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Chromosome 16p11.2 is one of the susceptible sites for recurrent copy number variations (CNVs) due to flanking near-identical segmental duplications. Five segmental duplications, named breakpoints 1 to 5 (BP1–BP5), have been defined as recombination hotspots within 16p11.2. Common CNVs on 16p11.2 include a proximal ~593 kb between BP4 and BP5, and a distal ~220 kb between BP2 and BP3. We performed a search for patients carrying 16p11.2 CNVs, as detected using chromosome microarray (CMA), in the Molecular Diagnostic Laboratory at the University of Texas Medical Branch (UTMB), in Galveston. From March 2013 through April 2018, a total of 1200 CMA results were generated for germline testing, and 14 patients tested positive for 16p11.2 CNVs, of whom 7 had proximal deletion, 2 had distal deletion, 4 had proximal duplication, and 1 had distal duplication. Herein, we provide detailed phenotype data for these patients. Our study results show that developmental delay, abnormal body weight, behavioral problems, and hypotonia are common phenotypes associated with 16p11.2 CNVs.

The Paleoproterozoic Ammassalik Intrusive Complex of south-east Greenland is located at the margin between the Rae Craton to the north and the North Atlantic Craton to the south. The complex is part of the Nagssugtoqidian Mobile Belt emplaced at mid- to lower-crustal level and represents an eastward continuation of the Nagssugtoqidian Orogen of western Greenland. We present new whole-rock geochemical and isotopic data (whole-rock Rb-Sr, Sm-Nd, U-Pb, Lu-Hf and zircon U-Pb, Lu-Hf, O) acquired from the Ammassalik Intrusive Complex, with the aim of reassessing its petrogenesis and evolution. The intrusive rocks and surrounding host-rocks show typical calc-alkaline signatures as well as negative Ta, Nb and Ti anomalies. The new U-Pb age data indicate a protracted magmatic history over ca. 40 Ma, involving at least three successive magmatic pulses between ca. 1910 and 1870 Ma emplaced into a granulite facies biotite-garnet paragneiss with ca. ≤ 1990 Ma protolith ages. Whole-rock trace element and isotope geochemistry indicate that the magmas that formed the Ammassalik Intrusive Complex were derived from a mildly enriched (eNd0 = −0.8 to –3.8; eSr0 = +24 to +42) and deep, garnet-bearing (MREE/HREE fractionated patterns) mantle source, possibly the sub-continental lithospheric mantle. However, the observed decreasing zircon eHf values with time (eHf0 = +2.8 to −12.5) indicate increasing degrees of crustal contamination involving variably 18O-enriched crustal material (δ18O = 7–9‰) into the mafic magmas of the complex. This interpretation suggests that the mantle-derived magma do not have a deep, garnet-bearing source, but that the continental arc signature in the Ammassalik Intrusive Complex igneous rocks may have been inherited from the assimilation of partial melts of the garnet-bearing host-rocks. In addition, based on the new crystallisation ages broadly coeval with the Columbia supercontinent build up, marked by the formation of world-class Ni-Cu deposits globally, it is proposed that the Ammassalik Intrusive Complex and the south-east Greenland region were in direct continuity of the mineralised Kotalahti belt (Svecofennian Province, Finland). Together with the recent discovery of Ni-Cu mineralisation in the Ammassalik Intrusive Complex, our study further supports the high prospectivity of south-east Greenland for orthomagmatic Ni-Cu deposits.

The Simorgh prospecting area is located in the central part of the Lut Block in eastern Iran. The Lut Block, the eastern part of the Central Iranian Microcontinent (CIM), has a complex tectonic evolution and is characterized by extensive magmatic activities with a range of geochemical signatures. The Simorgh intrusions have features typical of calc-alkaline to high-K calc-alkaline rocks, metaluminous to slightly peraluminous and formed in a volcanic arc setting. Mineralization in the area is believed to be related to these intrusions. To better understand the petrogenesis of these intrusions, we report zircon U-Pb dating and Hf isotopes and whole rock geochemistry of two granites and one diorite. Age dating using Zircon U-Pb method on pyroxene diorite porphyry stock and two granite porphyry dikes revealed 24.85 ± 0.51 Ma, 25.37 ± 0.56 Ma and 25.94 ± 0.76 Ma ages respectively (late Oligocene, Chattian). Field observations and U-Pb dating indicate that there are at least two stages of emplacement: pre-mineralization intrusions (diorite porphyry) and syn-mineralization intrusions (granite porphyry, granodiorite porphyry and pyroxene diorite porphyry). Mantle-normalized, trace-element spider diagrams display enrichment in large ion lithophile elements (LILE) and light rare earth elements ((La/Yb)N = 9.26–14.48), and depletion in high field strength elements (HFSE) and heavy rare earth elements, as well as negative Eu anomalies (Eu/Eu* = 0.49–0.91). Zircon Hf isotope data from the Simorgh intrusive rocks show largely positive eHf(t) (average = 3.6 ± 2.0). Hf model ages are ranging from 720 to 1320 Ma, indicating its derivation from a relatively juvenile source. The presence of zircon xenocrysts may prove its contamination by older continental crust (Siderian).

Stern, Robert/0000-0002-8083-4632; Santos, Jose Francisco/0000-0003-4997-8264; O'Reilly, Suzanne Y/0000-0002-3883-5498; Gain, Sarah/0000-0002-8459-0823; Murphy, Rosanna/0000-0003-3915-4341; Griffin, William L/0000-0002-0980-2566 WOS: 000429465900003 The Cadomian orogen of NW Iran includes a series of metamorphic rocks with zircon U-Pb ages between ca 562 and 505 Ma (Ediacaran to middle Cambrian). the Ediacaran-Cambrian basement is intruded by a series of Late Eocene-Late Oligocene I-type granitic rocks. U-Pb geochronology, integrated with geochemical and isotopic data for the basement rocks in NW Iran, provides further evidence of a Cadomian (562-505 Ma) arc-related magmatic event lasting similar to 60 Myr. Cadomian magmatism in Iran was a part of a similar to 100 Myr long episode of subduction-related arc magmatism at the northern margin of Gondwana. Zircon Hf-isotope compositions show that during Cadomian magmatic arc activity, juvenile arc magmas interacted with reworked Archean crust to generate the Ediacaran-Cambrian igneous rocks. Our results document both inheritance of old zircons and the presence of zircons with juvenile signatures in NW Iran, suggesting that the geotectonic setting for the Cadomian rocks was an Ediacaran continental magmatic arc and probably a neighboring back-arc basin. the occurrence of Ediacaran ophiolitic slices in NW Iran may provide evidence of back-arc basin opening at that time. Cenozoic plutonism in NW Iran is part of an Eocene-Oligocene magmatic 'flare-up' along the Urumieh-Dokhtar Magmatic Belt in central Iran, which lasted for ca 30 Myr. the melts responsible for the formation of these rocks had an essentially juvenile signature with minor contamination by Archean to Cadomian middle-lower continental crust. Continuous convergence between Arabia and Iran was accompanied by the transition of SW Eurasia from a compressional to an extensional convergent plate margin in Eocene-Oligocene times, leading to orogenic collapse, core-complex formation, exhumation of Cadomian crust and a major increase in arc magmatism. DEST Systemic Infrastructure GrantsAustralian GovernmentDepartment of Industry, Innovation and Science; ARC LIEFAustralian Research Council; NCRIS/AuScopeAustralian GovernmentDepartment of Industry, Innovation and Science; Macquarie University; Chinese Academy of SciencesChinese Academy of Sciences [XDB18000000, 2015VEC063]; FCT (Portugal)Portuguese Foundation for Science and Technology [UID/GEO/04035/2013] This is contribution 993 from the ARC Centre of Excellence for Core to Crust Fluid Systems (http://www.ccfs.mq.edu.au), 1168 from the GEMOC Key Centre (http://www.gemoc.mq.edu.au), and 1303 from UTD Geosciences. the LA-ICP-MS and zircon Hf-isotope data were obtained using instrumentation funded by DEST Systemic Infrastructure Grants, ARC LIEF, NCRIS/AuScope, industry partners and Macquarie University. SIMS U-Pb zircon dating was funded by the Chinese Academy of Sciences (grants XDB18000000 and 2015VEC063). Sr and Nd isotopic analyses were done in the scope of project Geobiotec (UID/GEO/04035/2013), funded by FCT (Portugal).

The Bruce Rise, a prominent bathymetric feature offshore the Bunger Hills (East Antarctica), has basement of unknown crustal affinity and age. In East Gondwana, the Bruce Rise is reconstructed near the Naturaliste Plateau (offshore SW Australia) and microcontinents now submerged in the eastern Indian Ocean. New zircon U-Pb-Hf data from two c. 1150 Ma granite samples dredged from the eastern escarpment of the Bruce Rise demonstrate that these rocks are dominated by xenocrystic cargo. Mesoproterozoic xenocrystic cores show textural evidence of melt-mediated coupled dissolution-precipitation to form rim domains with apparent ages that skew towards c. 1150 Ma. The zircon U-Pb-Hf signatures from the xenocrysts in the Bruce Rise granites, and from c. 1230 to 1180 Ma felsic-intermediate granites and orthogneisses from the conjugate Naturaliste Plateau basement, suggest late Mesoproterozoic magmatism occurred at a transition in the regional tectonic architecture between a reworked Archean cratonic margin and Proterozoic juvenile crust. On the basis of plate reconstructions, exhumation and thinning of the basement to the Bruce Rise/Naturaliste Plateau occurred predominantly during rifting of India (prior to c. 120 Ma). Minor further thinning likely occurred leading up to the onset of seafloor spreading between Australia/Naturaliste Plateau and Antarctica/Bruce Rise (from c. 90 to 84 Ma).

Time-constrained isotopic datasets permit the evaluation of tectonic processes, including continental collision, rifting, and the origins of terrane fragments. The Rudall Province, in the Paterson Orogen, is part of the West Australian Craton (WAC) and now lies to the east of the Archaean Pilbara Craton. Components within the Rudall Province have previously been linked to the Arunta Orogen of the North Australian Craton (NAC) based on similarities in timing of magmatism, deformation, and metamorphism and hence have been referred to as suspect terranes, with respect to the WAC. The Rudall Province is divided into three lithotectonic elements known as the Talbot, Connaughton, and Tabletop Terranes. The southern two terranes (Talbot and Connaughton) were affected by magmatism related to collision between the West and North Australian Cratons, during the 1800–1765 Ma Yapungku Orogeny. Zircon crystals in both Talbot and Connaughton terranes have a Hf isotopic and, in the case of inheritance, U–Pb age affinity to detritus that originated from the Capricorn Orogen basement in the WAC. Furthermore, the Hf isotopic composition of c. 1800 Ma magmatic zircons within the Rudall Province has similarity to components within the c. 1800 Ma Bridget Suite of the East Pilbara Terrane, which has an indubitable association to the Pilbara Craton. Hence, sources for all isotopic compositions preserved within the Rudall Province can be found within the proximal WAC. There is no necessity to invoke transfer of exotic NAC lithotectonic elements to the West Australian Craton margin and to suggest an accretionary style of orogenesis for the Rudall Province. The Tabletop Terrane has been regarded as a different far-travelled block with crust distinct from the other components of the Rudall Province. However, the currently available dataset implies that the Tabletop Terrane was derived from crust of similar composition to the Connaughton and Talbot terranes. A distinctive phase of crust formation at 1900 Ma is indicated by zircons, with mantle-like oxygen isotope ratios, within a c. 1450 Ma monzogranite of the Talbot Terrane. This timing of crust formation implies an affinity to a major deep lithospheric source of similar age recognized in the Musgrave Province and Edmund Basin. These data indicate that the major suture between the North and West Australian Cratons lies to the east of the Rudall Province.

The Hf and Nd isotopic evolution of the Musgrave Province, central Australia, is used to constrain the timing of crust formation and lithospheric organisation of Proterozoic Australia. The dataset from this region challenges two widely held tenets of Hf and Nd isotope systematics, namely; that crust formation events can only be identified as periods when crystallisation ages correspond to model ages, and that linear Hf evolution arrays away from depleted mantle (along crustal Lu/Hf or Sm/Nd slopes) reflect reworking of the same source. Hf isotopes in Musgrave Province zircon crystals indicate two major crust formation events at c. 1900 Ma and at 1600–1550 Ma. Although no juvenile rocks or crystals are known from c. 1900 Ma, radiogenic addition into the crust at this time is required to account for consistent Nd and Hf evolution patterns, which show no indication of an initially heterogeneous source. Oxygen isotopes in zircon grains confirm that much of the c. 1900 Ma Hf isotopic signal is not compromised by mixtures. Furthermore, the correspondence between mantle extraction and the commencement of reworking of Archean material supports new crust generation at c. 1900 Ma and a coupling between lower and upper crustal processes. The c. 1900 Ma timing of juvenile addition is dissimilar to that in the Albany–Fraser and Arunta Orogens and may reflect continental arc development on the margin of a southern continent. The general Hf isotopic evolution trend of the Musgrave Province apparently reflects reworking from a dominant c. 1900 Ma source with some additional unradiogenic and radiogenic input through time. However, in the 1220–1050 Ma interval this apparent isotopic evolution contrasts with geological observations that indicate input of voluminous mantle-derived material. Intracontinental rifts and other regions with sustained very-high temperature crustal recycling processes generate magmatic provinces with extreme HFSE-enrichment. This can have a profound influence on isotopic evolution trends, suppressing typical juvenile addition patterns. Isotopic mixture modelling indicates that a significant volume of mantle derived material can be accommodated within HFSE enriched magmas without diverging isotopic signatures from apparent reworking trends. In the Musgrave Province, the crust had become so HFSE enriched during the prolonged Musgrave Orogeny (1220–1150 Ma) that it was insensitive to mantle input, which is estimated to have been as much as 85% during this event.

Unmitigated oxidative stress is deleterious, as epitomized by CCl4 intoxication. In this well-characterized model of free radical-initiated damage, liver metabolism of CCl4 to CCl3. causes lipid peroxidation, F-ring isoprostane formation, and pathologic leukocyte activation. The nature of the mediator that couples oxidation to the hepatotoxic inflammatory response is uncharacterized. We found that oxidatively modified phosphatidylcholines were present in the livers of CCl4-exposed rats and not in livers from control animals, that CCl4 metabolism generated lipids that activated 293 cells stably transfected with the human platelet-activating factor (PAF) receptor, and that this PAF-like activity was formed as rapidly as isoprostane-containing phosphatidylcholine (iPC) during oxidation. iPC and the PAF-like activity also had similar chromatographic properties. The potential for iPC activation of the PAF receptor has been unexplored, but we conclude that iPC themselves did not activate the PAF receptor, as phospholipase A1 hydrolysis completely destroyed iPC, but none of the PAF-like bioactivity. Oxidatively fragmented phospholipids are potent agonists of the PAF receptor, but mass spectrometry characterized PAF as the major inflammatory component coeluting with iPC. Oxidatively fragmented phospholipids and iPC are markers of free radical generation in CCl4-intoxicated liver, but PAF generation by activated hepatic cells generated the inflammatory agent. —Marathe, G. K., K. A. Harrison, L. J. Roberts II, J. D. Morrow, R. C. Murphy, L. W. Tjoelker, S. M. Prescott, G. A. Zimmerman, and T. M. McIntyre. Identification of platelet-activating factor as the inflammatory lipid mediator in CCl4-metabolizing rat liver. J. Lipid Res. 2001. 42: 587–596.

Fast atom bombardment mass spectrometry of choline phospholipids produces negative ions characteristic of the intact molecule and tandem mass spectrometry of collision-induced decomposition of M-15 anions characterizes both the identity and substituent position of radyl groups. Certain choline phospholipid molecular species which may be of special interest in the generation of platelet activating factor contain a highly unsaturated fatty acyl substituent at sn-2 and an ether radyl group at sn-1; other choline phospholipid molecular species which contain esterified arachidonic acid are of interest as potential sources of arachidonate for eicosanoid biosynthesis. Collisional activated decomposition of 1-hexadecanoyl-2-arachidonoyl-sn-glycero-phosphocholine produce abundant carboxylate anions at m/z 303 (arachidonate) and m/z 255 (hexadecanoate) in a ratio of 3:1, diagnostic for the sn-2 arachidonoyl position. The ether analog, 1-O-hexadecyl-2-arachidonoyl glycerophosphocholine, produces only one collision-induced dissociation ion at m/z 303 and no product ions corresponding to the ether substituent at sn-1. Molecular weight information from the M-15 ion combined with the CID generated carboxylate anions completely characterize these important phospholipids. Precursor ion studies of M-15 anions from glycero-phosphocholine lipids indicate that this ion is derived directly from a unique adduct ion formed by attachment of the molecular species to a matrix alkoxide ion, neutralizing the positive charge of the quaternary choline nitrogen. Decomposition of this adduct ion yields a methylated matrix molecule and the nominal M-15 ion.

Radiolabeled leukotriene (LT) B4 was incubated with isolated rat hepatocytes in order to assess the metabolism of this chemotactic leukotriene by the liver. At least eight radioactive metabolites were observed, three of which were previously identified as 20-hydroxy-, 20-carboxy-, and 18-carboxy-19,20-dinor-LTB4. A less lipophilic major metabolite (designated HIV) was purified by two reverse phase high performance liquid chromatography separations and was found to exhibit maximal UV absorbance at 269 nm with shoulders at 260 and 280 indicating the presence of a conjugated triene chromophore. Negative ion electron capture gas chromatography/mass spectrometry analysis of the pentafluorobenzyl ester, trimethylsilyl ether derivative of HIV, and positive ion electron ionization mass spectra of the methyl ester trimethylsilyl derivative were consistent with a structure of this metabolite being 16-carboxy-14,15-dihydro-17,18,19,20-tetranor-LTB3. The appearance of this metabolite supports the concept of further beta-oxidation of LTB4 to the carbon 16 which requires the action of 2,4-dienoyl-CoA reductase to remove the 14,15-double bond located two carbon atoms removed from the CoA thioester moiety. One minor metabolite was analyzed by negative ion continuous flow fast atom bombardment mass spectrometry which revealed an ion at m/z 444 which by high resolution mass spectrometry was shown to contain both nitrogen and sulfur. Tandem mass spectrometry suggested the presence of SO3- as well as other fragments corresponding to the amino acid taurine. Incubation of isolated rat hepatocytes with [14C]taurine as well as [3H]LTB4 revealed the incorporation of both radioactive isotopes into this metabolite. The data supported the identification of this metabolite as tauro-18-carboxy-19,20-dinor-LTB4. Amino acid conjugation of leukotrienes has not been previously reported and suggests that such intermediates might participate in enterohepatic circulation of LTB4 metabolites in the intact animal and thus serve as an alternative metabolic route for LTB4 elimination.

Leukotriene B4 (LTB4) is a potent mediator of inflammation that recruits granulocytes to the site of injury during the inflammatory response. The biological activity of LTB4 is terminated by its metabolism into inactive metabolites. Recent studies have suggested that LTB4 may have additional activity as an endogenous ligand for the transcription factor peroxisome proliferator-activated receptor alpha (PPARalpha). Based on the data presented, a model was proposed in which LTB4 acts in a negative feedback manner by inducing the transcription of genes involved its own metabolism. In the present study the effect of PPARalpha activation on LTB4 metabolism was directly investigated. Primary cultures of rat hepatocytes were treated with LTB4 or the PPARalpha agonist WY-14,643, and LTB4 metabolism was assessed by measuring levels of LTB4 and the formation of LTB4 metabolites. In addition, the effect of PPARalpha activation on levels of acyl-CoA oxidase mRNA and expression of CYP4F proteins, which are specific omega-hydroxylases for LTB4, was determined. Treatment of hepatocytes with WY-14,643, but not LTB4, was found to increase acyl-CoA oxidase mRNA and enhance expression of rat hepatic CYP4F proteins and CYP4A1. Neither WY-14,643 nor LTB4 caused an increase of the basal levels of LTB4 metabolism, and no novel metabolites were observed. These results do not support the hypothesis that a pathway of negative feedback regulation of LTB4 metabolism involving PPARalpha exists in hepatocytes, because activation of PPARalpha by LTB4 or other PPARalpha agonists did not correlate with an increase in LTB4 metabolism.

Unmitigated oxidative stress is deleterious, as epitomized by CCl4 intoxication. In this well-characterized model of free radical-initiated damage, liver metabolism of CCl4 to CCl3. causes lipid peroxidation, F-ring isoprostane formation, and pathologic leukocyte activation. The nature of the mediator that couples oxidation to the hepatotoxic inflammatory response is uncharacterized. We found that oxidatively modified phosphatidylcholines were present in the livers of CCl4-exposed rats and not in livers from control animals, that CCl4 metabolism generated lipids that activated 293 cells stably transfected with the human platelet-activating factor (PAF) receptor, and that this PAF-like activity was formed as rapidly as isoprostane-containing phosphatidylcholine (iPC) during oxidation. iPC and the PAF-like activity also had similar chromatographic properties. The potential for iPC activation of the PAF receptor has been unexplored, but we conclude that iPC themselves did not activate the PAF receptor, as phospholipase A1 hydrolysis completely destroyed iPC, but none of the PAF-like bioactivity. Oxidatively fragmented phospholipids are potent agonists of the PAF receptor, but mass spectrometry characterized PAF as the major inflammatory component coeluting with iPC. Oxidatively fragmented phospholipids and iPC are markers of free radical generation in CCl4-intoxicated liver, but PAF generation by activated hepatic cells generated the inflammatory agent.

The metabolism of arachidonic acid by the 5-lipoxygenase pathway not only leads to the formation of leukotrienes but also to the biologically active eicosanoid 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE). The synthesis of 5-oxo-ETE was investigated in the elicited peritoneal macrophage and the formation of 5-hydroxyeicosatetraenoic acid (5-HETE) as well as 5-oxo-ETE was quantitated using stable isotope dilution tandem mass spectrometry. The metabolism of 5-oxo-ETE in these same cells led to the formation of a series of novel less lipophilic metabolites oxidized near the methyl terminus that were structurally characterized using electrospray LC/MS and LC/MS/MS. Five novel metabolites of 5-oxo-ETE were identified including 5,18-diHETE, 5,19-diHETE, 5-oxo-19-HETrE, 5-oxo-18-HETrE, and 5,19-diHETrE. These metabolites corresponded to omega-1 and omega-2 oxidation of 5-oxo-ETE presumably formed by a specific cytochrome P450. There was no evidence for the formation of omega-oxidation (20-hydroxy metabolites), which are known products of metabolism of 5-oxo-ETE in other cell types. None of the metabolites were found to elevate intracellular calcium release, suggesting that this metabolic pathway may result in inactivation of 5-oxo-ETE. This is the first report of the biosynthesis of 5-oxo-ETE by tissue resident cell outside of the blood and the formation of novel omega-1 and omega-2 oxidation of this eicosanoid.

Lysoglycerophosphocholine lipids (lyso-GPC) are important intermediates in the synthesis and metabolism of glycerophosphocholine lipids which are major components of the cellular lipid bilayer. Significant differences in the collisional induced decomposition (CID) behavior were observed for each of the four different subtypes of lyso-GPC in both positive and negative ions. A major difference was observed in the initial CID product ions derived from lyso-GPC [M + H]+ with the loss of water that was very abundant for acyl lyso-GPC which have a fatty acid ester substituent at either the sn-1 or sn-2 positions. Loss of neutral water was not very prominent in the case of plasmenyl and plasmanyl lyso-GPC species. The mechanism responsible for this difference in behavior of lyso-GPC subtypes was consistent with a higher proton affinity of carboxyl carbonyl oxygen atoms and vinyl ether oxygen atoms found in acyl and plasmenyl lyso-GPC lipids, respectively, as compared to the carbinol oxygen atom common to all lyso-GPC species. Collisional activation of lyso-GPC negative ions [M - 15]- also revealed distinctive differences in product ions derived from acyl and ether lyso-GPC species. The acyl compounds showed the facile elimination of a highly stable carboxylate anion, whereas plasmenyl species underwent fragmentation with loss of a neutral aldehyde, likely a result of rearrangement involving the double bond in the vinyl ether moiety. The alkyl ether species (plasmanyl lyso-GPC lipids) did not undergo either decomposition reaction observed for the other lyso-GPC subtypes which permitted differentiation of acyl, plasmenyl, and plasmanyl lyso-GPC subtypes.

Plasmenyl phospholipids are a structurally unique class of lipids that contain a vinyl ether substituent at the sn-1 position of the glycerol backbone, imparting unique susceptibility to oxidative reactions that may take place at the cell membrane lipid bilayer. Several studies have supported the hypothesis that plasmalogens may be antioxidant molecules that protect cells from oxidative stress. Because the molecular mechanism for the antioxidant properties of plasmenyl phospholipids is not fully understood, the oxidation of arachidonate-containing plasmalogen-glycerophosphocholine (GPC) was studied using electrospray tandem mass spectrometry after exposure to the free radical initiator 2, 2'-azobis(2-amidinopropane)hydrochloride (AAPH). Various oxidized GPC products involving the sn-1 position alone (1-formyl-2-arachidonyl lipids and lysophospholipid), oxidation products involving the sn-2 position alone (chain-shortened omega-aldehyde radyl substituents at sn-2) as well as products oxidized both at the sn-1 and sn-2 positions were observed and structurally identified. The results of these experiments suggest that oxidation of plasmenyl phospholipids esterified with polyunsaturated fatty acid groups at sn-2 likely undergo unique and specific free radical oxidation at the 1'-alkenyl position as well as oxidation of the double bond closest to the ester moiety at sn-2.

Oxidized low-density lipoprotein (LDL) is thought to play an important role in atherogenesis and cardiovascular disease in humans. Oxidized LDL is a complex mixture of many oxidized species, including numerous oxidized glycerophospholipids. Electrospray ionization and tandem mass spectrometry as well as microchemical derivatization of high-performance liquid chromatographically purified fractions derived from oxidized LDL were investigated as means to determine the structure of individual components present in oxidized LDL. One major oxidized phosphocholine lipid had an [M + H](+) ion at m/z 650. Derivatization to the trimethylsilyl ether and methoxime caused shifts in mass which, along with negative ion collision-induced dissociation mass spectra, were consistent with the presence of three species, 1-palmitoyl-2-(9-oxononanoyl)glycerophosphocholine and two isomeric 1-octadecanoyl-2-(hydroxyheptenoyl)glycerophosphocholines. These species were chemically synthesized. Trimethylsilylation of free hydroxyl groups increased the mass of the phospholipid acyl chains containing hydroxyl groups by 72 u. Conversion of carbonyl groups to the methoxylamine derivative increased the mass by 29 u. Ozonolysis of those products which contained double bonds proved to be a facile technique to determine the position and number of double bonds present. The use of these techniques was illustrated in the structural characterization of one major component (m/z 650, positive ions) in oxidized LDL as 1-octadecanoyl-2-(7-hydroxyhepta-5-enoyl)glycerophosphocholi ne. A possible mechanism for the formation of this unique chain-shortened glycerophospholipid is proposed.

Leukotriene B4 (LTB4) is a potent lipid mediator of the inflammatory response whose biological half-life is believed to be mediated principally by metabolism to inactive forms either in the tissue of origin or in the liver. Pathways of metabolic degradation of LTB4 along with structural identification of metabolites have been elucidated previously in isolated rat liver cells, human keratinocytes, human polymorphonuclear leukocytes, and cultured HepG2 cells. Research advances in human liver transplantation and preservation have made isolated human hepatocytes available for studying the metabolism of LTB4 in vitro. LTB4 was added to plated human hepatocytes from three different subjects for 24-h periods whereupon the substrate was analyzed by high-performance liquid chromatography coupled with scintillation counting, UV spectroscopy, and negative ion electrospray ionization tandem mass spectrometry. Each set of hepatocytes yielded a different distribution of metabolites, but several metabolites appeared in all three sets of cells. These central metabolites included the previously identified 20-carboxy-LTB4 and 18-carboxy-LTB4, implicating the presence in the liver of specific P-450-mediated omega-oxidation as well as the enzymes involved in beta-oxidation from the omega-terminus. Each set of hepatocytes produced the metabolite 10,11-dihydro-20-COOH-LTB4, a product of the 12-hydroxyeicosanoid dehydrogenase/Delta10 reductase pathway. Glucuronides of LTB4 and several metabolites were found, which represents the first description of glucuronidation as a pathway of LTB4 metabolism. Finally, a series of novel metabolites were observed corresponding to beta-oxidation from the carboxyl terminus of LTB4.

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.

Recent studies suggest that the lipid mediator platelet-activating factor (PAF) is involved in keratinocyte function and skin inflammation. Indeed, PAF is found in association with inflammatory skin diseases, intradermal injections of PAF induce inflammation, and keratinocytes express functional PAF receptors (PAF-R). One mechanism by which the keratinocyte PAF-R could contribute to epidermal functions and inflammatory states would be through the synthesis of inflammatory regulators, such as PAF, PGs, and cytokines. The ability of the epidermal PAF-R to induce the synthesis of these immunomodulators was tested using a model system created by transduction of the PAF-R-negative human epidermal cell line KB with the PAF-R. Activation of this epidermal PAF-R resulted in arachidonic acid release, and the biosynthesis of PAF and PGE2. In addition, the KB PAF-R triggered increased levels of mRNA and protein for the inducible isozyme of cyclooxygenase (COX-2) as well as IL-6 and IL-8, both of which have been implicated in skin inflammatory processes. Studies with the human keratinocyte-derived epidermal cell line HaCaT revealed that activation of the endogenous PAF-R led to the increased accumulation of COX-2, IL-6, and IL-8 mRNA similar to that seen with the KB PAF-R model system. Finally, treatment of HaCaT keratinocytes with IL-8 resulted in PAF biosynthesis, indicating the existence of a positive feedback loop between IL-8 and PAF in epidermal cells. These studies suggest involvement of PAF and the PAF-R in the epidermal cytokine network.

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.

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.

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.

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.

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.

Six previously unidentified leukotriene (LT) B4 metabolites formed during incubation of LTB4 with human keratinocytes in primary culture indicate the importance of the 12-hydroxyeicosanoid dehydrogenase pathway in LTB4 metabolism. The ultraviolet absorption spectra obtained for all keratinocyte metabolites revealed the presence of a conjugated diene structural moiety rather than the conjugated triene structure of LTB4. Metabolites were characterized using fast atom bombardment-mass spectrometry, gas chromatography-mass spectrometry of the pentafluorobenzyl ester, trimethylsilyl ether derivatives and specific degradation reactions. The previously identified 10,11-dihydro-LTB4 and 10,11-dihydro-12-epi-LTB4 were observed as well as 20-OH-10,11-dihydro-LTB4, consistent with the reductase pathway of LTB4 metabolism. This pathway involves initial formation of 12-oxo-LTB4 catalyzed by 12-hydroxyeicosanoid dehydrogenase followed by reduction by delta 10-reductase. The most lipophilic metabolite of LTB4 was identified as 10-hydroxy-4,6,12-octadecatrienoic acid which could result from beta-oxidation reactions of LTB4 following reduction of the 10,11-double bond. One of the most abundant metabolites was characterized as 3,7,14-trihydroxy-8,10,16- docosatrienoic acid which could result from fatty acid elongation following reduction of the 10,11-double bond. Additional abundant LTB4 metabolites were identified that result from glutathione conjugation of 12-oxo-LTB4. These were characterized using fast atom bombardment-mass spectrometry and by chemical degradation using hypochlorous acid as well as transpeptidases. These metabolites were identified as 5,12-dihydroxy-6-glutathionyl-7,9,14-eicosatrienoic acid (c-LTB3), 5,12-dihydroxy-6-cysteinyl-glycyl-7,9,14-eicosatrienoic acid (d-LTB3) and 5,12-dihydroxy-6-cysteinyl-7,9,14-eicosatrienoic acid (e-LTB3). We propose that these metabolites result from a 1,8 Michael-type addition of glutathione to the 12-oxo-LTB4 intermediate.

Arachidonic acid is oxidatively metabolized into numerous, structurally diverse molecules following cell activation. Considerable work has focused on two pathways of arachidonic acid metabolism, the cyclooxygenase pathway which leads to the formation of prostaglandins and thromboxanes (1) and the 5-lipoxygenase pathway which leads to the formation of leukotrienes (2). However, a third pathway exists for oxidative metabolism of arachidonic acid, that being the cytochrome P450 pathway which also leads to the formation of biologically active molecules (3). Much of our understanding of the formation of such eicosanoids has resulted from studies of arachidonic acid metabolism in purified cell populations. More recently, it has become clear that cells cooperate in the biochemical processing of chemically reactive intermediates synthesized from arachidonate by the cyclooxygenase and 5-lipoxygenase pathway in the ultimate formation of the biologically active compound. A particular example is the transcellular biosynthesis of LTC4 as a result of neutrophil-platelet interactions (4). There has been little evidence to suggest involvement of the cytochrome P450 pathway of arachidonic acid metabolism in such transcellular biosynthetic events.

Ionophore activation of the human polymorphonuclear neutrophil results in eicosanoid synthesis and the accumulation of inactive 5-lipoxygenase in a membrane compartment. We report here that inhibition of self-inactivation of 5-lipoxygenase in ionophore-treated neutrophils with the reversible inhibitor zileuton, results in the accumulation of active 5-lipoxygenase in the membrane fraction. In zileuton plus ionophore-treated cells, 77% of the specific activity of the cytosolic enzyme from resting cells was diverted to the membrane fraction compared to 22% of the activity translocated when ionophore alone was used to activate the neutrophils. Accumulation of active membrane-associated 5-lipoxygenase was inhibited and reversed by the 5-lipoxygenase translocation inhibitor MK-886. The membrane-associated 5-lipoxygenase was two times more efficient in the production of leukotriene A4 from arachidonate-derived 5-hydroperoxyeicosatetraenoic acid than the cytosolic enzyme. Unlike the cytosolic enzyme, membrane-associated 5-lipoxygenase could metabolize 12(S)- and 15(S)-hydroxyeicosatetraenoic acid to 5(S),12(S)- and 5(S),15(S)-dihydroxyeicosatetraenoic acid, respectively. The ability to metabolize hydroxy fatty acids was dependent upon 5-lipoxygenase-activating protein association, but was lost if 5-lipoxygenase was eluted from the membrane by MK-886. These studies reveal for the first time that significant quantities of active 5-lipoxygenase can be detected in the membrane fraction of activated neutrophils and show that membrane association can alter the substrate specificity of 5-lipoxygenase which is further evidence for the role of the membrane-associated enzyme in the synthesis of 5-lipoxygenase metabolites.

A mosquitocidal cyanobacterium has been developed by introducing the mosquito-toxic cryIVD gene from Bacillus thuringiensis subsp. israelensis into the unicellular cyanobacterium Agmenellum quadruplicatum PR-6 (Synechococcus sp. strain PCC 7002). The cryIVD gene was introduced into the cyanobacterium on a derivative of the PR-6 expression vector pAQE19 delta Sal in which the cryIVD gene was translationally fused to the initial coding sequence of the highly expressed PR-6 cpcB gene. Coomassie blue staining and immunoblot analysis of gel-fractionated cell extract polypeptides indicate that the cpcB-cryIVD gene fusion is expressed at high levels in the cyanobacterial cells, with little or no apparent degradation of the cryIVD gene product. Larvicidal assays revealed that freshly hatched Culex pipiens mosquito larvae readily ingested the transformed cyanobacteria and that the cells proved to be toxic to the larvae.

The different regioisomers of epoxyeicosatrienoic acids derived from cytochrome P-450 monooxygenase are readily esterified into phospholipids of mastocytoma cells. Incorporation of 14,15-epoxyeicosatrienoic acid was concentration-dependent, with Km = 1.1 microM and Vmax = 36 pmol/min/10(7) cells. Half-maximal incorporation occurred in 30 min, reaching a steady-state concentration of 470 pmol/10(6) cells. This was slightly lower than the values for arachidonic acid (665 pmol/10(6) cells) or 5-hydroxyeicosatetraenoic acid (554 pmol/10(6) cells). The distribution of 14,15-epoxyeicosatrienoic acid was preferential in the order phosphatidylethanolamine greater than phosphatidylcholine greater than phosphatidylinositol greater than phosphatidyl serine much greater than neutral lipids plus fatty acids. This contrasted with 5(S)-hydroxyeicosatetraenoic acid, which was distributed primarily into phosphatidylcholine. Fast atom bombardment/tandem mass spectrometry facilitated identification of molecular species containing epoxyeicosatrienoic acids without relying on radioisotopes. Phosphatidylethanolamine plasmalogens with 16:1 or 18:2 at the sn-1 position, or an 18:0 acyl group, and phosphatidylcholine with 16:0 alkyl ether or an acyl group at the sn-1 position incorporated all possible epoxyeicosatrienoic acid regioisomers. Under basal conditions, cells eliminated 14,15-cis-epoxyeicosatrienoic acid slowly with a half-life of 34.9 +/- 7 h. Cells stimulated with calcium ionophore A23187 eliminated 14,15-epoxyeicosatrienoic acid rapidly. It was notable that its rate of release from phosphatidylcholine and phosphatidylinositol exceeded that for arachidonic acid. A coenzyme A-independent transacylase also catalyzed the transfer of epoxyeicosatrienoic acids from mastocytoma cell membranes into 1-palmitoyl-2-lysophosphatidylcholine. The cellular incorporation, release, and distribution of epoxyeicosatrienoic acids is distinctive and contrasts with most other eicosanoids, suggesting that these compounds may have both autocoid and nonautocoid functions.

We measured the excretion of a major urinary metabolite of leukotriene (LT) C4, i.e., LTE4, during the infusion of exogenous LTC4 to enable estimation of the rate of entry of endogenous LTC4 into the bloodstream. Four healthy volunteers received 2-h intravenous infusions of vehicle alone and LTC4 at 0.063, 0.32, 1.6, and 2.9 pmol.kg-1.min-1 in random order. Urinary LTE4 was measured before, during, and up to 24 h after the infusions. The fractional elimination of LTE4 was independent of the rate of LTC4 infusion and averaged 4.3 +/- 0.9%. Calculation of the mean rate of entry of LTC4 into the circulation was found to be 0.06 pmol.kg-1.min-1. In addition, we characterized further metabolism of [14C]LTC4. The two major urinary metabolites were the omega- and beta-oxidation products (16-COOH-LTE4 and 14-COOH-LTE3), which accounted for 6-8% of the total infused amount of [14C]LTC4. We conclude that 1) LTC4 is produced at a low rate under physiological circumstances and is rapidly converted in the vasculature to LTE4, 2) changes in the urinary excretion of the latter may reliably reflect short-term changes in the rate of secretion of LTC4, and 3) measurement of the omega- and beta-oxidation products may reflect chronic changes in cysteinyl leukotriene biosynthesis.

The enzymatic steps involved in the synthesis of prostaglandins, thromboxanes, and leukotrienes have been elucidated in substantial detail in numerous biochemical studies (1,2). In most cases, these studies involved the addition of radiolabeled arachidonic acid to homogenous cell suspensions with characterization of the resultant radiolabeled metabolites. Thus, most of the studies during the 1960s and 1970s were designed to establish those oxidative pathways of arachidonic acid within a single cell type. As a result, the metabolic patterns of eicosanoids generated by a given cell type has been fairly well established, for example, those cells which are found in the blood have been studied in great detail and their primary cyclooxygenase and lipoxygenase metabolites are known (Table 1). While most cells in the blood do metabolize arachidonic acid by one or more cascades, there are some cells which do not participate in the oxidative conversion of arachidonic acid into reactive intermediates. Table 1 indicates that the red blood cell and the lymphocyte (3) cannot metabolize arachidonic acid by either the cyclooxygenase or lipoxygenase pathways. Normally, one considers most cells in the blood to have a Table 1 Arachidonic Acid Metabolites Generated by Stimulation of Isolated Blood Cells Cells Eicosanoid Oxidase Intermediate Secondary Enzyme Platelet TxB2 C.O. PGH2 Thromboxane Synthase HHT C.O. PGH2 Thromboxane Synthase 12-HETE 12-LO 12-HPETE --- Polymorphonuclear Leukocyte LTB4 5-LO LTA4 LTA4-Hydrolase Eosinophil LTC4 5-LO LTA4 LTC4-Synthase 5-HETE 15-LO 15-HPETE --- Basophil LTC4 5-LO LTA4 LTC4-Synthase Monocyte Macrophage TxA2 C.O. PGH2 Thromboxane Synthase LTB4 5-LO LTA4 LTA4-Hydrolase LTC4 5-LO LTA4 LTC4-Synthase Lymphocyte None None --- --- RBC None None --- --- substantial capacity for the production of active eicosanoids. For example, platelets are known to produce thromboxane A2, a pro-aggregatory substance in response to stimulation and that this eicosanoid plays an important role in thrombus formation (4). The neutrophil on the other hand appears only to have a 5-lipoxygenase pathway for metabolism of arachidonic acid and stimulation of this cell leads to the formation of LTB4, a chemotactic factor (5). The eosinophil and basophil also metabolize arachidonate by the 5-lipoxygenase pathway and convert the reactive LTA4 into the sulfidopeptide leukotriene LTC4, the potent smooth muscle contracting agent and substance which enhances capillary permeability (6). The studies in this manner have greatly expanded our understanding of the complexity of arachidonic acid metabolism and the role which the eicosanoid metabolites may play in health and disease. However, it appears that the synthesis of eicosanoids may be more complex than only occurring within one cell and may involve cell-cell interactions.

Radiolabeled leukotriene (LT) B4 was incubated with isolated rat hepatocytes in order to assess the metabolism of this chemotactic leukotriene by the liver. At least eight radioactive metabolites were observed, three of which were previously identified as 20-hydroxy-, 20-carboxy-, and 18-carboxy-19,20-dinor-LTB4. A less lipophilic major metabolite (designated HIV) was purified by two reverse phase high performance liquid chromatography separations and was found to exhibit maximal UV absorbance at 269 nm with shoulders at 260 and 280 indicating the presence of a conjugated triene chromophore. Negative ion electron capture gas chromatography/mass spectrometry analysis of the pentafluorobenzyl ester, trimethylsilyl ether derivative of HIV, and positive ion electron ionization mass spectra of the methyl ester trimethylsilyl derivative were consistent with a structure of this metabolite being 16-carboxy-14,15-dihydro-17,18,19,20-tetranor-LTB3. The appearance of this metabolite supports the concept of further beta-oxidation of LTB4 to the carbon 16 which requires the action of 2,4-dienoyl-CoA reductase to remove the 14,15-double bond located two carbon atoms removed from the CoA thioester moiety. One minor metabolite was analyzed by negative ion continuous flow fast atom bombardment mass spectrometry which revealed an ion at m/z 444 which by high resolution mass spectrometry was shown to contain both nitrogen and sulfur. Tandem mass spectrometry suggested the presence of SO3- as well as other fragments corresponding to the amino acid taurine. Incubation of isolated rat hepatocytes with [14C]taurine as well as [3H]LTB4 revealed the incorporation of both radioactive isotopes into this metabolite. The data supported the identification of this metabolite as tauro-18-carboxy-19,20-dinor-LTB4. Amino acid conjugation of leukotrienes has not been previously reported and suggests that such intermediates might participate in enterohepatic circulation of LTB4 metabolites in the intact animal and thus serve as an alternative metabolic route for LTB4 elimination.