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R C Murphy

Publications and source records attributed to R C Murphy.

At least 55 records · Page 3Linked to original sources
PubMed1998

Identification and pharmacological characterization of platelet-activating factor and related 1-palmitoyl species in human inflammatory blistering diseases.

Through its pro-inflammatory effects on leukocytes, endothelial cells, and keratinocytes, the lipid mediator platelet-activating factor (PAF) has been implicated in cutaneous inflammation. Although the 1-alkyl PAF species has been considered historically the most abundant and important ligand for the PAF receptor (PAF-R), other putative ligands for this receptor have been described including 1-acyl analogs of sn-2 acetyl glycerophosphocholines. Previous bioassays have demonstrated a PAF-like activity in lesions of the autoimmune blistering disease bullous pemphigoid. To assess the actual sn-2 acetyl glycerophosphocholine species that result in this PAF agonistic activity, we measured PAF and related sn-2 acetyl GPCs in fresh blister fluid samples from bullous pemphigoid and noninflammatory (suction-induced) bullae by mass spectrometry. We report the presence of 1-hexadecyl as well as the 1-acyl PAF analog 1-palmitoyl-2-acetyl glycerophosphocholine (PAPC) in inflammatory blister fluid samples. Because PAPC is the most abundant sn-2 acetyl glycerophosphocholine species found in all samples examined, the pharmacological effects of this species with respect to the PAF-R were determined using a model system created by transduction of a PAF-R-negative epidermoid cell line with the PAF-R. Radioligand binding and intracellular calcium mobilization studies indicated that PAPC is approximately 100x less potent than PAF. Though a weak agonist, PAPC could induce PAF biosynthesis and PAF-R desensitization. Finally, intradermal injections of PAF and PAPC into the ventral ears of rats demonstrated that PAPC was 100x less potent in vivo. These studies suggest possible involvement of PAF and related species in inflammatory bullous diseases.

Animals↗
PubMed1998

Electrospray mass spectrometric analysis of 5-hydroperoxy and 5-hydroxyeicosatetraenoic acids generated by lipid peroxidation of red blood cell ghost phospholipids.

Recent evidence suggests that generation of hydroxyl radicals in the presence of lipid membranes can lead to oxidation of arachidonic acid esterified to glycerophospholipids and the production of compounds isomeric to prostaglandins, thromboxanes, and leukotrienes. Liquid chromatography tandem mass spectrometry and multiple reaction monitoring were employed to quantitate the production of 5-hydroxyeicosatetraenoic acid (5-HETE), 5-hydroperoxyeicosatetraenoic acid (5-HPETE), and 5-oxo-eicosatetraenoic acid (5-oxo-ETE) in red blood cells ghosts treated with t-butylhydroperoxide (tBuOOH). Untreated red blood cell ghosts were found to contain low, but measurable quantities of these three 5-oxygenated eicosanoids as phospholipid esters. Following treatment, there was approximately a 53- and 22.5-fold increase in 5-HETE and 5-HPETE, respectively, and an 8.5-fold increase in 5-oxo-ETE. The formation of these compounds was inhibited nearly 90% by the antioxidants butylated hydroxytoluene, ascorbic acid, and resveratrol providing further evidence for free radical mediated oxidation of arachidonic acid. This analytical protocol provided sufficient sensitivity for detection of these compounds in studies in which previous analysis by high-pressure liquid chromatography with UV detection failed to detect their presence. These results reveal that the biologically active eicosanoids 5-HPETE, 5-HPETE, and 5-oxo-ETE are formed esterified to phospholipids following exposure of cellular membranes to reactive oxygen species and free radicals in a model system where intracellular antioxidant mechanisms were depleted.

Chemotactic Factors↗
PubMed1998

Activation of human polymorphonuclear leukocytes by products derived from the peroxidation of human red blood cell membranes.

Oxidation of red blood cell (RBC) ghost preparations initiated by tert-butyl hydroperoxide (tBuOOH) was employed to explore the formation of lipid products derived from endogenous phospholipids that specifically expressed biological activity toward the human polymorphonuclear leukocyte (PMN). Common measure of lipid peroxidation, thiobarbituric acid-reactive substances (TBARS) and the increased absorbance at 235 nm consistent with the formation of conjugated dienes, was observed following a 90-min incubation of RBC ghosts with tBuOOH. Saponification of phospholipids and separation of the resultant fatty acids by RP-HPLC permitted direct mass spectrometric analysis of oxidized fatty acids. Individual HPLC fractions were assayed for their ability to increase intracellular free calcium ion concentrations in human PMN to guide structural investigations. Two fractions were found to contain biologically active components, and tandem mass spectrometric analysis of the abundant ions observed in these fractions resulted in the characterization of several oxidized polyunsaturated fatty acids derived from arachidonic and linoleic acids. The major components in these fractions included 5-hydroxyeicosatetraenoic acid (5-HETE) and 5-hydroperoxyeicosatetraenoic acid (5-HpETE). The dose-dependent increases in intracellular calcium in the neutrophil using synthetic 5(rac)-HETE, 5(rac)-HpETE, and 5-oxo-ETE were found to have EC50's of 250, 6, and 3 nM, respectively. The quantity of 5-oxygenated arachidonate components present in oxidized RBC was consistent with the observed biological response elicited by fractions A and B. This study suggests that 5-HETE and 5-HpETE are abundant products of lipid peroxidation of cellular membranes and that these racemic products possess significant biological activity. Such compounds could play important roles as mediators of the cellular response to toxicologic stimuli that generate free radical species.

Arachidonic Acids↗
PubMed1998

The effects of ethanol and acetaldehyde on the metabolism of prostaglandin E2 and leukotriene B4 in isolated rat hepatocytes.

The effects of ethanol and acetaldehyde on the metabolism of leukotriene B4 (LTB4) and PGE2 were investigated in isolated cultures of rat hepatocytes. LTB4 undergoes initial cytochrome P450-dependent omega-oxidation leading to the principal metabolites 20-hydroxy-LTB4, 20-carboxy-LTB4 and the omega/beta-oxidation product 18-carboxy-LTB4. The addition of low concentrations of ethanol (25 mM) dramatically changes the relative amounts of these metabolite products by inhibiting the alcohol dehydrogenase-mediated oxidation of 20-hydroxy-LTB4. Addition of acetaldehyde to the incubation, up to 1 mM, had no significant effect on overall metabolism or distribution of metabolites. Above 1 mM acetaldehyde, beta-oxidation of LTB4 was inhibited. Thus the effect of ethanol on the metabolism of LTB4 appears to be due to ethanol itself and not to secondary effects from the metabolic transformation of ethanol to acetaldehyde in the cells. PGE2 is metabolized in isolated rat hepatocytes to produce chain-shortened products of beta-oxidation characterized as dinor-PGE1, dinor-PGE2, tetranor-PGE1, tauro-dinor-PGE1 and tauro-dinor-PGE2. Low concentrations of ethanol (25 mM) were found to increase the relative concentration of dinor-PGE1 in the metabolic distribution, with a corresponding decrease in concentration of tetranor-PGE1. The amount of dinor-PGE2 that was produced remained relatively unchanged in response to increasing concentrations of ethanol. Acetaldehyde concentrations from 0.1 mM to 1 mM did not affect metabolite distribution or the overall magnitude of PGE2 metabolism. Concentrations of acetaldehyde higher than 1 mM decreased all beta-oxidation metabolites. Ethanol, at physiologically relevant concentrations, could alter eicosanoid metabolism in the liver by inhibiting LTB4 metabolism and altering that of PGE2.

Acetaldehyde↗
PubMed1998

Exogenous leukotriene B4 (LTB4) inhibits human neutrophil generation of LTB4 from endogenous arachidonic acid during opsonized zymosan phagocytosis.

The effect of exogenous leukotriene B4 (LTB4) on opsonized zymosan-stimulated human neutrophil formation of 5-lipoxygenase products and arachidonic acid release was directly assessed using reverse-phase HPLC/tandem mass spectrometric methods for quantitation. Stable isotopically labeled LTB4, [1,2-13C2]LTB4, caused a dose-dependent inhibition of LTB4 production in isolated human neutrophils with significant inhibition (60 +/- 7% of control levels) when 0.12 nM [13C2]LTB4 was present. Production of 5-hydroxy-6,8,11,14-eicosatetraenoic acid and release of free arachidonic acid were also dose-dependently inhibited by exogenous LTB4. Metabolites of LTB4, 20-hydroxy-LTB4 and 3(S)-hydroxy-LTB4, also significantly reduced LTB4 production to levels as low as 10 +/- 6% and 10 +/- 7% of control levels, respectively, when present exogenously at 10 nM. Exogenous 5-hydroxy-6,8,11,14-eicosatetraenoic acid at concentrations as high as 10 nM produced no significant reduction in LTB4 biosynthesis during zymosan-stimulated human neutrophil production of LTB4. The inhibitory effect of LTB4 could be partially reversed by the LTB4 receptor antagonist U 75302. Furthermore, an alternative stimulus, N-formyl-methionyl-leucyl-phenylalanine (100 nM), did not inhibit the production of LTB4 in opsonized zymosan-stimulated human neutrophils. These results suggest that activation of the LTB4 receptor on the human neutrophil during phagocytosis limits the ultimate biosynthesis of LTB4. This autocrine effect is opposite to that observed when neutrophils have much of the signal transduction pathways bypassed when stimulated with calcium ionophore A23187 or treated with exogenous free arachidonic acid.

Arachidonic Acid↗
PubMed1997

Identification of novel metabolites of prostaglandin E2 formed by isolated rat hepatocytes.

The metabolism of prostaglandin E2 (PGE2) in isolated rat hepatocytes led to the formation of four major as well as several minor products which were structurally characterized using electrospray tandem mass spectrometry. The major metabolites identified included dinor-PGE1, dinor-PGE2, and tetranor-PGE1 and the taurine conjugates of dinor-PGE1 and dinor-PGE2. Several minor metabolites including the taurine conjugates of PGE2 and tetranor PGE1 along with a glucuronide conjugate of PGE2 were also identified. These taurine conjugates had not been previously identified in studies of PGE2 metabolism, yet comprised nearly 50% of the mixture of metabolites after 40-min incubations. Experiments carried out with deuterium-labeled PGE2 ([3,3,4,4-D4]PGE2) resulted in the complete loss of all deuterium atoms in dinor-PGE1, dinor-PGE2, and tetranor metabolites during incubation with hepatocytes. Metabolism via classic beta-oxidation pathways would predict one deuterium atom retained by dinor-PGE1 and two deuterium atoms retained by dinor-PGE2. When PGE2 was incubated with isolated rat hepatocytes in buffer containing 30% D2O, substantial incorporation (30%) of one deuterium atom could be observed in the dinor metabolites along with 10% incorporation into the tetranor and residual PGE2. Deuterium-labeled PGE1 ([3,3,4,4-D4]PGE1) was metabolized to D2-dinor-PGE1, tetranor-PGE1, and the taurine conjugate of D2-dinor-PGE1 by isolated rat hepatocytes. The loss of deuterium during metabolism of the deuterated substrates of PGE2, but not PGE1, as well as the incorporation of deuterium atoms from the aqueous solvent into PGE2 metabolites suggested that the delta 5 double bond and sequential isomerization reactions lead to eventual exchange of the protons from carbon atom 4 of PGE2 with water.

Alprostadil↗
PubMed1997

Pharmacological modulation of human platelet leukotriene C4-synthase.

The aim of this study was to test if human platelet leukotriene C4-synthase (LTC4-S) is pharmacologically different from cloned and expressed LTC4-S and, in light of the significant homologies between 5-lipoxygenase activating protein (FLAP) and LTC4-S, if different potencies of leukotriene synthesis inhibitors acting through binding with FLAP (FLAP inhibitors) reflect in different potencies as LTC4-S inhibitors. Leukotriene C4 (LTC4) synthesis by washed human platelets supplemented with synthetic leukotriene A4 (LTA4) was studied in the absence and presence of two different, structurally unrelated FLAP inhibitors (MK-886 and BAY-X1005) as well as a direct 5-lipoxygenase inhibitor (zileuton). LTC4 production was analyzed by RP-HPLC coupled to diode array detection. We report that human platelet LTC4-S was inhibited by MK-886 and BAY-X1005 (IC50 of 4.7 microM and 91.2 microM, respectively), but not by zileuton (inactive up to 300 microM); all 3 compounds were able to inhibit 5-lipoxygenase metabolite biosynthesis in intact human polymorphonuclear leukocytes (IC50 of 0.044 microM, 0.85 microM, and 1.5 microM, respectively). Platelet LTC4-S does not appear pharmacologically different from expression cloned LTC4-S. LTC4-S inhibition by FLAP inhibitors is in agreement with the significant homology reported for expression-cloned LTC4-S with FLAP, Furthermore, functional homology of the binding sites for inhibitors on LTC4-S and FLAP is suggested by the conservation of the relative potencies of MK-886 and BAY-X1005 vs FLAP-dependent 5-lipoxygenase activity and LTC4-S inhibition: MK-886 was 19.3-fold more potent than BAY-X1005 as FLAP inhibitor and 19.6-fold more potent than BAY-X1005 as LTC4-S inhibitor.

Blood Platelets↗
PubMed1997

Quantitation of 5-lipoxygenase products by electrospray mass spectrometry: effect of ethanol on zymosan-stimulated production of 5-lipoxygenase products by human neutrophils.

A reverse-phase-HPLC/tandem mass spectrometric method (LC/MS/MS) was developed for the quantitation of leukotriene B4 (LTB4), the 5-lipoxygenase product, 5-hydroxyeicosatetraenoic acid (5-HETE), as well as the omega-oxidation metabolites of LTB4, 20-hydroxy-LTB4, and 20-carboxy-LTB4. Electrospray-generated carboxylate anions were collisionally activated and decomposed to specific and abundant product ions and multiple reaction monitoring was used to analyze LTB4 (m/z 335-->195), 20-hydroxy-LTB4 (m/z 351-->195), 20-carboxy-LTB4 (m/z 365-->195), and 5-HETE (m/z 319-->115). A linear correlation was observed in comparison of results obtained from quantitation by LC/MS/MS with quantitation by gas chromatography/MS of the pentafluorobenzyl ester/trimethylsilyl ether derivative of LTB4 produced during opsonized zymosan stimulation of human neutrophils. Detection limits at the low picogram level were obtained for all metabolites. This method was applied to the quantitation of 5-lipoxygenase products produced from zymosan-stimulated human neutrophils in the presence of ethanol. At physiologically relevant concentrations of ethanol, production of all 5-lipoxygenase products generated by stimulated neutrophils was markedly attenuated.

Arachidonate 5-Lipoxygenase↗
PubMed1997

Analysis of epoxyeicosatrienoic and monohydroxyeicosatetraenoic acids esterified to phospholipids in human red blood cells by electrospray tandem mass spectrometry.

Electrospray ionization (ESI) and tandem mass spectrometry (MS/MS) were used to analyze epoxyeicosatrienoic acids (EETs) and monohydroxyeicosatetraenoic acids (HETEs) isolated from human red blood cell membranes following base hydrolysis. ESI results in the formation of an abundant isobaric carboxylate anion at m/z 319 for both of these oxidized metabolites of arachidonic acid. The product ion spectra from the collision-induced dissociation of this carboxylate anion could be used to identify each of the isomeric eicosanoids from the unique fragment ions of each eicosanoid. The observed product ion spectra were identical with those previously obtained by fast atom bombardment ionization; however, ESI required less EET and HETE for analysis. Both EET and HETE phospholipids were present in human red blood cells (RBCs) and their abundance could be substantially increased by treatment under conditions that would induce free radical oxidation of membrane phospholipids. Following incubation of human RBCs with tert-butyl hydroperoxide (tBuOOH), phospholipids were extracted and purified by normal-phase high-performance liquid chromatography (HPLC) as to glycerophospholipid class containing ethanolamine (GPE), serine (GPS) and choline (GPC) as the polar head group. Each class of phospholipid was hydrolyzed to yield the free carboxylic acid prior to on-line HPLC/ESI-MS/MS analysis. The formation of oxidized arachidonic acid esterified to phospholipids in treated RBCs was found to increase significantly for both esterified EETs in GPE, GPS and GPC which increased 49-, 34- and 59-fold, respectively, and also for esterified HETEs in GPE, GPS and GPC which increased 3-, 4- and 11-fold, respectively, compared with untreated RBCs. These results provide the first characterization of EETs formed non-enzymatically as intact phospholipids in a lipid peroxidation model system.

8,11,14-Eicosatrienoic Acid↗
PubMed1997

Identification and relative quantitation of F2-isoprostane regioisomers formed in vivo in the rat.

F2-isoprostanes are a complex mixture of isomers formed in four regioisomeric family types by free radical-initiated oxidation of arachidonic acid present in membrane phospholipids. F2-isoprostanes isolated from the livers of rats treated with carbon tetrachloride were separated by initial reverse phase HPLC and detected using electrospray ionization mass spectrometry with the characteristic loss of 44 u (C2H4O) from the common 1,3-diol cyclopropane ring found in these eicosanoids. Collision induced decomposition of the carboxylate anions from the separated F2-isoprostanes formed abundant ions characteristic for regioisomers of Type I (m/z 115), Type III (m/z 127), and Type IV (m/z 193), which made possible characterization of these three family subtypes by LC/MS/MS. Capillary GC/MS was employed to further identify the F2-isoprostane regioisomers using electron ionization mass spectrometry and to obtain characteristic mass spectra of the pentafluorobenzyl ester trimethylsilyl ether derivatives. Quantitation of the F2-isoprostanes separated by both reverse-phase HPLC and capillary GC/MS was carried out using negative ion chemical ionization mass spectrometry. The most abundant isomers identified were Type I and IV regioisomers constituting 33 and 25% of the total products, respectively. As expected, the Type II and III regioisomer products were of less abundance. Over 45 F2-isoprostanes could be separated in this complex mixture, suggesting random production of each regioisomeric subtype in this in vivo model.

Animals↗
PubMed1997

Identification of sn-2 acetyl glycerophosphocholines in human keratinocytes.

Evidence is accumulating suggesting that platelet-activating factor plays a role in inflammatory dermatoses. Mass spectrometric methods were used to examine the molecular species of sn-2 acetyl glycerophosphocholines (GPC) synthesized by primary cultures of human neonatal foreskin-derived keratinocytes. Ionophore-stimulated keratinocytes synthesize both 1-alkyl and 1-acyl sn-2 acetyl-GPC, and the relative amounts were as follows: hexadecyl > palmitoyl > octadecyl > stearoyl at the sn-1 position. PAF synthesis in the keratinocyte-derived cell line HaCaT was inhibited by dexamethasone, suggesting that the anti-inflammatory effects of glucocorticosteroids in inflammatory dermatoses might be in part related to the inhibition of the synthesis of mediators such as PAF.

Adrenal Cortex Hormones↗
PubMed1997

Copper-catalyzed oxidation mediates PAF formation in human LDL subspecies. Protective role of PAF:acetylhydrolase in dense LDL.

Free radical-mediated oxidation of cholesterol-rich LDL plays a key role in atherogenesis and involves the formation of oxidized phospholipids with proinflammatory biological activity. We evaluated the production of platelet-activating factor (PAF), a potent inflammatory mediator, in human LDL subspecies on copper-initiated oxidation (4 mumol/L CuCl2, 80 micrograms/mL for hours at 37 degrees C). PAF formation was determined by biological assay of HPLC-purified lipid extracts of copper-oxidized lipoproteins; chemical identity was confirmed by gas chromatographic and mass spectrometric analyses. PAF, characterized as the C16:0 molecular species, was preferentially produced in intermediate LDL (d = 1.029 to 1.039 g/mL) (8.6 +/- 5.7 pmol PAF/3 h per mg LDL protein) and light LDL (d = 1.019 to 1.029 g/mL), but was absent from dense LDL particles (d = 1.050 to 1.063 g/mL). As PAF:acetylhydrolase inactivates PAF and oxidized forms of phosphatidylcholine, we evaluated the relationship of lipoprotein-associated PAF:acetylhydrolase to PAF formation. We confirmed that PAF:acetylhydrolase activity was elevated in native, dense LDL (41.5 +/- 9.5 nmol/min per mg protein) but low in LDL subspecies of light and intermediate density (d 1.020 to 1.039 g/mL) (3.5 +/- 1.6 nmol/min per mg protein) [Tselepis et al, Arterioscler Thromb Vasc Biol. 1995;15:1764-1773]. On copper-mediated oxidation for 3 hours at 37 degrees C, dense LDL particles conserved 20 +/- 14% of their initial enzymatic activity; in contrast, PAF:acetylhydrolase activity was abolished in light and intermediate LDL subspecies. Clearly, the elevated PAF:acetylhydrolase activity of dense LDL efficiently diminishes the potential inflammatory role of endogenously formed PAF; nonetheless, formation of proatherogenic lysophospholipids results. In contrast, LDL particles of the light and intermediate subclasses can accumulate PAF on oxidative modification.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1997

Oxidative metabolism of a rexinoid and rapid phase II metabolite identification by mass spectrometry.

LGD1069 (Targretin), a retinoid "X" receptor-selective ligand, or rexinoid, is in clinical trials for treating cancer. Biologically-active oxidized LGD1069 metabolites have been observed in patient plasma samples, making corresponding structural characterizations necessary. Formation of multiple metabolite isomers in vivo has created technical challenges in metabolite structural analysis; however, mass spectrometry (MS) was able to pinpoint two sites of Phase I metabolism. A carbon-13 trideuterated analog was used as an isotopic marker to probe Phase II metabolism of LGD1069. Rats were orally gavaged with an equimolar mixture of LGD1069 and [13C2H3]LGD1069, then anesthetized prior to bile-duct cannulation. Bile was collected for 7 hr, extracted, and concentrated. Recovered metabolites were analyzed by narrow-bore, gradient liquid chromatography (LC) with negative ion, electrospray ionization MS detection. When resultant total ion chromatograms were interrogated for mass spectra exhibiting isotope clusters separated by 4 daltons, 13 such clusters corresponding to Phase II LGD1069 metabolites of nine different molecular weights were detected. Acyl-glucuronide and taurine conjugates of both parent compound and hydroxy-LGD1069 were observed. The sulfate and taurine conjugates of oxo-LGD1069 were also identified, as were 6,7-dihydroxy-LGD1069 taurine, LGD1069 ether glucuronide, and a secondary conjugate (taurine) of the latter. Identities of selected conjugates were confirmed by MS/MS. The results of this study demonstrate that when combined with traditional GC/MS and MS/MS data, the isotope cluster technique can provide powerful selectivity in identifying numerous Phase II drug metabolites during a single LC/MS analysis.

Animals↗
PubMed1996

Direct mass spectrometric analysis of ozonides: application to unsaturated glycerophosphocholine lipids.

The reaction of ozone with double bonds present in glycerophosphocholine lipids results in formation of ozonides that can be directly analyzed by mass spectrometry as either positive or negative molecular ion species generated by electrospray ionization. Polyunsaturated fatty acyl groups esterified to the phospholipid yielded a mixture of ozonide species with the maximum number of ozone molecules added equal to the total number of double bonds. Ozonide decomposition resulted in omega-aldehyde and omega-carboxylic acid products as revealed by ESI-MS. Collisional activation of the ozone adducts for mono- and polyunsaturated phospholipids gave rise to fragment ions indicative of the position of the double bonds in these molecules. The major decomposition pathway for either positive or negative ozonide ion species involved charge remote fragmentation of the ozonide initiated by homolytic cleavage of the peroxide bridge followed by rearrangement to form the omega-aldehyde and omega-carboxylate acyl species. The reaction of ozone with phospholipids containing polyunsaturated fatty acyl groups is a useful method to probe the position of double bonds by electrospray ionization mass spectrometry.

Lipids↗
PubMed1996

Activity of the mitochondrial multiple conductance channel is independent of the adenine nucleotide translocator.

The functional relationship between the adenine nucleotide translocator (ANT) and the mitochondrial multiple conductance channel (MCC) was investigated using patch-clamp techniques. MCC activity with the same conductance, ion selectivity, voltage dependence, and peptide sensitivity could be reconstituted from inner membrane fractions derived from mitochondria of ANT-deficient and wild-type Saccharomyces cerevisiae. In addition, the MCC activity of mouse kidney mitoplasts was unaffected by carboxyatractyloside, a known inhibitor of ANT and inducer of a permeability transition. These results suggest that MCC activity is independent of ANT.

Amino Acid Sequence↗
PubMed1996

Gas chromatographic/mass spectrometric analysis of oxo and chain-shortened leukotriene B4 metabolites. Leukotriene B4 metabolism in Ito cells.

Analysis by gas chromatography/mass spectrometry (GC/MS) of derivatized metabolites formed following incubation of leukotriene B4 (LTB4) incubation with Ito cells extends previous knowledge concerning fragmentation mechanisms for derivatized hydroxy-substituted unsaturated fatty acids. LTB4 was metabolized by rat Ito cells, a hepatic perisinusoidal stellate cell, by the delta 10- and delta 14-reductase pathways, resulting in the formation of 10,11-dihydro-LTB4 and 10,11,14,15-tetrahydro-LTB4. Formation of the intermediate metabolites, 12-oxo-10,11-dihydro-LTB4 and 12-oxo-10,11,14,15-tetrahydro-LTB4, was also observed. GC/electron impact (EI) MS analysis of the 12-oxo metabolites, derivatized as the pentafluorobenzyl ester/ trimethylsilyl ether compounds, resulted in unique fragmentations indicative of the oxo substituent and double bond positions. Further metabolism of 10,11-dihydro-LTB4 and 10,11,14,15-tetrahydro-LTB4 by carboxy terminus beta-oxidation resulted in chain-shortened monohydroxy metabolites. Possible intermediates in this metabolism, which resulted in loss of the original C-5 hydroxy substituent from LTB4, were identified as 2,4,6-conjugated triene-containing C-18 metabolites. The absence of a double bond allylic to the trimethylsiloxy ether in derivatized 10,11,14,15-tetrahydro LTB4 metabolites strikingly reduced the abundance of alpha-cleavage ions observed in the EI mass spectra of these compounds, thus suggesting the importance of formation of an allylic stabilized radical in such alpha-cleavage reactions. Lacking a favorable alpha-cleavage reaction, GC/EIMS analysis of 10-hydroxy-2,4,6-octadecatrienoic acid resulted in the formation of m/z 91, which may arise via cyclization of the conjugated triene moiety. In addition, GC/MS analysis of derivatized metabolites containing the 2,4,6 conjugated triene moiety resulted in a unique fragment ion in the electron capture ionization mass spectra that also may arise via cyclization of the conjugated triene with formation of m/z 121.

Animals↗
PubMed1995

Metabolism of 6-trans-isomers of leukotriene B4 in cultured hepatoma cells and in human polymorphonuclear leukocytes. Identification of a delta 6-reductase metabolic pathway.

The intermediate metabolic events which degrade hydroxy polyunsaturated fatty acids is largely unknown. Such molecules are common products of lipid peroxidation and lipoxygenase catalyzed oxidation of arachidonic acid. Metabolism of two 5,12-dihydroxyeicosatetraenoic acids, 6-trans-LTB4 (leukotriene B4), and 6-trans-12-epi-LTB4 was studied in HepG2 cells (a human-derived hepatoma cell line). Extensive metabolism was observed with a major metabolite identified as 4-hydroxy-6-dodecenoic acid for both epimers. Incubation of 6-trans-LTB4 epimers at shorter times revealed the formation of intermediate metabolites, including 6-hydroxy-4,8-tetradecadienoic acid and 8-hydroxy-4,6,10-hexadecatrienoic acid suggesting beta-oxidation as the major pathway leading to the formation of the common terminal metabolite. Two additional metabolites were structurally elucidated as 5-oxo-6,7-dihydro-LTB4 and 6,7-dihydro-LTB4 which have not been previously described. Formation of 5-oxo-6,7-dihydro-LTB4 and 6,7-dihydro-LTB4 were also observed during metabolism of 6-trans-12-epi-LTB4 in human polymorphonuclear leukocytes. Of particular interest is the metabolism of these compounds by beta-oxidation from the carboxyl terminus, a process which is not observed with leukotriene B4 or leukotriene C4. Identification of these metabolites suggested the operation of the 5-hydroxyeicosanoid dehydrogenase pathway followed by a delta 6-reductase metabolic pathway which has not been previously described. This pathway of beta-oxidation may limit the activity of various 5,12-diHETEs including nonenzymatic hydrolysis products of LTA4 and also the recently described B4-isoleukotrienes.

Carcinoma, Hepatocellular↗