubated with peroxisomes, 22-carbon acids with their first double bond at position 4 accumulated as did those with their first two double bonds at the 2-trans-4-cis-positions (D. L. Luthria, S. B. Mohammed, and H. Sprecher, J. Biol. Chem. 271, 16020-16025, 1996; and B. S. Mohammed, D. L. Luthria, S. P. Baykousheva, and H. Sprecher, Biochem. J., 326, 425-430, 1997). In the study reported here we analyzed the acyl-CoAs that accumulated when peroxisomes were incubated with 5,8,11,14-20:4 and 6,9,12-18:3, a metabolite that would be produced via one cycle of arachidonate degradation via the pathway requiring both NADPH-dependent 2,4-dienoyl-CoA reductase and delta 3,5, delta 2,4-dienoyl-CoA isomerase. With both substrates the acyl-CoAs of 2-trans-4-10:2, 4-10:1, 2-trans-4,7,10-16:4, and 4,7,10-16:3 accumulated. These results further establish that the reductase catalyzes a control step in the peroxisomal degradation of unsaturated fatty acids. It was not possible to detect any 18- or 12-carbon acyl-CoA when arachidonate was the substrate, nor did any 12-carbon catabolite accumulate from 6,9,12-18:3. The fractional amount of 5,8-14:2 and arachidonate catabolized via the pathway using only the enzymes of saturated fatty acid degradation versus the pathway that also uses the reductase and the isomerase could thus not be estimated.

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It is now established that fatty acid 7,10,13,16-22:4 is metabolized into 4,7,10,13,16-22:5 as follows: 7,10,13,16-22:4-->9,12,15, 18-24:4-->6,9,12,15,18-24:5-->4,7,10,13,16-22:5. Neither C24 fatty acid was esterified to 1-acyl-sn-glycero-3-phosphocholine (1-acyl-GPC) by microsomes, whereas the rates of esterification of 4, 7,10,13,16-22:5, 7,10,13,16-22:4 and 5,8,11,14-20:4 were respectively 135, 18 and 160 nmol/min per mg of microsomal protein. About four times as much acid-soluble radioactivity was produced when peroxisomes were incubated with [3-14C]9,12,15,18-24:4 compared with 6,9,12,15,18-24:5. Only [1-14C]7,10,13,16-22:4 accumulated when [3-14C]9,12,15,18-24:4 was the substrate, but both 4,7,10,13,16-22:5 and 2-trans-4,7,10,13,16-22:6 were produced from [3-14C]6,9,12,15, 18-24:5. When the two C24 fatty acids were incubated with peroxisomes, microsomes and 1-acyl-GPC there was a decrease in the production of acid-soluble radioactivity from [3-14C]6,9,12,15, 18-24:5, but not from [3-14C]9,12,15,18-24:4. The preferential fate of [1-14C]4,7,10,13,16-22:5, when it was produced, was to move out of peroxisomes for esterification into the acceptor, whereas only small amounts of 7,10,13,16-22:4 were esterified. By using 2H-labelled 9,12,15,18-24:4 it was shown that, when 7,10,13,16-22:4 was produced, its primary metabolic fate was degradation to yield esterified arachidonate. Collectively, the results show that an inverse relationship exists between rates of peroxisomal beta-oxidation and of esterification into 1-acyl-GPC by microsomes. Most importantly, when a fatty acid is produced with its first double bond at position 4, it preferentially moves out of peroxisomes for esterification to 1-acyl-GPC by microsomes, rather than being degraded further via a cycle of beta-oxidation that requires NADPH-dependent 2,4-dienoyl-CoA reductase.

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According to the revised pathways of polyunsaturated fatty acid biosynthesis three, rather than two acids, must be chain elongated for converting linoleate and linolenate, respectively, to 22:5(n-6) and 22:6(n-3) (Sprecher et al. (1995) J. Lipid Res. 36, 2471-2477). The present study was undertaken to determine whether microsomes contained chain-length specific chain-elongating enzymes and, secondly, whether reaction rates for any of these reactions might be rate limiting in the synthesis of 24:5(n-6) and 24:6(n-3), which are the immediate precursors of 22:5(n-6) and 22:6(n-3). Rates of total chain elongation products produced from both 18:4(n-3) and 20:5(n-3) were about 3 nmol/min/mg of microsomal protein while only about 0.5 nmol/min/mg of 24:5(n-3) plus 24:6(n-3) was synthesized from 22:5(n-3). The rate of 24:5(n-3) synthesis was similar to that for the desaturation of 24:5(n-3), at position 6, to yield 24:6(n-3) (Geiger et al. (1993) Biochim. Biophys. Acta 1170, 137-142). The results suggest that the last chain elongation step in unsaturated fatty acid biosynthesis may be equally regulatory in governing the synthesis of fatty acids as is desaturation at position 6. When an enzyme saturating level of [1-(14)C]18:4(n-3) was incubated with increasing amounts of 18:3(n-6) there was a decrease in the production [1-(14)C]20:4(n-3). In a similar way it was observed that 18:4(n-3) inhibited the chain elongation of [1-(14)C]18:3(n-6). Identical cross-over inhibitory studies, using 20:4(n-6) and 20:5(n-3), as well as 22:4(n-6) and 22:5(n-3) also suggested that microsomes contain chain length specific chain-elongating enzymes. This conclusion was further supported by the finding that neither 20:5(n-3) or 22:5(n-3) inhibited the chain elongation of [1-(14)C]18:4(n-3). However, 18:4(n-3), and to a lesser degree, 22:5(n-3) did inhibit the chain elongation of [1-(14)C]20:5(n-3). This latter finding suggests that 18:4(n-3) and 20:5(n-3) might interact with the enzyme that chain elongates 20:5(n-3) to depress its ability to synthesize 22:5(n-3). Our results are most consistent with the presence of multiple chain-elongating enzymes, but a more definitive answer requires the purification of these membrane-bound proteins. In addition our results suggest that the channeling of acids between enzymes in the endoplasmic reticulum may play an important role in regulating the biosynthesis of unsaturated fatty acids.

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When [1-(14)C]4,7,10-16:3, a product produced after two cycles of arachidonate beta-oxidation, was incubated with rat liver peroxisomes and microsomes it was metabolized to 2-trans-4,7,10-16:4, a catabolic product; 6,9,12-18:3 and 8,11,14-20:3, anabolic products made via microsomal chain elongation of the substrate; and 7,10-16:2 and 9,12-18:2. Analysis of the acyl-CoAs produced when 6,9,12-18:3 and its catabolic product, 4,7,10-16:3, where incubated under the above conditions showed that the acyl-CoAs of all of the above compounds, as well as 5,8-14:2-CoA and 6:0-CoA accumulated. Our results show that when 5,8-14:2 and 4,7,10-16:3 are produced by peroxisomal beta-oxidation they can be further degraded to hexanoyl-CoA or move to microsomes for conversion back to linoleate, which is a precursor of arachidonate.

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The synthesis of 4,7,10,13,16,19-docosahexaenoic acid (22:6(n-3)) requires that when 6,9,12,15,18,21-tetracosahexaenoic acid (24:6(n-3)) is produced in the endoplasmic reticulum, it preferentially moves to peroxisomes for one cycle of beta-oxidation rather than serving as a substrate for membrane lipid synthesis. Both 24:6(n-3) and its precursor, 9,12,15,18,21-tetracosapentaenoic acid (24:5(n-3)), were poor substrates for acylation into 1-acyl-sn-glycero-3-phosphocholine (1-acyl-GPC) by rat liver microsomes. When peroxisomes were incubated with 1-14C- or 3-14C-labeled 7,10,13,16,19-docosapentaenoic acid (22:5(n-3)), [1-14C]22:6(n-3), [3-14C]24:5(n-3), or [3-14C]24:6(n-3), only small amounts of acid-soluble radioactivity were produced when double bond removal at positions 4 and 5 was required. When microsomes and 1-acyl-GPC were included in incubations, the preferred metabolic fate of acids, with their first double bond at either positions 4 or 5, was to move out of peroxisomes for esterification into the acceptor rather than serving as substrates for continued beta-oxidation. When [1-14C]22:6(n-3) or [3-14C]24:6(n-3) was incubated with peroxisomes, 2-trans-4,7,10,13,16,19-22:7 accumulated. The first cycle of 20:5(n-3) beta-oxidation proceeds through 2-trans-4,8,11,14,17-20:6 and thus requires both Delta3,5,Delta2, 4-dienoyl-CoA isomerase and 2,4-dienoyl-CoA reductase. The accumulation of the substrate for 2,4-dienoyl-CoA reductase, as generated from 22:6(n-3), but not from 20:5(n-3), suggests that this enzyme distinguishes between subtle structural differences. When 22:6(n-3) is produced from 24:6(n-3), its continued degradation is impaired because of low 2,4-dienoyl-CoA reductase activity. This slow reaction rate likely contributes to the transport of 22:6(n-3) out of peroxisomes for rapid acylation into 1-acyl-GPC by microsomes.

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Recent studies refute the commonly accepted, but untested, hypothesis that 7,10,13,16-22:4 and 7,10,13,16,19-22:5 are desaturated at position 4 by a microsomal acyl-CoA-dependent desaturase. The synthesis of 4,7,10,13,16,19-22:6 occurs via the following reaction sequence: 7,10,13,16,19-22:5-->9,12,15,18,21-24:5-->6,9,12,15,18,21-24:6 4,7,10,13,16,19-22:6. The synthesis of 4,7,10,13,16-22:5 from 7,10,13,16-22:4 takes place via an analogous pathway. According to these pathways the 24-carbon acids that are made in the endoplasmic reticulum move to a site for partial beta-oxidation, which is most likely peroxisomes. The products of partial beta-oxidation, 4,7,10,13,16-22:5 and 4,7,10,13,16,19-22:6, then move back to the endoplasmic reticulum where they are used as substrates for membrane lipid biosynthesis. The ability of a fatty acid to serve as a substrate for continued peroxisomal beta-oxidation, versus its transfer out of peroxisomes for subsequent endoplasmic reticulum-associated esterification reactions, may be an important control for regulating membrane lipid fatty acid composition. Indeed, the revised pathways of polyunsaturated fatty acid biosynthesis imply that there is considerable intracellular movement and recycling of fatty acids between peroxisomes and the endoplasmic reticulum. In addition, these revised pathways require that two 18-carbon and two 24-carbon acids are substrates for desaturation at position 6. Also, as linoleate and linolenate are metabolized, respectively, to 6,9,12,15,18-24:5 and 6,9,12,15,18,21-24:6, three n-6 acids and three n-3 acids are substrates for malonyl-CoA dependent chain elongation. It remains to be determined how many microsomal enzymes are required to carry out these reactions and whether other ancillary enzymes are expressed in tissues whose membrane lipids accumulate very long-chain polyunsaturated acids with up to 36 carbon atoms.

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Male weanling rats were fed a diet that contained 2.1% ethyl oleate, 1% ethyl linoleate, and 0.2% ethyl linolenate. After 4 weeks all the linoleate was replaced by the deuterium-labeled analog and the animals were killed 4 days later. The molar fraction of total 20:4(n-6) in liver, heart, and kidney phospholipids containing deuterium was 33.9, 8.9, and 13.3%, respectively. Second, animals were preconditioned by incorporating either 0.2% of 18:3(n-6), 20:3(n-6), or 20:4(n-6) into the above diet and again after 4 weeks all the linoleate was replaced with the labeled analog. Now the molar fraction of labeled 20:4(n-6) in liver phospholipids from these three groups of animals was reduced from 33.9 to 27.1, 23.9, and 24.1% respectively. In contrast, there was little change in the specific activity of 20:4(n-6) in heart and kidney phospholipids. The third protocol was a direct crossover study in that again unlabeled linoleate was fed during the entire period. Four days prior to killing the unlabeled 18:3(n-6), 20:3(n-6), and 20:4(n-6) were replaced with the deuterium-labeled analogs. The mole % of total esterified 20:4(n-6) in liver phospholipids was now 24.6, 32.0, and 26.2%, respectively. Even though 18:3(n-6), 20:3(n-6), and 20:4(n-6) were all fed at only 20% of the level of 18:2(n-6), it can be calculated that the molar fraction of esterified 20:4(n-6) in liver phospholipids was between 65 to 77% of that found when 18:2(n-6) was the only dietary (n-6) acid as under these conditions 33.9 mol % of the 20:4(n-6) was labeled. Interestingly, when deuterium-labeled 18:3(n-6), 20:3(n-6), or 20:4(n-6) was fed, the specific activity of esterified 20:4(n-6) in kidney and heart phospholipids was always equal to or greater than what was derived from deuterium-labeled 18:2(n-6). The results show that under steady-state dietary conditions, (n-6) dietary fatty acids are processed in different ways by liver, heart, and kidney.

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The pathway for the peroxisomal beta-oxidation of arachidonic acid (5,8,11,14-20:4) was elucidated by comparing its metabolism with 4,7,10-hexadecatrienoic acid (4,7,10-16:3) and 5,8-tetradecadienoic acid (5,8-14:2) which are formed, respectively, after two and three cycles of arachidonic acid degradation. When [1-14C]4,7,10-16:3 was incubated with peroxisomes in the presence of NAD+ and NADPH, it resulted in a time-dependent increase in the production of acid-soluble radioactivity which was accompanied by the synthesis of 2-trans-4,7,10-hexadecatetraenoic acid and two 3,5,7,10-hexadecatetraenoic acid isomers. The formation of conjugated trienoic acids suggests that peroxisomes contain delta 3,5,delta 2,4-dienoyl-CoA isomerase with the ability to convert 2-trans-4,7,10-hexadecatetraenoic acid to 3,5,7,10-hexadecatetraenoic acid. When 1-14C-labeled 6,9,12-octadecatrienoic acid or 7,10,13,16-docosatetraenoic acid was incubated without nucleotides, the 3-hydroxy metabolites accumulated, since further degradation requires NAD(+)-dependent 3-hydroxyacyl-CoA dehydrogenase. When [1-14C]5,8,11,14-20:4 was incubated under identical conditions, no polar metabolite was detected, but 2-trans-4,8,11,14-eicosapentaenoic acid accumulated. When NADPH was added to incubations, 3-hydroxy-8,11,14-eicosatrienoic, 2-trans-4,8,11,14-eicosapentaenoic, 2-trans-8,11,14-eicosatetraenoic, and 8,11,14-eicosatrienoic acids were produced. Analogous compounds were formed from [1-14C]5,8-14:2. Our results show that the removal of double bonds from odd-numbered carbons in arachidonic acid thus requires both NADPH-dependent 2,4-dienoyl-CoA reductase and delta 3,5,delta 2,4-dienoyl-CoA isomerase. One complete cycle of 5,8-14:2 and 5,8,11,14-20:4 beta-oxidation yields, respectively, 6-dodecenoic and 6,9,12-octadecatrienoic acids.

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The addition of 1-acyl-sn-glycero-3-phosphocholine (1-acyl-GPC) to peroxisomes decreased the production of acid-soluble radioactivity formed by beta-oxidation of [1-(14)C]arachidonate due to substrate removal by esterification into the acceptor. This peroxisomal-associated acyl-CoA:1-acyl-GPC acyltransferase activity was due to microsomal contamination. The production of acid-soluble radioactivity from [1-(14)C]7,10,13,16-22:4, but not from [3-(14)C]7,10,13,16-22:4 was independent of 1-acyl-GPC, with and without microsomes. By comparing rates of peroxisomal beta-oxidation with those for microsomal acylation, it was shown that the preferred metabolic fate of arachidonate, when added directly to incubations, or generated via beta-oxidation, was esterification by microsomal 1-acyl-GPC acyltransferase, rather than continued peroxisomal beta-oxidation.

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Peroxisomal beta-oxidation of linoleic acid and arachidonic acid was depressed when 1-palmitoyl-sn-glycero-3-phosphocholine and microsomes were included in incubations. This reduction was due to the esterification of the substrate into the acceptor by microsomal 1-acyl-sn-glycero-3- phosphocholine acyltransferase. The first cycle of the beta-oxidation of 7,10,13,16-docosatetraenoic acid was independent of 1-acyl-sn-glycero-3-phosphocholine and microsomes. However, when arachidonate was produced it was esterified rather than serving as a substrate for continued beta-oxidation. When arachidonate and linoleate were incubated with peroxisomes alone, 2-trans-4,7,10-hexadecatetraenoic acid and 2-trans-4-decadienoic acid were the respective end products of beta-oxidation. 2-Oxo-8,11-heptadecadienone, a catabolite produced from linoleate, was most likely a nonenzymatic decarboxylation product of 3-oxo-9,12-octadecadienoic acid. In addition to the termination of beta-oxidation by microsomal-peroxisomal communication, our results with linoleate and arachidonate suggest that the reaction catalyzed by 2-trans-4-cis-dienoyl-CoA reductase is the control step in double bond removal. In addition, the beta-ketothiolase step may play a regulatory role in the peroxisomal beta-oxidation of linoleate but not arachidonate or 7,10,13,16-docosatetraenoic acid.

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In order to determine how dietary linoleate is metabolized, rats were maintained on a chemically defined diet containing 1.6% ethyl linoleate. After 5 weeks the linoleate was replaced by an equal amount of ethyl 9,10,12,13-d4-linoleate. The animals were killed 3 days later and the molar percentage of d4-linoleate and d4-arachidonate were quantified in liver, heart and kidney phospholipids. In liver, 54 and 22.8 mol% respectively of the esterified linoleate and arachidonate was deuteriated. The lower specific activity of arachidonate versus linoleate suggests that desaturation of linoleate, by a 6-desaturase, is not only rate limiting for synthesis of arachidonate but that the amount of newly synthesized arachidonate is insufficient by itself to maintain steady state levels of esterified arachidonate. The molar fraction of deuteriated linoleate in heart and kidney phospholipids was respectively 35 and 37.4%. These values are lower than for liver phospholipids but it appears there is adequate dietary linoleate available in these tissues for the synthesis of arachidonate. However, of the esterified arachidonate in heart and kidney phospholipids only 4.2 and 8.6 mol% respectively was deuteriated. Our results suggest that arachidonate is made in liver and transported to heart and kidney.

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Ethyl 5,8,11,14-eicosatetraenoate-19,19,20,20-d4 and ethyl 8,11,14-eicosatrienoate-19,19,20,20-d4 were synthesized by Grignard coupling of the methanesulfonyl ester of 2,5-undecadiyn-1-ol-10,10,11,11-d4 with 5,8-nonadiynoic acid and 8-nonynoic acid, respectively. The coupled products upon Lindlar reduction, followed by the preparation of their ethyl esters, yielded deuteriated ethyl arachidonate and ethyl dihomo-gamma-linolenate, which were completely characterized by 13C and 1H nuclear magnetic resonance and mass spectral analysis.

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Several types of unsaturated fatty acid methyl esters were converted into 4,4-dimethyloxazoline (DMOX) derivatives and analyzed by mass spectrometry to further evaluate the feasibility of using this derivative for locating the positions of double bonds in isomeric fatty acids. Five isomeric 20-carbon tetraenoic acids were analyzed in which the four cis double bonds were systematically moved from the 4,7,10,13- to the 8,11,14,17-positions. It was possible to locate the positions of all four double bonds in the 7,10,13,16- and 8,11,14,17-isomers by appropriate ions differing by 12 atomic mass units. In a similar way the three terminal double bonds in the 4,7,10,13-, 5,8,11,14- and 6,9,12,15-isomers could be assigned. Odd-numbered ions at m/z 139, 153 and 167 which are accompanied by an even mass ion at 138, 152 and 166, respectively, are diagnostic for DMOX derivatives of acids with their first double bond, respectively, at positions 4, 5 and 6. It was thus possible to assign the location of all four double bonds in these three isomers. A comparison of the spectra of the DMOX derivatives of 17,17,18,18-d4 vs. 9,10,12,13-d4 linoleic acid suggests that double bonds preferentially migrate toward the polar end of the molecule prior to fragmentation. The merit of using DMOX derivatives to locate double-bond positions in mono- and dicarboxylic acids, produced during beta-oxidation of polyunsaturated fatty acids, was evaluated. The spectra of 3-cis- and 4-cis-decenoic acids differ as do the spectra of 8-carbon dicarboxylic acids with their double bonds at positions 3 and 4.

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