Platelet-neutrophil interactions. 12S,20- and 5S,12S-dihydroxyeicosapentaenoic acids: two novel neutrophil metabolites from platelet-derived 12S-hydroxyeicosapentaenoic acid

Dietary marine n-3 polyunsaturated fatty acids have demonstrated an antiinflammatory potential in epidemiologic and intervention studies in humans. Proposed mechanisms, involving only leukocytes, fall short of explaining this potential completely. Enriched by dietary means with eicosapentaenoic acid (EPA), stimulated human platelets release substantial amounts of eicosapentaenoic acid and 12S-hydroxyeicosapentaenoic acid (12S-HEPE) in addition to 12S-hydroxyeicosatetraenoic acid (12S-HETE) derived from arachidonic acid. Human neutrophils metabolize 12S-HETE to 5S,12S-DiHETE when stimulated, whereas unstimulated neutrophils produce 12S,20-DiHETE. This study was undertaken to characterize metabolism of 12S-HEPE in human neutrophils. We demonstrate herein for the first time that 12S-HEPE is metabolized by human neutrophils. In unstimulated neutrophils 20-hydroxylation to 12S,20-DiHEPE occurs, whereas in stimulated neurtrophils 5-lipoxygenation to 5S,12S-DiHEPE takes place. The structures of these metabolites were characterized by their relative retention times on reversed-phase high pressure liquid chromatography, by their UV absorbance spectra, and by gas-liquid chromatography-mass spectrometry. With increasing amounts of 12S-HEPE, stimulated neutrophils produced increasing amounts of 5S,12S-DiHEPE, which is virtually inactive biologically. Concomitantly, production of the potent chemokinetic and chemoattractant arachidonic acid derivative leukotriene B4 decreased. Thus, 12S-HEPE can compete with endogenous arachidonic acid for 5-lipoxygenation in stimulated human neutrophils. 12,20-DiHEPE, LTB5, and 5S,12S-DiHEPE were detectable after coincubating EPA-enriched platelets with unenriched neutrophils, and arachidonic acid-derived 5-lipoxygenase products were decreased. We conclude that 12S-HEPE can participate in platelet-neutrophil interactions in a manner similar to 12S-HETE. By providing competing substrates for neutrophil 5-lipoxygenase, platelets might contribute to the antiinflammatory potential of dietary n-3 fatty acids through platelet-neutrophil interaction.     

Hydroxylation of leukotriene B4 in leukocytes from various species: identification of a metabolite to authentic 5S,12R,19-trihydroxy-6Z,8E,10E,14Z-eicosatetra enoic acid and relative importance of 19- and 20-hydroxylations

The major hydroxylated metabolite of leukotriene B4 in rat PMNL was found identical (UV spectrum and retention times in 3 different HPLC systems) to a synthetic compound of known stereochemistry, 19-hydroxy-LTB4. PMNL from various species exhibited 3 different types of behaviour for LTB4 hydroxylation. Human and monkey PMNL showed a high hydroxylating activity and a high regioselectivity with almost exclusive formation of products from 20-hydroxylation. Rat and mini-pig PMNL exhibited a very different regioselectivity with major formation of 19-OH-LTB4 (3:1 ratio). Finally, pig and beef PMNL were found almost devoid of any hydroxylating activity toward LTB4.     

Platelet-neutrophil interactions. (12S)-hydroxyeicosatetraen-1,20-dioic acid: a new eicosanoid synthesized by unstimulated neutrophils from (12S)-20-dihydroxyeicosatetraenoic acid

In the course of a cell-cell interaction, 12-HETE (12-hydroxy-5,8,10,14-eicosatetraenoic acid), the arachidonic acid lipoxygenase product released from stimulated platelets, is metabolized by a cytochrome P-450 enzyme system in unstimulated neutrophils to 12,20-DiHETE (12,20-dihydroxy-5,8,10,14-eicosatetraenoic acid). This report describes time-dependent formation of a new eicosanoid by unstimulated neutrophils exposed to 12-HETE, which is more polar than 12,20-DiHETE (reversed-phase high performance liquid chromatography). Time course studies indicated that the precursor compound of this new eicosanoid was 12,20-DiHETE. This was determined by incubation of purified 12,20-DiHETE with neutrophils, which resulted in a progressive decrease in 12,20-DiHETE as formation of the polar metabolite increased. In the absence of neutrophils, 12,20-DiHETE was quantitatively unchanged. The new metabolite of 12,20-DiHETE was identified as 12-hydroxyeicosatetraen-1,20-dioic acid, based upon its UV spectrum, co-chromatography with a chemically synthesized standard in both high performance liquid chromatography and thin layer chromatography systems, and gas chromatography-mass spectrometry. Formation of 12-HETE-1,20-dioic acid was partially inhibited by 20-hydroxy-LTB4. This indicated that the neutrophil dehydrogenase responsible for further metabolism of 12,20-DiHETE may also be involved in conversion of 20-hydroxy-LTB4 to 20-carboxy-LTB4. The 12,20-DiHETE dehydrogenase enzyme system specifically requires NAD as cofactor and has subcellular components in both cytosolic and microsomal fractions which are synergistic in their activity. These results provide additional evidence for the occurrence of multicellular metabolic events during hemostasis, thrombosis, and the inflammatory response.     

NAD(P)H-dependent reduction of 12-ketoeicosatetraenoic acid to 12(R)- and 12(S)-hydroxyeicosatetraenoic acid by rat liver microsomes

The possibility that 12-keto-5,8,10,14 eicosatetraenoic acid (12-KETE) could be used as substrate by reductase(s) to generate 12-hydroxyeicosatetraenoic acid (12-HETE) was investigated using rat liver microsomes as a source of enzyme activity. Microsomes catalyzed the time-dependent reduction of 12-KETE to 12-HETE in a reaction that required NAD(P)H. The maximal specific activity of 12-HETE formation was 1.7 nmol/min/mg of protein in the presence of NADH. The reaction could not be detected in the absence of cofactor or by using heat inactivated microsomes. The identity of the 12-HETE product was established by U.V. spectroscopy and co-elution with 12-HETE in two different systems of RP-HPLC. Resolution of the methyl esters of reaction products by chromatography on chiral columns also indicated that the reduction of 12-KETE with either NADPH or NADH generated a mixture of 12(S)- and 12(R)-HETE in a ratio of about 2:1. The results demonstrate the presence of a 12-KETE reductase activity in rat liver microsomes which can form both the R and S isomers of 12-HETE.     

Synthesis of hydroxy fatty acids from 4, 7, 10, 13, 16, 19-[1-14C] docosahexaenoic acid by human platelets

Human platelets incubated in the presence of 54 microM [1-14C]22:6 produced hydroxydocosahexaenoic acid (HDHE) at about half the rate with which 12-hydroxy-5,8,10,14-eicosatetraenoic acid is produced from [1-14C]arachidonic acid. More than 90% of the radioactivity in HDHE was distributed among two major isomers, 14-HDHE and 11-HDHE. The production of HDHEs was unaffected by indomethacin but completely inhibited by 5,8,11,14-heneicosatetraynoic acid, which suggests that the hydroxy fatty acids are produced by lipoxygenase. The proportions of HDHE isomers varied with the concentration of 22:6. The ratio 14-HDHE/11-HDHE was higher at 6.8 microM 22:6 than when platelets were incubated with 54 microM 22:6. It is suggested that the amounts of these isomers produced will depend both on the availability of 22:6 as well as by competition of this acid with other acids for lipoxygenase.     

C20 steroids: The metabolism of 3-hydroxy-19-nor[21-14C]pregna-1,3,5(10)-trien-20-one in the rabbit

1. The metabolism of 3-hydroxy-19-norpregna-1,3,5(10)-trien-20-one, a possible product of the aromatization of progesterone or pregnenolone, has been studied. 2. After oral administration of this C(20) steroid as the 21-(14)C-labelled compound to two groups of rabbits, the excretion pattern of metabolites in the urine was examined. 3. At 14 days after administration, 3.3-6.5% of the radioactivity had appeared in the urine, 71-79% in the faeces and approx. 10% remained in the gut. 4. A metabolite, isolated from urine mainly as the unconjugated steroid, was identified as 19-norpregna-1,3,5(10)-triene-3,20alpha-diol and constituted 18.5-22% of the total urinary radioactivity. 5. A minor component of the urinary unconjugated steroids was identified as 19-norpregna-1,3,5(10)-triene-3,17alpha,20alpha-triol. 6. A further 2-7.5% of the total urinary radioactivity, isolated only from the urinary sulphate fraction, was tentatively identified as an 18-oxygenated derivative of the administered steroid.     

Stereoselective synthesis of (6Z,10E,12Z)-octadeca-6,10,12-trienoic acid, (8Z,12E,14Z)-eicosa-8,12,14-trienoic acid,and their [1-14C]-radiolabeled analogs

To study the metabolic fate of conjugated linoleic acid isomers, we synthesized, in seven steps, from 1-heptyne, (6Z,10E,12Z)-octadeca-6,10,12-trienoic acid, (8Z,12E,14Z)-eicosa-8,12,14-trienoic acid, and their [1-(14)C]-analogs. In the case of (6Z,10E,12Z)-octadecatrienoic acid, a series of palladium-catalyzed cross-coupling reactions between 1-heptyne and (E)-1,2-dichloro-ethene, a coupling reaction with a Grignard reagent and cleavage of the dioxolane gave (E)-dodec-4-en-6-ynal 3. Stereoselective Wittig reaction between aldehyde 3 and triphenyl-[5-(tetrahydro-pyran-2-yloxy)-pentyl]-phosphonium provided a dienyne. Stereocontrolled reduction of the triple bond and replacement of the tetrahydropyranyl group by a bromine gave (5Z,9E,11Z)-1-bromo-heptadeca-5,9,11-triene 10. Formation of the alkenyl lithium derivative and carbonation with CO(2) furnished (6Z,10E,12Z)-octadecatrienoic acid. (8Z,12E,14Z)-eicosa-8,12,14-trienoic acid was obtained by the same route but using triphenyl-[5-(tetrahydro-pyran-2-yloxy)-heptyl]-phosphonium iodide for the Wittig reaction. [1-(14)C]-analogs were obtained from the bromides by carbonation with (14)CO2. In all cases, chemical or radiochemical purities were found to be better than 95% after purification by flash chromatography on silica gel (>99% after additional purification by RP-HPLC). Metabolism studies in animals are in progress.     

Resolution by DEAE-cellulose chromatography of the enzymatic steps in the transformation of arachidonic acid into 8, 11, 12- and 10, 11, 12-trihydroxy-eicosatrienoic acid by the rat lung

The 30-50% ammonium sulfate fraction of the high speed supernatant (100,000 xg) of a rat lung homogenate is capable of catalysing the conversion of arachidonic acid into 8,11,12- and 10,11, 12-trihydroxyeicosatrienoic acids. This enzyme preparation was resolved through DEAE cellulose chromatography into three stages which were assayed with precursors specific for each stage. Thus in the first stage arachidonic acid is converted by 12-lipoxygenase into 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE) detected as the corresponding 12-hydroxy product (12-HETE). 12-HPETE in turn is converted into 8-hydroxy-11,12-epoxy-5,9,14-eicosatrienoic acid and 10-hydroxy-11,12-epoxy-5,8,14-eicosatrienoic acid. These epoxides are in turn selectively converted through an epoxide hydrase into the respective triols. While the first and third stages were carried out by distinct fractions from the DEAE columns, the second i.e. conversion of 12-HPETE into epoxides, was detected in all fractions as was the reduction of 12-HPETE into 12-HETE.     

Identification of 17-methyl-18-norandrosta-5,13(17-dien-3beta-ol, the C19 fragment formed by adrenal side chain cleavage of a 20-aryl analog of (20S)-20-hydroxycholesterol.

Incubation of (20R)-20-phenyl-5-pregnene-3beta,20-diol, an aromatic analog of (23S)-20-hydroxycholesterol, with an adrenal mitochondrial preparation leads to the formation of four compounds: pregnenolone, phenol, a C8 ketone, acetophenone, and a nonpolar C19 compound. This latter compound has now been identified by reverse isotope dilution analysis and by gas chromatography/mass spectrometry as 17-methyl-18-norandrosta-5,13(17)-dien-3beta-ol. From these results it is evident that enzymatic fission of the C-17,20 bond of this synthetic derivative occurs. On the other hand, when (20S)-20-hydroxy[21-14C]cholesterol was used as substrate, the analogous cleavage did not take place. Thus, substitution of an aromatic group on C-20 facilitates side chain cleavage between that carbon atom and the nucleus whereas neither of the naturally occuring precursors, cholesterol or its 20-hydroxylated counterpart, are metabolized to a C8 fragment.     

Purification of Drosophila ribosomal proteins. Isolation of proteins S8, S13, S14, S16, S19, S20/L24, S22/L26, S24, S25/S27, S26, S29, L4, L10/L11, L12, L13, L16, L18, L19, L27, 1, 7/8, 9, and 11

The proteins of Drosophila melanogaster embryonic ribosomes were separated into seven groups (A80 through G80) by stepwise elution from carboxymethylcellulose with lithium chloride at pH 6.5 by procedures previously described [Chooi, W. Y., Sabatini, L. M., MacKlin, M. D., & Fraser, W. (1980) Biochemistry 19, 1425-1433]. Three relatively acidic proteins, S14, S25/S27, and 7/8, have now been isolated from group A80 by ion-exchange chromatog raphy on carboxymethylcellulose eluted with a linear gradient of lithium chloride at pH 4.2. Fractions containing the relatively basic proteins (groups B80 through G80) were furher combined into a total of 24 "pools". The criterion for combination was the migration patterns in one-dimensional polyacrylamide gels containing sodium dodecyl sulfate (NaDodS04) of every fifth fraction from the carboxymethylcellulose column. Each pool contained between 1 and 12 major proteins. Proteins S8, S13, S16, S19, S20/L24, S22/L26, S24, S26, S29, L4, L10/L11, L12, L13, L16, L18, L19, L27, 1, 9, and 11 have now been isolated from selected pools by gel filtration through Sephadix G-100. The amount of each protein recovered from a starting amount of 1.8 g of total 80S proteins varied form 0.2 to 10.8 mg. Five proteins had no detectable contamination, and in each of the others the impurities were no greater than 9%. The amino acid composition of the individual purified proteins was determined. The molecular weights of the proteins were estimated by polyacrylamide gel electrophoresis in NaDodSO4.     

Biological activities of conjugated fatty acids: conjugated eicosadienoic (conj. 20:2delta(c11,t13/t12,c14)), eicosatrienoic (conj. 20:3delta(c8,t12,c14)), and heneicosadienoic (conj. 21:2delta(c12,t14/c13,t15)) acids and other metabolites of conjugated linoleic acid

The elongated form of conjugated linoleic acid (CLA), conjugated eicosadienoic acid (CEA, conj. 20:2delta(c11,t13/t12,c14)), was generated from CLA by liver microsomal fractions. Subsequent testing showed that dietary CEA significantly reduced body fat, and increased lean mass similar to CLA when compared to controls. CEA also decreased lipoprotein lipase activity and triacylglyceride, and increased glycerol release in 3T3-L1 adipocytes, correlated with the trans-12,cis-14 isomer, but CEA required a longer incubation period than cells treated with CLA. Based on the fact that CEA fed animals had CLA in tissue, we suggest that the effect of CEA is due to the CLA converted from CEA in the system. The delta-6 desaturated and elongated form of trans-10,cis-12 CLA (conjugated eicosatrienoic acid, CETA, conj. 20:3delta(c8,t12,c14)) inhibited LPL activity and increased glycerol release but was less active than trans-10,cis-12 CLA or CEA. The 21-carbon conjugated fatty acid, conjugated heneicosadienoic acid (CHDA, conj. 21:2delta(c12,t14/c13,t15)), was not active on LPL inhibition, triacylglyceride, or glycerol release in 3T3-L1 adipocytes. We also provide evidence that CLA was metabolized to conjugated dodecadienoic acid (conj. 12:2delta(c3,t5/t4,c6)). In addition, there were indications of the presence of conjugated tetradecadienoic acid (conj. 14:2delta(c5,t7/t6,c8)), suggesting that CLA can be metabolized through fatty acid beta-oxidation. This is the first work to report the presence of conjugated 12 and 14 carbon fatty acids, originated from CLA, and the biological activities of CEA, CETA and CHDA.     

Four novel Loci (19q13, 6q24, 12q24, and 5q14) influence the microcirculation in vivo

There is increasing evidence that the microcirculation plays an important role in the pathogenesis of cardiovascular diseases. Changes in retinal vascular caliber reflect early microvascular disease and predict incident cardiovascular events. We performed a genome-wide association study to identify genetic variants associated with retinal vascular caliber. We analyzed data from four population-based discovery cohorts with 15,358 unrelated Caucasian individuals, who are members of the Cohort for Heart and Aging Research in Genomic Epidemiology (CHARGE) consortium, and replicated findings in four independent Caucasian cohorts (n = 6,652). All participants had retinal photography and retinal arteriolar and venular caliber measured from computer software. In the discovery cohorts, 179 single nucleotide polymorphisms (SNP) spread across five loci were significantly associated (p<5.0×10(-8)) with retinal venular caliber, but none showed association with arteriolar caliber. Collectively, these five loci explain 1.0%-3.2% of the variation in retinal venular caliber. Four out of these five loci were confirmed in independent replication samples. In the combined analyses, the top SNPs at each locus were: rs2287921 (19q13; p = 1.61×10(-25), within the RASIP1 locus), rs225717 (6q24; p?=?1.25×10(-16), adjacent to the VTA1 and NMBR loci), rs10774625 (12q24; p = 2.15×10(-13), in the region of ATXN2,SH2B3 and PTPN11 loci), and rs17421627 (5q14; p?=?7.32×10(-16), adjacent to the MEF2C locus). In two independent samples, locus 12q24 was also associated with coronary heart disease and hypertension. Our population-based genome-wide association study demonstrates four novel loci associated with retinal venular caliber, an endophenotype of the microcirculation associated with clinical cardiovascular disease. These data provide further insights into the contribution and biological mechanisms of microcirculatory changes that underlie cardiovascular disease.     

Metabolism of 18:2(n - 6), 18:3(n - 3), 20:4(n - 6) and 20:5(n - 3) by the fungus Gaeumannomyces graminis: identification of metabolites formed by 8-hydroxylation and by w2 and w3 oxygenation.

The present study was aimed at developing a cell-free preparation of Gaeumannomyces graminis to biosynthesize w2-hydroxy, w3-hydroxy and related metabolites of essential fatty acids. 14C-labelled linoleic acid (18:2(n - 6)), linolenic acid (18:3(n - 3)), arachidonic acid (20:4(n - 6)) and eicosapentaenoic acid (20:5(n - 3)) were incubated with the cytosolic and microsomal fractions and NADPH. Significant metabolism was only found in the cytosol. The main products were purified by high-performance liquid chromatography and identified by gas chromatography-mass spectrometry (GC-MS). 18:2(n - 6) was metabolized mainly to 8-hydroxy-9,12-octadecadienoic acid (8-HODE), while the w2 and the w3 alcohols were formed in relatively small amounts. The absolute configuration of the 8-hydroxyl was found to be R by ozonolysis of the diastereoisomeric (-)-menthoxycarbonyl derivative of 8-HODE and GC-MS analysis. In analogy, 18:3(n - 3) was converted to 8-hydroxy-9,12,15-octadecatrienoic acid and to smaller amounts of the 15,16-diol (15,16-DiHODE). In contrast, 8-hydroxy metabolites of 20:4(n - 6) or 20:5(n - 3) could not be detected. 20:4(n - 6) was efficiently converted to 18(R)-hydroxyeicosatetraenoic acid (18(R)-HETE) and 19(R)-HETE and to traces of 17-HETE, while 20:5(n - 3) was mainly metabolized to the 17,18-diol (17,18-DiHETE) and to smaller amounts of the w2 alcohol. In conclusion, the cytosol of G. graminis can be used for stereoselective biosynthesis of some hydroxy metabolites of essential fatty acids.     

Thermodynamic and structural characterization of cis-trans isomerization of 12-(S)-hydroxy-(5Z, 8E, 10E)-heptadecatrienoic acid by high-performance liquid chromatography and gaschromatography-mass spectrometry.

It is shown that 12-(S)-hydroxy-(5Z, 8E, 10E)-heptadecatrienoic acid (5-cis-HHT)--a physiological metabolite of arachidonic acid--is acid-catalyzed converted into a less polar substance with its maximum UV-absorption at (1)max=232 nm and a molar absorptivity of about epsilon=26600 +/- 200 M(-1)cm(-1). Using a reversed-phase high-performance liquid chromatographic (HPLC) method this equilibrium reaction (K(c) = 1.78 +/- 0.05 at pH 1.10 and 298 K) could be thermodynamicly characterized as a pH dependent, exergonic and exothermic reaction according to kinetics of a first order reaction (at pH 1.10 and 298 K: delta(R)G(o) = -1.42 +/- 0.07 kJ mol(-1), delta(R)H(o) = -3.50 +/- 0.9 kJ mol(-1), delta(R)S(o) = 7.0 +/- 3.0 J mol(-1)*K, delta(R)H*f = 100.0 +/- 4.0 kJ mol(-1)). Kinetic data for several pH-values and temperatures are presented. These data and structural characterization by gaschromatography-mass spectrometry (GC/MS) lead to the conclusion that 5-cis-HHT is isomerized to 12-(S)-hydroxy-(5E, 8E, 10E)-heptadecatrienoic acid (5-trans-HHT).     

Conversion of [14C]gibberellin A12-aldehyde to C19- and C20-gibberellins in a cell-free system from immature seed of Phaseolus coccineus L.

A cell-free system prepared from developing seed of runner bean (Phaseolus coccineus L.) converted [¹⁴C]gibberellin A₁₂-aldehyde to several products. Thirteen of these were identified by capillary gas chromatography-mass spectrometry as gibberellin A₁ (GA₁), GA₄, GA₅, GA₆, GA₁₅, GA₁₇, GA₁₉, GA₂₀, GA₂₄, GA₃₇, GA₃₈, GA₄₄ and GA₅₃-aldehyde, all giving mass spectra with ¹⁴C-isotope peaks. GA₈ and GA₂₈ were also identified but contained no ¹⁴C. All the [¹⁴C]GA₁₂-aldehyde metabolites, except GA₁₅, GA₂₄ and GA₅₃-aldehyde, are known endogenous GAs of P. coccineus.Abbreviations GA_n gibberellin A_n - GC-MS combined gas chromatography-mass spectrometry - HPLC highperformance liquid chromatography - MVA mevalonic acid - S-2 2000-g supernatant     

In Vitro Metabolism of 20(R)-25-Methoxyl-Dammarane-3, 12, 20-Triol from Panax notoginseng in Human,Monkey, Dog,Rat, and Mouse Liver Microsomes

The present study characterized in vitro metabolites of 20(R)-25-methoxyl-dammarane-3β, 12β, 20-triol (20(R)-25-OCH₃-PPD) in mouse, rat, dog, monkey and human liver microsomes. 20(R)-25-OCH₃-PPD was incubated with liver microsomes in the presence of NADPH. The reaction mixtures and the metabolites were identified on the basis of their mass profiles using LC-Q/TOF and were quantified using triple quadrupole instrument by multiple reaction monitoring. A total of 7 metabolites (M1–M7) of the phase I metabolites were detected in all species. 25(R)-OCH₃-PPD was metabolized by hydroxylation, dehydrogenation, and O-demethylation. Enzyme kinetic of 20(R)-25-OCH₃-PPD metabolism was evaluated in rat and human hepatic microsomes. Incubations studies with selective chemical inhibitors demonstrated that the metabolism of 20(R)-25-OCH₃-PPD was primarily mediated by CYP3A4. We conclude that 20(R)-25-OCH₃-PPD was metabolized extensively in mammalian species of mouse, rat, dog, monkey, and human. CYP3A4-catalyzed oxygenation metabolism played an important role in the disposition of 25(R)-OCH₃-PPD, especially at the C-20 hydroxyl group.