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To evaluate the endocannabinoid system in an animal model of Parkinson's disease, in-tube solid-phase microextraction (in-tube SPME) was directly coupled to a tandem mass spectrometry (MS/MS) system for determination of the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in rat brain samples. In-tube SPME-which consisted of a microtube of restricted access material (RAM) with a hydrophilic diol external surface and a hydrophobic octyl inner surface-efficiently excluded (up to 95%) macromolecules from the biological samples and selectively pre-concentrated the analytes. In-tube SPME parameters, such as sample volume, mobile phases, flow rate, and pre-concentration time, were evaluated to improve the extraction efficiency and throughput performance. The selectivity of the in-tube SPME and MS/MS (MRM mode) techniques allowed them to be directly coupled online, which dismissed the need for the chromatographic separation step. The in-tube SPME-MS/MS method was validated and shown to be linear from 6.0 to 30.0 ng mL -1 for AEA and from 10.0 to 100.0 ng mL -1 for 2-AG; the intra- and inter-assay accuracy and precision were lower than 15%. Parallelism between the calibration curves constructed in the matrix and aqueous solution confirmed that there was no matrix effect. The method allowed endogenous concentrations of AEA and 2-AG to be determined in rat brain striatum from unilaterally 6-hydroxydopamine-lesioned animals. The concentrations of these endocannabinoids in striatum ipsilateral and contralateral to the lesion differed significantly (p<0.001)., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

We investigated the synthesis of N- docosahexaenoylethanolamine (synaptamide) in neuronal cells from unesterified docosahexaenoic acid (DHA) or DHA-lysophosphatidylcholine (DHA-lysoPC), the two major lipid forms that deliver DHA to the brain, in order to understand the formation of this neurotrophic and neuroprotective metabolite of DHA in the brain. Both substrates were taken up in Neuro2A cells and metabolized to N- docosahexaenoylphosphatidylethanolamine (NDoPE) and synaptamide in a time- and concentration-dependent manner, but unesterified DHA was 1.5 to 2.4 times more effective than DHA-lysoPC at equimolar concentrations. The plasmalogen NDoPE (pNDoPE) amounted more than 80% of NDoPE produced from DHA or DHA-lysoPC, with 16-carbon-pNDoPE being the most abundant species. Inhibition of N- acylphosphatidylethanolamine-phospholipase D (NAPE-PLD) by hexachlorophene or bithionol significantly decreased the synaptamide production, indicating that synaptamide synthesis is mediated at least in part via NDoPE hydrolysis. NDoPE formation occurred much more rapidly than synaptamide production, indicating a precursor-product relationship. Although NDoPE is an intermediate for synaptamide biosynthesis, only about 1% of newly synthesized NDoPE was converted to synaptamide, possibly suggesting additional biological function of NDoPE, particularly for pNDoPE, which is the major form of NDoPE produced.

A simple and reliable method was developed and validated to determine the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in rat brain samples by micro salting-out assisted liquid-liquid extraction combined with ultra-high performance liquid chromatography tandem mass spectrometry (SALLLE/UHPLC-MS/MS). The SALLE parameters (brain homogenate volume, salting-out agent, salt concentration, salt solution volume, organic solvent, organic solvent volume, and centrifugation temperature) were optimized to improve sensitivity and selectivity of the method. The SALLE/UHPLC-MS/MS method presented linear ranges from 2.00 to 20.00 ng mL -1 for AEA and from 0.300 to 10.00 μg mL -1 for 2-AG, no significant matrix effect, and inter- and intra-assay precision and accuracy with CV and RSE values lower than 15%, respectively. This innovative method was successfully applied to determine AEA and 2-AG in brain hemispheres from a 6-OHDA animal model of Parkinson's disease (PD)., (Copyright © 2020 Elsevier B.V. All rights reserved.)

This work describes the development and validation of an ultra-high performance liquid chromatography tandem mass spectrometry method that uses disposable pipette extraction (DPX-UHPLC-MS/MS) to determine the endocannabinoid anandamide (AEA) in cerebrospinal fluid samples (CSF). The DPX parameters sorption equilibrium time, sample volume, number of draw-eject cycles, washing solvent volume, and elution solvent volume were optimized by design of experiments (DOE) techniques. The simple DPX protocol proposed herein required a reduced amount of CSF sample and organic solvent. The DPX-UHPLC-MS/MS method presented linear range from 0.10 ng mL -1 (LLOQ) to 3.0 ng mL -1 , inter- and intra-assay accuracy with EPR values varying from -8.2% to 9.6%, inter- and intra-assay precision with CV values ranging from 1.3% to 14.8% (except for the LLOQ), and no significant matrix effect. The innovative DPX-UHPLC-MS/MS method was successfully applied to determine AEA in CSF samples from Parkinson's disease (PD) patients and should therefore be used in clinical studies., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: ω-3 polyunsaturated fatty acids (PUFAs) are synthesized from α-Linolenic acid (ALA, C18:3ω3) and play important roles in anti-inflammatory and antioxidant responses in mammal cells. ALA is an essential fatty acid which cannot be produced within the human body and must be acquired through diet. The purpose of this study was to evaluate the potential of a novel microalgal strain (HDMA-20) as a source of ω-3 PUFAs including ALA and eicosatetraenoic acid (ETA, C20:4ω3)., Method: Phylogenetic Neighbor-Joining analysis based on 18S ribosomal DNA sequence was used to identify the microalga strain HDMA-20. Autotrophic condition was chosen to cultivate HDMA-20 to reduce the cultivation cost. GC-MS was used to determine the fatty acid composition of HDMA-20 lipid., Results: A microalgal strain (HDMA-20) from Lake Chengfeng (Daqing, Heilongjiang province, China) was found to accumulate high content of ω-3 PUFAs (63.4% of total lipid), with ALA and eicosatetraenoic acid (ETA, C20:4ω3) accounting for 35.4 and 9.6% of total lipid, respectively. Phylogenetic analysis based on 18S ribosomal DNA sequences suggested that the HDMA-20 belonged to genus Monoraphidium (Selenastraceae, Sphaeropleales) and its 18S rDNA sequence information turned out to be new molecular record of Monoraphidium species. The biomass productivity and lipid content of HDMA-20 were also investigated under autotrophic condition. The biomass productivity of HDMA-20 reached 36.3 mg L - 1  day - 1 , and the lipid contents was 22.6% of dry weight., Conclusion: HDMA-20 not only represent an additional source of ALA, but also a totally new source of ETA. The high content of ω-3 PUFAs, especially ALA, of HDMA-20, makes it suitable as a source of nutrition supplements for human health. In addition, HDMA-20 exhibited good properties in growth and lipid accumulation, implying its potential for cost-effective ω-3 PUFAs production in future.

Δ5-Olefinic acids have been characterized in gymnosperm plants and have been reported to have several biological health benefits. Δ5-Olefinic acids from pine nut oil were effectively concentrated by repeated lipase-catalyzed esterification. The pine nut oil contained three major Δ5-olefinic acids, namely taxoleic acid (C18:2 Δ5,9), pinolenic acid (C18:3 Δ5,9,12), and sciadonic acid (C20:3 Δ5,11,14). The fatty acids present in pine nut oil were selectively esterified with ethanol using Lipozyme RM IM from Rhizomucor miehei as a biocatalyst. The Δ5-olefinic acids were concentrated in the unesterified fatty acid fraction. The optimum molar ratio of the substrates (fatty acid:ethanol), temperature, the enzyme loading, and the reaction time were 1:7, 25°C, 5% of total substrate weight, and 6 h, respectively. There was no significant effect in the concentration of Δ5-olefinic acids when water was added in the reaction mixture. The same protocol and optimum conditions were employed for two times repeated lipase-catalyzed esterifications. In first lipase-catalyzed esterification, the Δ5-olefinic acids content in the pine nut oil increased from 17 mol% to 51 mol% with a yield of 40 mol%. In a second lipase-catalyzed esterification, with the Δ5-olefinic acids-concentrated fatty acids obtained from the first reaction as the substrate, the Δ5-olefinic acids content increased to 86 mol% with a yield of 15 mol%. Finally, a maximum Δ5-olefinic acids content of ca. 96 mol% with a yield of 6 mol% was obtained via a third lipase-catalyzed esterification.

LC-MS/MS is a powerful analytical technique that provides unequivocal identification and reliable quantification of the analytes, using Selected Reaction Monitoring or Multi Reaction Monitoring acquisition mode.2-Arachidonoylglycerol (2-AG) is the most abundant endocannabinoid, which plays a major role in a wide variety of physiological and pathological processes. Analysis of 2-AG by means of LC-MS/MS allows the detection of very low concentrations in biological samples. Here, we describe how to determine 2-AG levels in tiny samples of tissues and plasma through LC-MS/MS, by using very quick and easy to perform extraction procedures, with reduced solvent consumption.

This article represents a timely opportunity to express my affection, admiration and gratitude to Professor David Triggle. David was my Ph.D. advisor as well as a key consultant in the 1980s and early 1990s for research programs at Miles Institute for Preclinical Pharmacology in West Haven, CT, the U.S. research operation of Bayer AG, in the areas of Ca(2+) and K(+) channel ligands. The binding methodology developed in his laboratory was used to search for an endogenous ligand for L-type Ca(2+) channels. We did not find the substance that we were searching for, a genetically-determined, competitive inhibitor for the 1,4-dihydropyridine binding site, but instead isolated the endogenous ligand for the brain's own marijuana, anandamide. Devane, Mechoulam and coworkers first discovered that this compound was the endogenous ligand for delta-9-tetrahydrocannabinol, the active substance in cannabis. The endogenous endocannabinoid system is now the target of many exciting new approaches to drug discovery., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Anandamide (AEA) is an endocannabinoid present in human plasma that is associated with several physiological functions and disease states. However, low AEA plasma levels pose challenges in terms of analytical characterization. Classical liquid-based lipid extraction and solid-phase extraction require complicated procedures and the drying down of relatively large volumes of solvents, making them unsuitable for high-throughput analysis. Here a high-throughput salting-out assisted liquid-liquid extraction (SALLE) method with acetonitrile and mass spectrometry compatible salts for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of AEA in human plasma has been developed and validated. The seamless interface of SALLE and LC-MS eliminated the drying-down step, only 100 μL of plasma is required and minimal volumes of organic solvent are used. Good reproducibility, accuracy and precision were demonstrated during the method validation. The method is linear up to 10 ng/mL with a lower limit of quantitation of 0.1 ng/mL for AEA, the accuracy for AEA was from 93.3 to 96.7% and the precision was <8.57%. This new methodology was successfully applied to analysis of clinical samples from maintenance hemodialysis patients., (Copyright © 2015 John Wiley & Sons, Ltd.)

The lipids of gymnosperms frequently feature unusual polyunsaturated fatty acids (PUFAs) such as sciadonic acid (20:3Δ5,11,14) and juniperonic acid (20:4Δ5,11,14,17) showing a first double bond on C-5 which is separated from the next double bond by five methylene units. Compared to "classic" fatty acids, these fatty acids are not easily commercially available and their prices are quite high. For this reason, we wished to isolate those fatty acids from the seed oil of Podocarpus falcatus by countercurrent chromatography (CCC) after conversion of the fatty acids to methyl esters (FAMEs). The contribution of sciadonic acid (20:3Δ5,11,14) and juniperonic acid (20:4Δ5,11,14,17) in the unfractionated sample was 10% and 6% respectively, while oleic acid (18:1Δ9) and linoleic acid (18:2Δ9,12) were the major fatty acids. After a first CCC run with FAMEs from Podocarpus falcatus, fractions enriched in the target compounds were chosen for subsequent isolation by means of two subsequent CCC runs. Initially, 13mg of juniperonic acid was recovered with a purity of 92% according to analysis by gas chromatography with mass spectrometry (GC/MS). Further purification of this fraction yielded 2.7mg with a purity of 99% according to GC/MS. The isolation of sciadonic acid was hampered by high amounts of linoleic acid with the same equivalent chain length in suitable fractions of the first CCC separation. After an enrichment step by CCC, the critical pair sciadonic acid and linoleic acid was finally separated as free fatty acids. After this step, 4.4mg of sciadonic acid was recovered with 99% purity. The methodology could also be applied to isolate larger amounts of those fatty acids or for the isolation of other minor fatty acids., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Truffles are the fruiting body of fungi, members of the Ascomycota phylum endowed with major gastronomic and commercial value. The development and maturation of their reproductive structure are dependent on melanin synthesis. Since anandamide, a prominent member of the endocannabinoid system (ECS), is responsible for melanin synthesis in normal human epidermal melanocytes, we thought that ECS might be present also in truffles. Here, we show the expression, at the transcriptional and translational levels, of most ECS components in the black truffle Tuber melanosporum Vittad. at maturation stage VI. Indeed, by means of molecular biology and immunochemical techniques, we found that truffles contain the major metabolic enzymes of the ECS, while they do not express the most relevant endocannabinoid-binding receptors. In addition, we measured anandamide content in truffles, at different maturation stages (from III to VI), through liquid chromatography-mass spectrometric analysis, whereas the other relevant endocannabinoid 2-arachidonoylglycerol was below the detection limit. Overall, our unprecedented results suggest that anandamide and ECS metabolic enzymes have evolved earlier than endocannabinoid-binding receptors, and that anandamide might be an ancient attractant to truffle eaters, that are well-equipped with endocannabinoid-binding receptors., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The discovery of the interaction of plant-derived N-alkylamides (NAAs) and the mammalian endocannabinoid system (ECS) and the existence of a plant endogenous N-acylethanolamine signaling system have led to the re-evaluation of this group of compounds. Herein, the isolation of seven NAAs and the assessment of their effects on major protein targets in the ECS network are reported. Four NAAs, octadeca-2E,4E,8E,10Z,14Z-pentaene-12-ynoic acid isobutylamide (1), octadeca-2E,4E,8E,10Z,14Z-pentaene-12-ynoic acid 2'-methylbutylamide (2), hexadeca-2E,4E,9Z-triene-12,14-diynoic acid isobutylamide (3), and hexadeca-2E,4E,9,12-tetraenoic acid 2'-methylbutylamide (4), were identified from Heliopsis helianthoides var. scabra. Compounds 2-4 are new natural products, while 1 was isolated for the first time from this species. The previously described macamides, N-(3-methoxybenzyl)-(9Z,12Z,15Z)-octadecatrienamide (5), N-benzyl-(9Z,12Z,15Z)-octadecatrienamide (6), and N-benzyl-(9Z,12Z)-octadecadienamide (7), were isolated from Lepidium meyenii (Maca). N-Methylbutylamide 4 and N-benzylamide 7 showed submicromolar and selective binding affinities for the cannabinoid CB1 receptor (Ki values of 0.31 and 0.48 μM, respectively). Notably, compound 7 also exhibited weak fatty acid amide hydrolase (FAAH) inhibition (IC50 = 4 μM) and a potent inhibition of anandamide cellular uptake (IC50 = 0.67 μM) that was stronger than the inhibition obtained with the controls OMDM-2 and UCM707. The pronounced ECS polypharmacology of compound 7 highlights the potential involvement of the arachidonoyl-mimicking 9Z,12Z double-bond system in the linoleoyl group for the overall cannabimimetic action of NAAs. This study provides additional strong evidence of the endocannabinoid substrate mimicking of plant-derived NAAs and uncovers a direct and indirect cannabimimetic action of the Peruvian Maca root.

For the first time a new fluorescent analogue of anadamide incorporating BODIPY®-FL-fluorophore, attached to arachidonic acid via 2,2'-(ethylenedioxy)-bis(ethylenediamine), was prepared. Using rat glioma C6 cells it was demonstrated that the fluorescent analogue is a substrate of the cellular anandamide uptake system (Km 4.5 ± 0.9 µM, Vmax 20 ± 1 amol/(min x cell)).

Endocannabinoids (ECs) are endogenous compounds that interact with type-1 and type-2 cannabinoid receptors (CB(1) and CB(2)), as well as non-cannabinoid receptors. The multitude of roles attributed to ECs makes them an emerging target of pharmacotherapy for a number of disparate diseases. Here a high-throughput bioanalytical method based on micro SPE (μ-SPE) followed by LC-MS/MS analysis for the simultaneous determination of the two major endocannabinoids 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (anandamide, AEA) in human plasma is presented. The chromatographic conditions obtained with the fused-core column allowed a good separation in 10 min also of the AG isomers. A very simple and reliable extraction has been optimised by means of C18-modified tips: it requires only 100 μL of plasma and allows the use of minimal volumes of organic solvent. The present method allows a rapid and effective clean-up, which also minimises the isomerisation of 2-AG. The whole procedure has been validated following the FDA guidelines for bioanalytical methods validation: the satisfactory recovery values, the negligible matrix effect and the good values of accuracy and reproducibility make it a simple and high-throughput analytical tool for clinical and biochemical studies on endocannabinoid signaling in humans.

It has been previously shown that the positional isomers of triacylglycerol (TAG) containing palmitic acid (P) and highly unsaturated fatty acids (HUFAs) such as DHA (D) and EPA (E) vary between fishes and marine mammals. However, it has not yet been understood why in marine mammals HUFAs are located only at the α position when two palmitic acid chains combine, and not in fishes. In order to gain further understanding of the biosynthetic pathways involved in the formation of these asymmetric TAGs, we investigated whether the HUFA in the TAG of marine mammals exists predominantly at the sn-1 or sn-3 position. We examined the TAG positional isomers and enantiomers in marine organisms in detail. As a result, while PDP and PEP were not detected, sn-PPD and sn-PPE were found in abundance in marine mammals. For fishes, on the other hand, PDP, PEP, sn-PPD, and sn-PPE were all identified. In the case of TAGs that contain two HUFAs and one palmitic acid, marine mammals were rich in DPD and EPE whereas fishes were rich in sn-PDD and sn-PEE.

Analysis of the endocannabinoid (EC) system's key molecules 2-arachidonoyl glycerol (2AG) and arachidonoyl ethanolamide (anandamide, AEA) is challenging due to several peculiarities. 2AG isomerizes spontaneously to its biologically inactive analogue 1-arachidonoyl glycerol (1AG) by acyl migration and it is only chromatographically distinguishable from 1AG. Matrix-effects caused primarily by co-extracted phospholipids may further compromise analysis. In addition, 2AG and 1AG are unstable under certain conditions like solvent evaporation or reconstitution of dried extracts. We examined effects of different organic solvents and their mixtures, such as toluene, ethyl acetate, and chloroform-methanol, on 2AG/1AG isomerisation, 2AG/1AG stability, and matrix-effects in the UPLC-MS/MS analysis of 2AG and AEA in human plasma. Toluene prevented, both, 2AG isomerisation to 1AG and degradation of 2AG/1AG during evaporation. Toluene extracts contain only 2% of matrix-effect-causing plasma phospholipids compared to extracts from the traditionally used solvent mixture chloroform-methanol. Toluene and all other tested organic solvents provide comparable 2AG and AEA extraction yields (60-80%). Based on these favourable toluene properties, we developed and validated a UPLC-MS/MS method with positive electrospray ionization (ESI+) that allows for simultaneous accurate and precise measurement of 2AG and AEA in human plasma. The UPLC-MS/MS method was cross-validated with a previously described fully-validated GC-MS/MS method for AEA in human plasma. A close correlation (r(2)=0.821) was observed between the results obtained from UPLC-MS/MS (y) and GC-MS/MS (x) methods (y=0.01+0.85x). The UPLC-MS/MS method is suitable for routine measurement of 2AG and AEA in human plasma samples (1 mL) in clinical settings as shown by quality control plasma samples processed over a period of 100 days. The UPLC-MS/MS method was further extended to human urine. In urine, AEA was not detectable and 2AG was detected in only 3 out of 19 samples from healthy subjects at 160, 180 and 212 pM corresponding to 12.3, 14.5 and 9.9 pmol/mmol creatinine, respectively., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Introduction: Heterotheca inuloides Cass., also known as "arnica", is used in traditional medicine in Mexico., Objective: Development of fast methods for the extraction of lipidic and phenolic fractions from arnica plants and their subsequent characterization., Methodology: Ultrasound was applied to accelerate extraction of the target compounds from this plant and reduce the use of organic solvents as compared with conventional methods. Gas chromatography-ion trap mass spectrometry and liquid chromatography with diode-array detection were used for the characterization of the lipidic and phenolic fractions, respectively., Results: Under optimal extraction conditions, 9 and 55 min were necessary to complete extraction of the lipidic and phenolic fractions, respectively. The fatty acids present at the highest concentrations in H. inuloides were eicosatetraenoic n3 (24.6 μg/g), cis-9-hexadecenoic n7 (23.1 μg/g), exacosanoic (22.7 μg/g) and cis-9-octadecenoic acid (21.3 μg/g), while the rest were in the range 7.6-1.3 μg/g. The most concentrated phenols were guaiacol (41.5 μg/g), catechin (38.7 μg/g), ellagic acid (35.9 μg/g), carbolic acid (24.2 μg/g) and p-coumaric acid (19.5 μg/g), while the rest were in the range 5.1-0.4 μg/g., Conclusion: Ultrasound reduces the time necessary to complete the extraction 160 and 26 times, the extraction volume 2.5 and 4 times, and increases the extraction efficiency 5 and 3 times for lipidic and phenolic fractions, respectively, in comparison with conventional extraction methods. In addition, the characterization of the lipidic and phenolic fractions constitutes a first approach to the H. inuloides metabolome., (Copyright © 2011 John Wiley & Sons, Ltd.)

Anandamide (N-arachidonoylethanolamide), a bioactive lipid, is reported to play a role in pregnancy maintenance and parturition. Our aims were to (1) evaluate AEA levels at the human maternal:fetal interface and (2) validate the use of solid-phase extraction of AEA from tissues. AEA was analyzed in cord and maternal blood, amniotic fluid, placenta, and fetal membranes collected during Caesarean section (n=14). Extraction efficiencies were 42 and 36% for the placenta and the fetal membranes, respectively. Tissue AEA was quantified using an isotope-dilution method and UPLC-ESI-MS/MS giving intra- and inter-day variability for tissues spiked with 0.2, 1, and 5pmol/g AEA of less than 12%. Accuracy for these spiked samples was between 95% and 103% for fetal membranes and between 99% and 114% for placenta. Mean AEA concentrations were 2.72 + or - 1.04 pmol/g for placenta and 1.19 + or - 0.68 pmol/g for fetal membranes, and 0.93 + or - 0.28, 0.88 + or - 0.33, 0.77 + or - 0.30, and 0.06 + or - 0.04nM for maternal, umbilical vein, and umbilical artery plasma and amniotic fluid. Higher AEA concentrations were found in placenta compared to fetal membranes (P<0.0001), in umbilical vein compared with umbilical artery (P=0.0015), and in plasma from maternal circulation compared with umbilical artery (P=0.0152). The relevance of these changes in AEA concentrations at the maternal:fetal interface requires further investigation., (Copyright 2010 Elsevier Inc. All rights reserved.)

N-Arachidonoylethanolamine (AEA, anandamide) was the first endocannabinoid to be identified and has since become associated with the mediation of several physiological functions and disease states. AEA has been isolated from numerous tissues and biofluids, in the low nanomolar range, using lipid extraction techniques with organic solvents. These techniques require the drying down of relatively large volumes of solvents, making them unsuitable for high-throughput analysis. Here we describe a solid-phase extraction (SPE) method for the investigation of AEA concentrations in human plasma, serum, milk, urine, amniotic fluid, peritoneal fluid, saliva, follicular fluid, and fluid from an ovarian cyst. AEA was detected in serum and plasma from blood isolated from 20 adult women (means+/-standard deviations: 0.68+/-0.29 and 0.64+/-0.28 nM, respectively), from pregnant women at term (1.37+/-0.42 nM), and from umbilical vein (1.26+/-0.33 nM) and umbilical artery (1.14+/-0.35nM), in milk (0.12+/-0.05 nM) and from amniotic (0.03+/-0.02 nM), peritoneal (0.93+/-0.27 nM), follicular (1.17+/-0.51 nM), and ovarian cyst (0.32+/-0.01 nM) fluids. AEA was undetectable in saliva and urine. The 60% AEA extraction efficiency achieved with SPE from plasma was superior to the 19% efficiency achieved using the existing organic solvent extraction method. Limits of quantification and detection for AEA were also improved dramatically using SPE (8 and 4 fmol/ml) compared with organic extraction (25 and 18.75 fmol/ml plasma). These improvements allow the use of smaller plasma samples with SPE. Intra- and interday variability were comparable, and the mean AEA concentration of pooled plasma samples (1.18 nM, n=15) was identical with the two techniques. Similarly, when 56 plasma samples from laboring and nonlaboring women were analyzed using both techniques, no extraction method-dependent differences were observed. Consequently, we provide evidence for a robust SPE technique for the extraction of AEA from biomatrices to replace the existing liquid extraction methods, with the SPE technique being superior in terms of speed, extraction efficiency, and sample size required.

The endocannabinoids anandamide and 2-arachidonoylglycerol, as well as several anandamide-related N-acylethanolamines, belong to a family of lipid transmitter that regulate fundamental physiological processes, including neurotransmission and neuroinflammation. Their precise quantification in biological matrices can be achieved by gas chromatography-mass spectrometry (GC-MS), but this method typically requires multiple time-consuming purification steps such as solid-phase extraction followed by HPLC. Here we report a novel solid-phase extraction procedure allowing for single-step, and thus higher throughput, purification of endocannabinoids and N-acylethanolamines before GC-MS quantification. We determined the minimal amount of mouse brain tissue required to reliably detect endocannabinoids and N-acylethanolamines when using this approach and provide direct evidence for quantification accuracy by using radioactive and deuterated standards spiked into mouse brain samples. Using this method, we found that mouse brain contains much higher levels of anandamide (>1 nmol/g tissue) than previously reported, whereas levels of 2-arachidonoylglycerol and other N-acylethanolamines are well within the range of previous reports. In addition, we show that mouse brain amounts of endocannabinoids and N-acylethanolamines differ depending on animal gender as well as on whether the tissue was fixed or not. Our study shows that endocannabinoid and N-acylethanolamine levels quantified in mouse brain by GC-MS depend closely on tissue amount and preparation as well as on animal gender and that, depending on such parameters, anandamide levels could be underestimated.

The endocannabinoid N-arachidonoyl ethanolamine (anandamide), found both in the CNS and in the periphery, plays a role in numerous physiological systems. One might expect that the chemically related N-arachidonoyl-L-serine (ARA-S) could also be formed alongside anandamide. We have now isolated ARA-S from bovine brain and elucidated its structure by comparison with synthetic ARA-S. Contrary to anandamide, ARA-S binds very weakly to cannabinoid CB1 and CB2 or vanilloid TRPV1 (transient receptor potential vanilloid 1) receptors. However, it produces endothelium-dependent vasodilation of rat isolated mesenteric arteries and abdominal aorta and stimulates phosphorylation of p44/42 mitogen-activated protein (MAP) kinase and protein kinase B/Akt in cultured endothelial cells. ARA-S also suppresses LPS-induced formation of TNF-alpha in a murine macrophage cell line and in wild-type mice, as well as in mice deficient in CB1 or CB2 receptors. Many of these effects parallel those reported for abnormal cannabidiol (Abn-CBD), a synthetic agonist of a putative novel cannabinoid-type receptor. Hence, ARA-S may represent an endogenous agonist for this receptor.

Purpose: Direct hemoperfusion (DHP) with polymixin B-immobilized fiber (PMX) has been reported to be effective for patients with septic shock. The aim of this study was to clarify the mechanism of PMX-DHP effect on septic shock., Methods: The following parameters were measured in septic shock patients who were treated with PMX-DHP: survival rate, sepsis-related organ failure assessment (SOFA) score, acute physiology and chronic health evaluation II (APACHE-II) score, and plasma concentrations of cannabinoids [anandamide (ANA) and 2-arachidonyl glyceride (2-AG)], cytokines [interleukin (IL)-6, IL-8, IL-10], transforming growth factor beta (TGF-beta), and calcitonin gene-related peptide (CGRP)]. The primary end point was mortality from all causes at day 28 after intensive care unit (ICU) admission or discharge., Results: The survival rate of all patients at 28 days after ICU admission was 37.5% (9/24). The survival group showed significantly lower SOFA and APACHE-II scores than the nonsurvival group after PMX-DHP treatment (P = 0.008 and 0.028, respectively). The improved SOFA score group showed a better survival rate than the nonimproved SOFA score group (71.4% versus 23.5%, P = 0.028). Plasma ANA level significantly decreased after PMX-DHP treatment both in the improved SOFA score group and in the survival group. The level of 2-AG, however, showed no significant change in either group., Conclusion: ANA, an intrinsic cannabinoid that induces hypotension in septic shock, is inferred to be the main mechanism of the PMX-DHP effect. Removal of ANA by PMX-DHP could be key to successful septic shock treatment.

Mammalian cells produce both N-arachidonoylethanolamine (20:4n-6 NAE, anandamide) and 2-arachidonoylglycerol (2-AG), lipid signaling molecules that activate cannabinoid receptors. Because both agonists occur in the presence of receptor-inactive congeners, we have developed a sensitive method for the simultaneous assay of N-acylethanolamines (NAEs) and 2-monoacylglycerols (2-MAG). These lipid classes are isolated from total lipids by solid phase extraction and converted to tert-butyldimethylsilyl (tBDMS) derivatives in the presence of deuterated analogs. The tBDMS derivatives are analyzed by gas chromatography/mass spectrometry using selected ion monitoring programs specific for NAE and 2-MAG. Individual NAEs and 2-MAGs can be quantified in the nanogram and subnanogram range. The NAE and 2-MAG compositions of rat organs and cultured JB6 cells are reported.

The background knowledge leading to the isolation and identification of anandamide and 2-arachidonoyl glycerol, the principal endocannabinoids is described. The structure-activity relationships of these lipid derivatives are summarized. Selected biochemical and pharmacological topics in this field are discussed, the main ones being levels of endocannabinoids in unstimulated tissue and cells, biosynthesis, release and inactivation of endocannabinoids, the effects of 'entourage' compounds on the activities of anandamide and 2-arachidonoyl glycerol, their signaling mechanisms and effects in animals.

Eicosanoids are a large group of biologically active metabolites of arachidonic acid and related C20 fatty acids. Many of these compounds contain hydroxyl groups which can be converted to oxo groups by a variety of substrate-specific dehydrogenases. In many cases, this results in a reduction in potency, but in others, such as the oxidation of 5-hydroxyeicosatetraenoic acid to its oxo metabolite 5-oxo-eicosatetraenoic acid, there is a dramatic increase in biological activity. Thus, it is often very important to analyze the relative amounts of oxo- and hydroxy-eicosanoids formed by various cells and tissues. The present study was designed to compare the chromatographic behavior of oxo-eicosanoids and their hydroxy counterparts in commonly used mobile phases for reversed-phase and normal-phase HPLC. We examined three groups of eicosanoids: prostaglandins, leukotriene B4 and some of its metabolites, and monohydroxy-eicosanoids and their oxo metabolites. We found that in reversed-phase HPLC, the retention times of oxo-eicosanoids were longer than those of the corresponding hydroxy-eicosanoids in mobile phases containing acetonitrile as the major organic component, whereas the reverse was true for mobile phases containing methanol. Normal-phase HPLC using mobile phases containing hexane, isopropanol, and acetic acid gave excellent separation of oxo- and hydroxy-eicosanoids. Increasing the concentration of acetic acid in the mobile phase selectively reduced the retention times of oxo-eicosatetraenoic acids compared to monohydroxy-eicosatetraenoic acids, whereas the reverse was true for isopropanol. Differences in the chromatographic behavior of oxo- and hydroxy-eicosanoids can be useful clues in the structural characterization of these compounds, as illustrated by the chromatographic properties of a complex series of LTB4 metabolites formed by rat neutrophils.

Anandamide (arachidonoylethanolamide, AnNH) has been recently proposed as the endogenous ligand at the brain cannabinoid receptor CB1. Two alternative pathways have been suggested for the biosynthesis of this putative mediator in the central nervous system. Here we present data (1) substantiating further the mechanism by which AnNH is produced by phospholipase D (PLD)-catalysed hydrolysis of N-arachidonoylphosphatidylethanolamine in mouse neuroblastoma N18TG2 cells, and (2) suggesting for the first time that AnNH is biosynthesized via the same mechanism in a non-neuronal cell line, mouse J774 macrophages, together with other acylethanolamides and is possibly involved in the control of the immune/inflammatory response. Lipids from both neuroblastoma cells and J774 macrophages were shown to contain a family of N-acylphosphatidylethanolamines (N-aPEs), including the possible precursor of AnNH, N-arachidonoyl-PE. Treatment with exogenous PLD, but not with exogenous phospholipase A2 and ethanolamine, resulted in the production of a series of acylethanolamides (AEs), including AnNH, from both cell types. The formation of AEs was accompanied by a decrease in the levels of the corresponding N-aPEs. Enzymically active homogenates from either neuroblastoma cells or J774 macrophages were shown to convert synthetic N-[3H]arachidonoyl-PE into [3H]AnNH, thus suggesting that in both cells an enzyme is present which is capable of catalysing the hydrolysis of N-aPE(s) to the corresponding AE(s). Finally, as previously shown in central neurons, on stimulation with ionomycin, J774 macrophages also produced a mixture of AEs including AnNH and palmitoylethanolamide, which has been proposed as the preferential endogenous ligand at the peripheral cannabinoid receptor CB2 and, consequently, as a possible down-modulator of mast cells. On the basis of this as well as previous findings it is now possible to hypothesize for AnNH and palmitoylethanolamide, co-synthesized by macrophages, a role as peripheral mediators with multiple actions on blood cell function.

To gain some insight into the mechanisms involved in the opposing effects of arachidonic acid and docosahexaenoic acid on the growth of rat uterine stromal cells (UIII cells), the dynamics of the uptake, conversion, and incorporation of labeled 18:2(n-6), 18:3(n-3), 20:4(n-6), 20:5(n-3), and 22:6(n-3) into lipid pools and phospholipid subclasses were examined. A very active and time-dependent conversion of [14C]18:3(n-3) to higher homologs was observed; 64.7 +/- 0.7 and 11.5 +/- 0.4% of the [14C] radioactivity incorporated in cellular lipids was recovered as 22:5(n-3) and 22:6(n-3) after 72 h incubation, respectively. The distribution of labeled fatty acids obtained after 72 h incubation with [3H]20:5(n-3) was not significantly different from that observed with 18:3(n-3). Arachidonic acid was the major fatty acid formed from [14C]18:2(n-6) and only trace amounts of 22:5(n-6) were detected. When cells were incubated for 72 h with 20:4(n-6), more than 75% of the radioactivity was recovered as arachidonate and slightly higher amounts of 22:4(n-6) and 22:5(n-6) were formed compared to those obtained after incubation with 18:2(n-6). Using both [14C]- and [3H]22:6(n-3), no significant retroconversion of labeled 22:6(n-3) occurred in the cells. More than 90% of labeled 20:4(n-6) and 22:6(n-3) taken up by the cells were esterified into phospholipids, but significant differences in their distribution among phospholipid classes and subclasses were observed. Docosahexaenoic acid was more rapidly and efficiently incorporated into phosphatidylethanolamine than 20:4(n-6) and was principally recovered in plasmalogens. Arachidonic acid was mainly incorporated in the diacyl subclasses of phosphatidylcholine and phosphatidylethanolamine and in phosphatidylinositol. The divergent profiles of these two fatty acids within the phospholipid compartments provide some information for the mechanisms of their opposite effects on UIII cell growth.

Recent reports have suggested that N-arachidonoylethanolamine (anandamide) acts as an endogenous ligand for cannabinoid receptors in mammalian brain. Here we describe methods for the extraction, purification and analysis of anandamide and related N-fatty acyl-ethanolamines (NAEs). Liquid-phase extraction, silica gel G column chromatography and thin-layer chromatography (TLC) were employed for sample fractionation. Three analytical high-performance liquid chromatography (HPLC) methods for purification of NAEs were developed. Finally, analyses of NAEs by gas chromatography/mass spectrometry (GC/MS) are described. The applications of these analytical methods to the identification of anandamide and related NAEs in cell cultures as well as of artifacts in biosynthetic studies are described.

Two types of mechanisms have been proposed to account for the combination of peroxidase and cyclooxygenase activities in prostaglandin H synthase (PGHS). One, a branched-chain mechanism [Dietz, R., et al. (1988) Eur. J. Biochem. 171, 321-328], postulates that the cyclooxygenase reaction propagates essentially independently of peroxidase catalysis. The second, a tightly coupled mechanism [Bakovic, M., & Dunford, H. B. (1994) Biochemistry 33, 6475-6482], postulates that peroxidase catalysis is an integral part of cyclooxygenase propagation. Qualitative and quantitative predictions from the two mechanisms have been compared with several observed characteristics of the PGHS reaction with arachidonate, including the ability to accumulate PGG2 and oxidized enzyme intermediates, the stoichiometry between cosubstrate and fatty acid consumption, and the hydroperoxide activator requirement. The observed characteristics, particularly the accumulation of micromolar levels of PGG2 even in the presence of cosubstrate and the stoichiometry between cosubstrate oxidation and fatty acid oxygenation of less than 1.3 (compared to a theoretical maximum of 2.0), were largely consistent with predictions from the branched-chain mechanism, but contradicted important predictions of the tightly coupled mechanism. These results indicate that PGHS catalysis is more accurately described by the branched-chain mechanism than by the tightly coupled mechanism.

There is evidence that iodoarachidonates are mediators of iodide in thyroid autoregulation, however, their occurrence in vivo has not yet been demonstrated. We therefore tried to identify delta-iodolactone (5-Hydroxy-6-iodo-8,11,14-eicosatrienoic delta-lactone, IL-delta) in thyroid tissue from a patient with Graves' disease treated with high doses of iodide. Lipids were extracted from thyroid tissue, purified by reversed phase chromatography and analyzed by gas chromatography--tandem mass spectrometry (GC-MSMS). The retention time in gas chromatography and fragmentation pattern in tandem mass spectrometry were determined with biochemically synthesized non-deuterated and deuterated IL-delta. According to retention time (13.44 min) and specific fragments (m/z 303, m/z 259) the occurrence of IL-delta could be demonstrated in the extract of iodide treated goiter. In vitro, potassium iodide (40 microM) as well as IL-delta (1.0 microM) significantly inhibited the proliferation of human thyroid follicular cells induced by phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate). These results demonstrate for the first time that Il-delta is present in iodide treated human thyroid. As cell proliferation is under negative control of IL-delta, a crucial role in thyroid involution following iodide treatment may be possible.

Anandamide, an endogenous eicosanoid derivative (arachidonoylethanolamide), binds to the cannabinoid receptor, a member of the G protein-coupled superfamily. It also inhibits both adenylate cyclase and N-type calcium channel opening. The enzymatic synthesis of anandamide in bovine brain tissue was examined by incubating brain membranes with [14C]ethanolamine and arachidonic acid. Following incubation and extraction into toluene, a radioactive product was identified which had the same Rf value as authentic anandamide in several thin-layer chromatographic systems. When structurally similar fatty acid substrates were compared, arachidonic acid exhibited the lowest EC50 and the highest activity for enzymatic formation of the corresponding ethanolamides. The concentration-response curve of arachidonic acid exhibited a steep slope, and at higher concentrations arachidonate inhibited enzymatic activity. When brain homogenates were separated into subcellular fractions by sucrose density gradient centrifugation, anandamide synthase activity was highest in fractions enriched in synaptic vesicles, myelin, and microsomal and synaptosomal membranes. When several areas of brain were examined, anandamide synthase activity was found to be highest in the hippocampus, followed by the thalamus, cortex, and striatum, and lowest in the cerebellum, pons, and medulla. The ability of brain tissue to enzymatically synthesize anandamide and the existence of specific receptors for this eicosanoid suggest the presence of anandamide-containing (anandaergic) neurons.

This study was part of a broad search for endogenous regulators of L-type calcium channels. The screening for active fractions was done by measuring inhibition [3H]1,4-dihydropyridine (DHP) binding to rat cardiac and cortex membranes. An inhibitory fraction, termed lyophilized brain hexane-extractable inhibitor (LBHI), was isolated from hexane extracts of lyophilized calf brain. The active substance was purified by a series of chromatographic steps. 13C nuclear magnetic resonance (NMR), 1H coherence spectroscopy (COSY) NMR and fast atom bombardment (FAB) mass spectroscopy suggested that LBHI was N-arachidonic acid-2-hydroxyethylamide. Synthesis of this substance and subsequent high performance liquid chromatography (HPLC) and NMR analysis confirmed this structure. Synthetic LBHI (SLBHI) inhibited [3H]DHP binding to rat cortex membranes with an IC50 value of congruent to 15 microM and a Hill coefficient of congruent to 2. Saturation analysis in the presence of SLBHI showed a change in KD (equilibrium dissociation constant), but not maximal binding capacity (Bmax). SLBHI produced an increased dissociation rate, which, along with the Hill slope of > 1, suggested a non-competitive interaction with the DHP binding site. The results suggest that arachidonic acid derivatives may be endogenous modifiers of the DHP calcium antagonist binding site.

Phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-diphosphate [PtdIns(4,5)P2] were isolated from the pancreas of rats fed an ethanol-containing liquid diet for 24 days and from the corresponding pair-fed controls. The isolation involved chromatography on a lipophilic anion exchanger in the phosphate form. The species composition was determined by fast-atom bombardment mass spectrometry. The compositions of PtdIns4P and PtdIns(4,5)P2 were similar to that of PtdIns, with the stearoyl/arachidonoyl species constituting about 32% of the total, compared with 38% in PtdIns. PtdIns(4,5)P2 contained a larger fraction of fully saturated species than PtdIns. The PtdIns species having an arachidonoyl group were about half as abundant in the ethanol-treated as in the control rats. The differences between ethanol-fed and control rats were qualitatively similar for PtdIns, PtdIns4P and PtdIns(4,5)P2, but were less marked for PtdIns4P and PtdIns(4,5)P2. The results indicate that the species compositions of PtdIns4P and PtdIns(4,5)P2 reflect that of PtdIns, and that the changes of PtdIns4P and PtdIns(4,5)P2 in ethanol-treated rats are secondary to changes in PtdIns.

In this work it was demonstrated that the incubation of hepatoma cultured cells (HTC 7288 c) with 11-deoxycorticosterone (DOC) ranging from 0 to 10(-4) M concentration provoked a dose-dependent inhibition in the conversion of [1-14C] eicosatrienoic acid to arachidonic acid. This steroid also produced an increase in the uptake of exogenous 20:3 (n-6) acid. The depressive effect evoked by DOC on delta 5 desaturating activity was reflected on the fatty acid composition changes of the hepatoma cells. The delta 5 desaturase activity was inhibited by a soluble factor that would be induced by the hormone and that was present in the cytosol fraction from DOC-treated cells, corresponding to a low molecular mass below 25 kDa. Presently we report that an 11-beta-OH group on the steroid molecule is not an essential requirement for the production of a delta 5 desaturase inhibitory factor.

Conversion of tritiated arachidonic acid (AA) into metabolites of the cyclo- and lipoxygenase pathways by bovine fetal placental tissue (200 mg) and fetal plus maternal placental tissue (400 mg) of Days 255, 265, 275 of gestation and at parturition (n = 5) during a 30 min incubation was measured using reverse-phase high pressure liquid chromatography. Fetal placental tissue produced 13,14-dihydro-15-keto-prostaglandin E2 (PGEM) as the major metabolite, the synthesis of which increased from Day 265 to Day 275 and parturition by 150% and 475%, respectively. In tissues collected at parturition, PGE2 synthesis was also detected. On Day 275 and at parturition fetal placental tissue synthesized the metabolite 12-hydroxyheptadecatrienoic acid (HHT), and throughout the experimental period the lipoxygenase product 15-HETE was detected with synthesis rates increasing over time of gestation. In addition, an unidentified metabolite was regularly found in the radiochromatograms which eluted at 1 h and 1 min (U101), between HHT and 15-HETE. The synthesis of this metabolite decreased as pregnancy progressed. Furthermore, various other polar and nonpolar metabolites pooled under the heading UNID were eluted, the production of which increased over time of gestation. The presence of maternal placental tissue did not influence the synthesis of PGEM, 15-HETE and U101, but the production of HHT was decreased when maternal tissue was present. Also, as pregnancy progressed, maternal placental tissue seemed to contribute to the pool of unidentified metabolites. In conclusion, fetal placental tissue seems to be the major source of the AA metabolites when compared with maternal placental tissue, and AA metabolism by bovine placental tissue is markedly increased throughout the last month of pregnancy, suggesting a role for AA metabolites in mechanisms controlling parturition.

Sonicates of freshly isolated Clara cells produced thromboxane B2 (TxB2), prostaglandin (PG) D2, PGE2, PGF2 alpha, hydroxyheptadecatrienoic acid (HHT), and 12-hydroxyeicosatetraenoic acid (12-HETE) as detected using high-performance liquid chromatography (HPLC). Sonicates of Clara cells cultured on collagen matrices produced the same metabolites. Rates of [3H]arachidonic acid metabolism increased in culture, but the changes were not associated with changes in cell number. Sonicates of freshly isolated tracheal cells produced mainly 12-HETE. Cyclooxygenase products were not produced consistently. Sonicates of tracheal cultures produced significant quantities of TxB2, PGD2, PGE2, PGF2 alpha, and HHT, but 12-HETE remained the major metabolite. Equivalent short-circuit current (Ieq) across cultured Clara cell epithelia was unaffected by bilateral exposure to TxB2, PGD2, PGE2, PGF2 alpha, HHT, or 12-HETE. A minor (1%) decrease in transepithelial resistance (Rt) followed exposure to PGD2. Indomethacin had no significant effect on Rt or Ieq, but exposure of indomethacin-pretreated preparations to PGE2 revealed a minor (2%) increase in Ieq. In contrast, tracheal cell epithelia exhibited significant changes in Rt and Ieq in response to PGF2 alpha, PGE2, and HHT. These results indicate that Clara cells metabolize arachidonic acid to biologically active eicosanoids, but the resulting products do not play a major role in regulation of transepithelial ion transport by this cell type.

Epithelial inflammation may play an obligatory role in the pathogenesis of a number of chronic pulmonary diseases such as asthma or bronchitis and has been implicated during the promotion phase of multistage carcinogenesis. At sites of inflammation, bioactive lipid mediators are released and activate a wide range of pathophysiological responses including bronchospasm. Previous studies suggest that one class of inflammatory mediators, the eicosanoids, can also influence cell growth. Epithelial cell proliferation and hyperplasia are common sequelae to irritation and inflammation, and because the lung has a high capacity to produce eicosanoids, we investigated the effects of a group of these compounds, the cysteinyl leukotrienes, on growth of human airway epithelial cells. Leukotrienes were found to be mitogenic in a concentration-dependent manner and exhibit a structure-activity relationship, with leukotriene C4 being more potent than its sequential metabolites leukotriene D4 and E4. The potency of leukotriene C4 is striking, stimulating colony-forming efficiency in concentrations as low as 10 fM. These findings suggest a new physiological role for leukotrienes in the lung that links inflammation with epithelial cell proliferation.

In rat alveolar macrophages treated with 100 microM t-butyl hydroperoxide (tBOOH), leukotriene B4 (LTB4) synthesis was significantly lower than the basal level while levels of cyclooxygenase pathway products were increased. LTB4, 5,6-dihydroxyeicosatetraenoic acid (5,6-DiHETEs), and 5-hydroxyeicosatetraenoic acid (5-HETE) production in macrophages was significantly stimulated by 2 microM A23187, but this was suppressed 40% by simultaneous addition of 10 microM tBOOH and completely abolished by 100 microM tBOOH. Basal and A23187-stimulated macrophage production of chemotactic agents were similarly suppressed by addition of tBOOH; this effect paralleled depression of cellular LTB4 synthesis. In contrast to the significant depression of A23187-stimulated formation of 5-lipoxygenase products by 10 microM tBOOH, cellular adenosine triphosphate (ATP) was unchanged. Macrophages pretreated with KCN led to a 42% decline in ATP levels; however, LTB4, 5,6-DiHETEs, and 5-HETE production in response to A23187 was not suppressed. The results indicate that inhibition of 5-lipoxygenase pathway products in macrophages treated with tBOOH did not occur by depletion of cellular ATP levels.

Incubation of rat liver microsomal fractions with arachidonic acid in the presence of NADPH results in the formation of three novel monohydroxylated fatty acid metabolites. Utilizing chromatographic and mass spectral techniques, these metabolites have been identified as 16-, 17-, and 18-hydroxyeicosatetraenoic acids. The NADPH-dependent microsomal metabolism of arachidonic acid to 16-, 17-, 18-, and 19-hydroxyeicosatetraenoic acids is induced after animal treatment with beta-naphthoflavone. Reconstitution of the arachidonic acid oxygenase utilizing individual purified cytochrome P-450 enzymes demonstrates regioselectivity, controlled by the protein catalyst, for the hydroxylation of the sp3 carbon atoms adjacent to the methyl end of the fatty acid.

12-Lipoxygenases oxygenate arachidonic acid producing its 12S-hydroperoxy derivative and are well known as platelet and leukocyte enzymes. When a peroxidase-linked immunoassay of the enzyme according to the avidin-biotin method was applied to the cytosol fractions from various parts of porcine brain, a considerable amount of the enzyme was found in the anterior pituitary. The enzyme level (about 200 ng/mg cytosol protein) corresponded to about 6% of the enzyme content in porcine peripheral leukocytes. Posterior and intermediate lobes showed about one-tenth of the enzyme level of anterior pituitary. Other parts of porcine brain contained the 12-lipoxygenase in amounts below 7 ng/mg cytosol protein. The cytosol fraction (0.7 mg of protein) of anterior pituitary produced 12S-hydroxy-5,8,10,14-eicosatetraenoic acid from 25 microM arachidonic acid in about 34% conversion at 24 degrees C for 5 min, giving a specific enzyme activity about 3 nmol/min/mg protein. Furthermore, various octadecapolyenoic acids were oxygenated almost as fast as the arachidonate 12-oxygenation. When anterior pituitary was investigated immunohistochemically with anti-12-lipoxygenase antibody, most of the immunostained cells were certain parenchymal cells with granules, which were not blood cells. These biochemical and immunohistochemical results provide a good reason for considering that 12-lipoxygenase does play an important role in pituitary function.