Synthesis and characterization of 5-alkoxycarbonyl-4-hydroxymethyl-5-alkyl-pyrroline N-oxide derivatives

The syntheses, analytical properties, and spin trapping behavior of four novel EMPO derivatives, namely 5-ethoxycarbonyl-4-hydroxymethyl-5-methyl-pyrroline N-oxide (EHMPO), 5-ethoxycarbonyl-5-ethyl-4-hydroxymethyl-pyrroline N-oxide (EEHPO), 4-hydroxymethyl-5-methyl-5-propoxycarbonyl-pyrroline N-oxide (HMPPO), and 4-hydroxymethyl-5-methyl-5-iso-propoxycarbonyl-pyrroline N-oxide (HMiPPO), towards different oxygen- and carbon-centered radicals are described.     

Synthesis and characterization of several carbamoyl- and methylcarbamoyl-substituted EMPO derivatives

The spin trapping behavior of four novel carbamoyl-substituted EMPO derivatives, namely 5-carbamoyl-3,5-dimethyl-pyrroline N-oxide (CADMPO), 3,5-dimethyl-5-methylcarbamoyl-pyrroline N-oxide (DMMCAPO), 5-carbamoyl-3-ethyl-5-methyl-pyrroline N-oxide (CAEMPO), and 3-ethyl-5-methyl-5-methylcarbamoyl-pyrroline N-oxide (EMMCAPO), towards different oxygen- and carbon-centered radicals is described, the half lives of the respective superoxide adducts ranging from about 10 to 20 min. The most characteristic adducts were, however, formed from methyl, hydroxymethyl, hydroxyethyl, and carbon dioxide anion radicals.     

Spin trapping experiments with different carbamoyl-substituted EMPO derivatives

The spin trapping behavior of five carbamoyl-substituted EMPO derivatives, 5-aminocarbonyl-5-methyl-pyrroline N-oxide (CAMPO (AMPO)), 5-aminocarbonyl-5-ethyl-pyrroline N-oxide (CAEPO), 5-aminocarbonyl-5-propyl-pyrroline N-oxide (CAPPO), 5-aminocarbonyl-5-n-butyl-pyrroline N-oxide (CABPO), and 5-aminocarbonyl-5-n-pentyl-pyrroline N-oxide (CAPtPO), toward different oxygen- and carbon-centered radicals is described, the stabilities of the superoxide adducts ranging from about 8 to 17min.     

Synthesis and characterization of 5-hydroxymethyl-5-methyl-pyrroline N-oxide and its derivatives

Synthesis and spin trapping behavior of three novel DMPO derivatives, namely 5-hydroxymethyl-5-methyl-pyrroline N-oxide (HMMPO), 5-(2-furanyl)-oxymethyl-5-methyl-pyrroline N-oxide (FMMPO), and 5-(2-pyranyl)-oxymethyl-5-methyl-pyrroline N-oxide (PMMPO) towards different oxygen- and carbon-centered radicals are described. The stabilizing effect of a series of cyclodextrins on the superoxide adducts was tested.     

Detection of superoxide anion using an isotopically labeled nitrone spin trap: potential biological applications

We describe the synthesis and biological applications of a novel nitrogen-15-labeled nitrone spin trap, 5-ethoxycarbonyl-5-methyl-1-pyrroline N-oxide ([(15)N]EMPO) for detecting superoxide anion. Superoxide anion generated in xanthine/xanthine oxidase (100 nM min(-1)) and NADPH/calcium-calmodulin/nitric oxide synthase systems was readily detected using EMPO, a nitrone analog of 5,5'-dimethyl-1-pyrroline N-oxide (DMPO). Unlike DMPO-superoxide adduct (DMPO-OOH), the superoxide adduct of EMPO (EMPO-OOH) does not spontaneously decay to the corresponding hydroxyl adduct, making spectral interpretation less confounding. Although the superoxide adduct of 5-(diethoxyphosphoryl)-5-methyl-pyrroline N-oxide is more persistent than EMPO-OOH, the electron spin resonance spectra of [(14)N]EMPO-OOH and [(15)N]EMPO-OOH are less complex and easier to interpret. Potential uses of [(15)N]EMPO in elucidating the mechanism of superoxide formation from nitric oxide synthases, and in ischemia/reperfusion injury are discussed.     

Metabolic origins of urinary unsaturated dicarboxylic acids

Previously, we [Jin, S.-J., & Tserng, K.-Y. (1989) J. Lipid Res. 30, 1611-1619] reported the structures of urinary octenedioic acids occurring in patients with dicarboxylic aciduria. We proposed that these unsaturated octenedioic acids were derived from the oxidation of oleic and linoleic acids. By comparison with synthetic decenedioic acids, we have further identified the higher homologues of unsaturated dicarboxylic acids in urine as cis-5-decenedioic (c5DC10), cis-4-decenedioic (c4DC10), cis-3-decenedioic (cDC10), trans-4-decenedioic, trans-3-decenedioic, cis-5-dodecenedioic (c5DC12), cis-3-dodecenedioic (c3DC12), and trans-3-dodecenedioic acids. The presence of these isomeric decenedioic and dodecenedioic acids in urine is consistent with the proposed metabolic origins. In vitro studies using synthetic unsaturated fatty acids and rat liver homogenates support the proposed metabolic origins of these acids. The following metabolic sequences are proposed for metabolites derived from oleic acid: (route A) cis-5-tetradecenoic acid----cis-5-tetradecenedioic acid----c5DC12----c5DC10----suberic (DC8)----adipic (DC6); (route B) cis-3-dodecenoic acid----c3DC12----c3DC10----c3DC8 (cis-3-octenedioic)----DC6. A similar route is derived from linoleic acid: cis-4-decenoic acid----c4DC10----c4DC8 (cis-4-octenedioic)----DC6. The presence of a double bond at position 3, 4, or 5 of fatty acid appears to be rate limiting for further beta-oxidation; therefore, metabolic products with cis-3, cis-4, or cis-5 structure accumulate. Urinary DC8 and DC6 are derived partially from the metabolic degradation of these unsaturated dicarboxylic acids.     

The line asymmetry of electron spin resonance spectra as a tool to determine the cis:trans ratio for spin-trapping adducts of chiral pyrrolines N-oxides: the mechanism of formation of hydroxyl radical adducts of EMPO, DEPMPO, and DIPPMPO in the ischemic-reperfused rat liver

Nonstereospecific addition of free radicals to chiral nitrones yields cis/trans diastereoisomeric nitroxides often displaying different electron spin resonance (ESR) characteristics. Glutathione peroxidase-glutathione (GPx-GSH) reaction was applied to reduce the superoxide adducts (nitrone/*OOH) to the corresponding hydroxyl radical (HO*) adducts (nitrone/*OH) of two nitrones increasingly used in biological spin trapping, namely 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) and 5-ethoxycarbonyl-5-methyl-1-pyrroline N-oxide, and of 5-diisopropoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DIPPMPO), a sterically hindered DEPMPO analogue. The method offered improved conditions to record highly resolved ESR spectra and by accurate simulation of line asymmetry we obtained clear evidence for the existence of previously unrecognized isomer pairs of cis- and trans-[DEPMPO/*OH] and [DIPPMPO/*OH]. Additional nitrone/*OH generation methods were used, i.e. photolysis of hydrogen peroxide and the Fenton reaction. We developed a kinetic model involving first- and second-order decay and a secondary conversion of trans to cis isomer to fully account for the strongly configuration-dependent behavior of nitrone/*OH. In the reductive system and, to a lower extent, in the Fenton or photolytic systems cis-nitrone/*OH was the more stable diastereoisomer. In various biologically relevant milieu, we found that the cis:trans-nitrone/*OH ratio determined right after the spin adduct formation significantly differed upon the GPx-GSH vs (Fenton or photolytic) systems of formation. This new mechanistic ESR index consistently showed for all nitrones that nitrone/*OH signals detected in the postischemic effluents of ischemic isolated rat livers are the reduction products of primary nitrone/*OOH. Thus, ESR deconvolution of cis/trans diastereoisomers is of great interest in the study of HO* formation in biological systems.     

Synthesis of radiolabeled stilbene derivatives as new potential PET probes for aryl hydrocarbon receptor in cancers

New carbon-11 and fluorine-18 labeled stilbene derivatives, cis-3,5-dimethoxy-4'-[11C]methoxystilbene (4'-[11C]8a), cis-3,4',5-trimethoxy-3'-[11C]methoxystilbene (3'-[11C]8b), trans-3,5-dimethoxy-4'-[11C]methoxystilbene (4'-[11C]10a), trans-3,4',5-trimethoxy-3'-[11C]methoxystilbene (3'-[11C]10b), cis-3,5-dimethoxy-4'-[18F]fluorostilbene (4'-[18F]12a), and trans-3,5-dimethoxy-4'-[18F]fluorostilbene (4'-[18F]13a), were designed and synthesized as potential PET probes for aryl hydrocarbon receptor (AhR) in cancers.     

Cytotoxicity of the novel spin trapping compound 5-ethoxycarbonyl-3,5-dimethyl-pyrroline N-oxide (3,5-EDPO) and its derivatives

ESR spin trapping allows detection of superoxide radicals. Novel spin traps forming more stable superoxide adducts (t(1/2) ca. 12-55 min) were tested for their toxicity to cultured cells. The following toxicity ranking was obtained: 4,5-DPPO>4-BEMPO approximately 3-BEMPO>trans-3,5-EDPO>3,5-DPPO approximately 4,5-DiPPO approximately 4,5-EDPO>cis-3,5-EDPO approximately 3,5-DiPPO>DEPMPO. In conclusion, 4,5-EDPO, cis-3,5-EDPO and 3,5-DiPPO can be recommended for further investigation of superoxide in biological systems.     

Replication inhibition and translesion synthesis on templates containing site-specifically placed cis-diamminedichloroplatinum(II) DNA adducts.

A series of site-specifically plantinated, covalently closed circular M13 genomes (7250 bp) was constructed in order to evaluate the consequences of DNA template damage induced by the anticancer drug cis-diamminedichloroplatinum(II) (cis-DDP). Here are reported the synthesis and characterization of genomes containing the intrastrand cross-linked adducts cis-[Pt(NH3)2[d(ApG)-N7(1),-N7(2)]], cis-[Pt-(NH3)2[d(GpCpG)-N7(1),-N7(3)]], and trans-[Pt(NH3)2[d(CpGpCpG)-N3(1),-N7(4)]]. These constructs, as well as the previously reported M13 genome containing a site-specifically placed cis-[Pt(NH3)2[d-(GpG)-N7(1),-N7(2)]] adduct, were used to study replication in vitro. DNA synthesis was initiated from a position approximately 177 nucleotides 3' to the individual adducts, and was terminated either by the adducts or by the end of the template, located approximately 25 nucleotides on the 5' side of the adducts. Analysis of the products of these reactions by gel electrophoresis revealed that, on average, bypass of the cis-DDP adducts occurred approximately 10% of the time and that the cis-[Pt(NH3)2[d(GpG)-N7(1),-N7(2)]] intrastrand cross-link is the most inhibitory lesion. The cis-[Pt(NH3)2[(GpCpG)-N7(1),-N7(3)]] adduct allowed a higher frequency of such translesion synthesis (ca. 25%) for two of the polymerases studied, modified bacteriophage T7 polymerase and Escherichia coli DNA polymerase I (Klenow fragment). These enzymes have either low (Klenow) or no (T7) associated 3' to 5' exonuclease activity. Bacteriophage T4 DNA polymerase, which has a very active 3' to 5' exonuclease, was the most strongly inhibited by all three types of cis-DDP adducts, permitting only 2% translesion synthesis. This enzyme is therefore recommended for replication mapping studies to detect the location of cis-DDP-DNA adducts in a heterologous population. The major replicative enzyme of E. coli, the DNA polymerase III holoenzyme, allowed less than 10% adduct bypass. Postreplication restriction enzyme cleavage studies established that the templates upon which translesion synthesis was observed contained platinum adducts, ruling out the possibility that the observed products were due to a small amount of contamination with unplatinated DNA. The effects on in vitro replication of a recently characterized adduct of trans-DDP [Comess, K. M., Costello, C. E., & Lippard, S. J. (1990) Biochemistry 29, 2102-2110] were also evaluated. This adduct provided a poor block both to DNA polymerases and to restriction enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Reaction of 2'-deoxyribonucleosides with cis- and trans-1,4-dioxo-2-butene

cis-1,4-Dioxo-2-butene is a toxic metabolite of furan, while the trans-isomer is a product of deoxyribose oxidation in DNA. It has recently been reported that both cis- and trans-1,4-dioxo-2-butene react with the 2'-deoxynucleosides dC, dG, and dA to form novel diastereomeric oxadiazabicyclo(3.3.0)octaimine adducts. We have now extended these studies with kinetic and spectroscopic analyses of the reactions of cis- and trans-1,4-dioxo-2-butene, as well as the identification of novel adducts of dA. The reaction of dC with trans-1,4-dioxo-2-butene was observed to be nearly quantitative and produced two interchanging diastereomers with a second-order rate constant of 3.66 x 10(-2)M(-1)s(-1), which is nearly 10-fold faster than the reaction with the cis-isomer (3.74 x 10(-3)M(-1)s(-1)). HPLC analyses of reactions of 1,4-dioxo-2-butene with both dA and 9-methyladenine (pH 7.4, 37 degrees C) revealed multiple products including a novel pair of closely eluting fluorescent species of identical mass ([M+H] m/z 420), each of which contains two molecules of 1,4-dioxo-2-butene, and a more abundant but unstable and non-fluorescent species ([M+H] m/z 414). Partial structural characterization of the fluorescent adducts of dA revealed the presence of the oxadiazabicyclo(3.3.0)octaimine ring system common to the dC adducts. These results support the genotoxic potential of furan metabolites and products of deoxyribose oxidation.     

Isolation and characterization of phytotoxic compounds produced by Phomopsis helianthi

The isolation, chemical characterization and biological activity of two phytotoxic metabolites of Phomopsis helianthi Munt-Cvet et al. is reported. These compounds were identified by spectroscopic methods (UV, IR, 1H and 13C NMR, and MS) as trans-4,6-dihydroxymellein (trans-3-methyl-4,6,8-trihydroxy-3,4-dihyroisocoumarin) and cis-4,6-dihydroxymellein (cis-3-methyl-4,6,8-trihydroxy-3,4-dihydroisocoumarin). This is the first report of the isolation of trans-4,6-dihydroxymellein from fungal cultures and of the production of cis- and trans-4,6-dihydroxymelleins by P. helianthi. Rice was found to be a good substrate for the production of the dihydroxymelleins. Culture extracts of some Italian and French strains of P. helianthi showed different degrees of phytotoxicity towards sunflower leaves and seedlings. The minimum effective doses of trans- and cis-4,6-dihydroxymelleins with different bioassays were 76 and 135 microg per spot (leaf puncture bioassay), 3 and 5 micromol g(-1) fresh tissue (absorption by leaf cutting) and 5 and 2 micromol g(-1) fresh tissue (absorption by cut seedlings), respectively. These compounds may contribute to the severity of the sunflower disease caused by P. helianthi.     

Chemo- and stereoselective synthesis of benzocycloheptene and 1-benzoxepin derivatives as alpha-sympathomimetic and anorexigenic agents

The synthesis and pharmacological evaluation of cis- and trans-6-amino-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ols 4a-c and 5a-c and cis- and trans-4-amino-2,3,4,5-tetrahydro-1-benzoxepin-5-ols 4d-f and 5d-f were carried out. Chemo- and stereoselective synthesis of 5a-f was achieved by reduction of corresponding alpha-amino ketones 3a-f with LiAl(t-BuO)3H. cis-4-Amino-2,3,4,5-tetrahydro-1-benzoxepin-5-ol 4d and trans-4-amino-2,3,4,5-tetrahydro-1-benzoxepin-5-ol 5d exhibited marked anorexigenic activity in mice at a dose of LD50 800 and 500 mg/kg and ED50 75 and 55 mg/kg, respectively, while the analog cis-2,3-dihydroxy-6-amino-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol 8 showed typical alpha-sympathomimetic activity.     

Gas chromatography-mass spectrometry determination of metabolites of conjugated cis-9,trans-11,cis-15 18:3 fatty acid

Structural determination of polyunsaturated fatty acids by gas chromatography-mass spectrometry (GC-MS) requires currently the use of nitrogen containing derivatives such as picolinyl esters, 4,4-dimethyloxazoline or pyrrolidides derivatives. The derivatization is required in most cases to obtain low energy fragmentation that allows accurate location of the double bonds. In the present work, the following metabolites of rumelenic (cis-9,trans-11,cis-15 18:3) acid, from rat livers, were identified: cis-8,cis-11,trans-13,cis-17 20:4, cis-5,cis-8,cis-11,trans-13,cis-17 20:5, cis-7,cis-10,cis-13,trans-15,cis-19 22:5, and cis-4,cis-7,cis-10,cis-13,trans-15,cis-19 22:6 acids by GC-MS as their 4,4-dimethyloxazoline and methyl esters derivatives. Specific fragmentation of the methyl ester derivatives revealed some similarity with their corresponding DMOX derivatives. Indeed, intense ion fragments at m/z=M+-69, corresponding to a cleavage at the center of a bis-methylene interrupted double bond system were observed for all identified metabolites. Moreover, intense ion fragments at m/z=M+-136, corresponding to allylic cleavage of the n-12 double bonds were observed for the C20:5, C22:5, C22:6 acid metabolites. For the long chain polyunsaturated fatty acids from the rumelenic metabolism, we showed that single methyl esters derivatives might be used for both usual quantification by GC-FID and identification by GC-MS.     

Dietary conjugated linoleic acid does not adversely affect bone mass in obese fa/fa or lean Zucker rats

Conjugated linoleic acid (CLA) elevates body ash in healthy animals. The objective of the present study was to determine if single or mixed CLA isomers improve bone mass in an obese and hyperinsulinemic state. Male (n = 120) lean and obese fa/fa Zucker rats (age, 6 weeks) were randomized to 8 weeks on a control diet or to 0.4% (w/w) cis-9, trans-11 CLA (Group 1); 0.4% (w/w) trans-10, cis-12 CLA (Group 2); 0.4% (w/w) cis-9, trans-11 CLA and 0.4% (w/w) trans-10, cis-12 CLA (Group 3); 0.4% (w/w) cis-9, trans-11 CLA, 0.4% (w/w) trans-10, cis-12 CLA, and traces of other CLA isomers (Group 4); and 0.4% (w/w) cis-9, trans-11 CLA, 0.4% (w/w) trans-10, cis-12 CLA, and 0.3% (w/w) other CLA isomers (Group 5). Bone area (BA), bone mineral content (BMC), and bone mineral density (BMD) of the whole body, spine, and femur were measured at baseline (6 weeks) and at 14 weeks of age. Effects of genotype, diet, and genotype x diet interactions were assessed using factorial analysis of variance. At 6 and 14 weeks, whole-body BA and BMC were lower in lean rats compared with fa/fa rats. Similarly, at 14 weeks, fa/fa rats had a higher spine and femur BMD despite a lower femur weight. The fa/fa rats in Groups 4 and 5 had higher adjusted whole-body BMC compared with Group 3, but not with Group 1, Group 2, or the control. In lean rats, Group 3 had a greater adjusted whole-body BMC than Groups 1 and 2, but not Group 4, Group 5, or the control. Thus, commercially available CLA mixtures and single CLA isomers do not affect bone mass in a hyperinsulinemic, obese state.     

The metabolites of cyclohexylamine in man and certain animals

1. [1-(14)C]Cyclohexylamine hydrochloride was synthesized and given orally or intraperitoneally to rats, rabbits and guinea pigs (dose 50-500mg/kg) and orally to humans (dose 25 or 200mg/person). The (14)C is excreted mainly in the urine, most of the excretion occurring in the first day after dosing. Only small amounts (1-7%) are found in the faeces. 2. In the rat, guinea pig and man, the amine is largely excreted unchanged, only 4-5% of the dose being metabolized in 24h in the rat and guinea pig and 1-2% in man. In the rabbit about two-thirds of the dose is excreted unchanged and about 30% is metabolized. 3. In the rat, five minor metabolites were found, namely cyclohexanol (0.05%), trans-3- (2.2%), cis-4- (1.7%), trans-4- (0.5%) and cis-3-aminocyclohexanol (0.1% of the dose in 24h). 4. In the rabbit, eight metabolites were identified, namely cyclohexanol (9.3%), trans-cyclohexane-1,2-diol (4.7%), cyclohexanone (0.2%), cyclohexylhydroxylamine (0.2%) and trans-3- (11.3%), cis-3- (0.6%), trans-4- (0.4%) and cis-4-aminocyclohexanol (0.2%). 5. In the guinea pig, six minor metabolites were found, namely cyclohexanol (0.5%), trans-cyclohexane-1,2-diol (2.5%) and trans-3- (1.2%), cis-3- (0.2%), trans-4- (0.2%) and cis-4-aminocyclohexanol (0.2%). 6. In man only two metabolites were definitely identified, namely cyclohexanol (0.2%) and trans-cyclohexane-1,2-diol (1.4% of the dose), but man had been given a smaller dose (3mg/kg) than the other species (50mg/kg). 7. The hydroxylated metabolites of cyclohexylamine were excreted in the urine in both free and conjugated forms. 8. Although cyclohexylamine is metabolized to only a minor extent, in rats the metabolism was mainly through hydroxylation of the cyclohexane ring, in man by deamination and in guinea pigs and rabbits by ring hydroxylation and deamination.     

The impact of spacer structure on 5-HT7 and 5-HT1A receptor affinity in the group of long-chain arylpiperazine ligands

New cis-, trans-2-butene and 1,2-bismethylbenzene analogues of MM77 and NAN-190 (1-[4-[4-(2-methoxyphenyl)-piperazin-1-yl]-butyl]-pyrrolidine-2,5-dione and isoindole-1,3-dione, respectively) were synthesized. The differences in their in vitro affinity for serotonin 5-HT(7) and 5-HT(1A) receptors were explained using a conformational analysis. A bioactive conformation of those compounds for the 5-HT(7) receptor, different from that established for 5-HT(1A), was proposed.     

5-Fluorouracil-cisplatin adducts with potential antitumor activity

Using 5-fluorouracil (5-FU) and cis-diamminedichloroplatinum(II) (cisplatin, CDDP) as starting compounds, 5-FU-cisplatin adducts cis-[Pt(NH(3))(2)(HFU)Cl] (1) and cis-[Pt(NH(3))(2)(HFU)(2)] (2) were prepared. The obtained complexes were characterized by IR, ES-MS and 1H NMR spectroscopy. Complex 1 reacted with guanosine-5'-monophosphate (5'-GMP) and gave rise to a stable mixed-ligand complex cis-[Pt(NH(3))(2)(HFU)(GMP)] (3), whereas 2 did not undergo a similar reaction. In vitro cell growth inhibition tests of complexes 1 and 2 exhibited moderate antitumor activities against the melanoma B16-BL6 cell line. This work provides the basis for a potential alternative for the combinational use of 5-FU and CDDP in cancer therapy.     

Chlorogenic acid biosynthesis: characterization of a light-induced microsomal 5-O-(4-coumaroyl)-D-quinate/shikimate 3'-hydroxylase from carrot (Daucus carota L.) cell suspension cultures

Microsomal preparations from carrot (Daucus carota L.) cell suspension cultures catalyze the formation of trans-5-O-caffeoyl-D-quinate (chlorogenate) from trans-5-O-(4-coumaroyl)-D-quinate. trans-5-O-(4-Coumaroyl)shikimate is converted to about the same extent to trans-5-O-caffeoylshikimate. trans-4-O-(4-Coumaroyl)-D-quinate, trans-3-O-(4-coumaroyl)-D-quinate, trans-4-coumarate, and cis-5-O-(4-coumaroyl)-D-quinate do not act as substrates. The reaction is strictly dependent on molecular oxygen and on NADPH as reducing cofactor. NADH and ascorbic acid cannot substitute for NADPH. Cytochrome c, Tetcyclacis, and carbon monoxide inhibit the reaction suggesting a cytochrome P-450-dependent mixed-function monooxygenase. Competition experiments as well as induction and inhibition phenomena indicate that there is only one enzyme species which is responsibl for the hydroxylation of the 5-O-(4-coumaric) esters of both D-quinate and shikimate. The activity of this enzyme is greatly increased by in vivo irradiation of the cells with blue/uv light. We conclude that the biosynthesis of the predominant caffeic acid conjugates in carrot cells occurs via the corresponding 4-coumaric acid esters. Thus, in this system, 5-O-(4-coumaroyl)-D-quinate can be seen as the final intermediate in the chlorogenic acid pathway.     

The role of Delta(9)-desaturase in the production of cis-9, trans-11 CLA

Biomedical studies with animal models have demonstrated that cis-9, trans-11 conjugated linoleic acid (CLA), the predominant isomer found in milk fat from dairy cows, has anticarcinogenic effects. We recently demonstrated endogenous synthesis of cis-9, trans-11 CLA from ruminally derived trans-11 C18:1 by Delta(9)-desaturase in lactating dairy cows. The present study further examined endogenous synthesis of cis-9, trans-11 CLA and quantified its importance by increasing substrate supply using partially hydrogenated vegetable oil (PHVO) as a source of trans-11 C18:1 and blocking endogenous synthesis using sterculic oil (SO) as a source of cyclopropene fatty acids which specifically inhibit Delta(9)-desaturase. Four cows were abomasally infused with 1) control, 2) PHVO, 3) SO, and 4) PHVO+SO in a 4 x 4 Latin square design. With infusion of PHVO, cis-9, trans-11 CLA was increased by 17% in milk fat. Consistent with inhibition of desaturase, SO treatments increased milk fat ratios for the fatty acid pairs effected by Delta(9)-desaturase, C14:0/cis-9 C14:1, C16:0/cis-9 C16:1, and C18:0/cis-9 C18:1. The role of endogenous synthesis of CLA was evident from the 60-65% reduction in cis-9, trans-11 CLA which occurred in milk fat with SO treatments. cis-9 C14:1 originates from desaturation of C14:0 by Delta(9)-desaturase and can be used to estimate the extent of SO inhibition of Delta(9)-desaturase. When this correction factor was applied, endogenous synthesis was estimated to account for 78% of the total cis-9, trans-11 CLA in milk fat. Thus, endogenous synthesis was the major source of cis-9, trans-11 CLA in milk fat of lactating cows.