Targeted profiling of arachidonic acid and eicosanoids in rat tissue by UFLC-MS/MS: Application to identify potential markers for rheumatoid arthritis.

We describe a method for the targeted analysis of bioactive arachidonic acid metabolites through cyclooxygenase (COX) and lipoxygenase (LOX) pathway in knee joint, liver, kidney, spleen and heart using an ultra-fast liquid chromatography-tandem mass (UFLC-MS/MS) method. Method validation was investigated, including linearity, precision, accuracy, matrix effect, extraction recovery and stability for the simultaneous analysis of prostaglandins (PGs), thromboxanes (TXs), leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs). The method enables us to chromatographically separate branched-chain species from their straight-chain isomers as well as separate biologically important eicosanoids. The concentrations of the following major eicosanoids were significantly increased in rheumatoid arthritis model rats than in normal ones: 5-HETE, 8-HETE, 12-HETE, 15-HETE, PGF2α, TXB2, 5-HpETE, LTE4, PGE2, PGD2, LTB4. Further multivariate data analysis (partial least square-discriminant analysis) showed COX products (PGs, TXs) were readily distributed towards liver and kidney, LOX products (LTs, HETEs) towards knee joint and spleen, and heart had no characteristic metabolites. The method described here offers a useful tool for the evaluation of complex regulatory eicosanoids responses in RA disease states and provides support for use of dual inhibitors of COX and LOX enzymes on RA treatment.

Enantioseparation of hydroxyeicosatetraenoic acids by hydroxypropyl-γ-cyclodextrin-modified micellar electrokinetic chromatography.

Complete resolution of hydroxyeicosatetraenoic acid (HETE) enantiomers was achieved using hydroxypropyl-γ-cyclodextrin (HP-γ-CD)-modified MEKC. The optimum running conditions were determined to be utilizing a 30 mM phosphate-15 mM borate buffer (pH 9.0) containing 30 mM HP-γ-CD and 75 mM SDS as the BGE, application of +30 kV as the effective voltage, and carrying out the experiment at 15°C. The eluents were detected at 235 nm. The method was used successfully for the simultaneous separations of (S)- and (R)-enantiomers of regioisomeric 8-, 11-, 12-, and 15-HETEs. Subsequently, the optimized method was applied to evaluate the stereochemistry of 8- and 12-HETEs from the marine red algae, Gracilaria vermiculophylla and Gracilaria arcuata, respectively. The 8-HETE was found to be a mixture of 98% (R)-enantiomer and 2% (S)-enantiomer, while the 12-HETE was a mixture of 98% (S)-enantiomer and 2% (R)-enantiomer. The present study demonstrates that the HP-γ-CD-modified MEKC method is simple and sensitive and provides unambiguous information on the configuration of natural and synthetic HETEs.

Stable isotopic internal standard correction for quantitative analysis of hydroxyeicosatetraenoic acids (HETEs) in serum by on-line SPE-LC-MS/MS in selected reaction monitoring mode.

The influence of the inclusion of a stable isotopic labeled internal standard (SIL-IS) on the quantitative analysis of hydroxyeicosatetranoic acids (HETEs) in human serum is evaluated in this research. A solid-phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) platform, one of the preferred approaches for targeted analysis of biofluids through the selected reaction monitoring (SRM) operational mode, was used to determine HETEs. These compounds were chosen as targeted metabolites because of their involvement in cardiovascular disease, cancer and osteoporosis. 15HETE-d8 was chosen as internal standard to evaluate matrix effects. Thus, the physico-chemical properties of the SIL-IS were the basis to evaluate the analytical features of the method for each metabolite through four calibration models. Two of the models were built with standard solutions at different concentration levels, but one of the calibration sets was spiked with an internal standard (IS). The other two models were built with the serum pool from osteoporotic patients, which was spiked at different concentrations with the target analytes. In this case, one of the serum calibration sets was also spiked with the IS. The study shows that the IS allowed noticeable correction of matrix effects for some HETE isomers at certain concentration levels, while accuracy was decreased at low concentration (15ng/mL) of them. Therefore, characterization of the method has been properly completed at different concentration levels.

New N-acyl taurine from the sea urchin Glyptocidaris crenularis.

A new N-acyl taurine (1), together with a new natural product, l-(beta-D-ribofuranosyl)-1,2,4-triazole (4), and two known compounds (2 and 3), were isolated from the sea urchin, Glyptocidaris crenularis. The new N-acyl taurine was elucidated as 2-(5R,15S-dihydroxyeicosanoylamino) ethanesulfonic acid on the basis of spectroscopic (NMR, MS) analyses and the modified Mosher ester method. Compound 2 showed significant toxicity against brine shrimp larvae.

HPLC quantification of 5-hydroxyeicosatetraenoic acid in human lung cancer tissues.

A simple and cost-effective HPLC method was established for quantification of 5-hydroxyeicosatetraenoic acid (5-HETE) in human lung cancer tissues. 5-HETE from 27 patients' lung cancer tissues were extracted by solid-phase extraction and analyzed on a Waters Symmetry C(18) column (4.6 x 250 mm, 5 microm) with a mobile phase consisting of methanol, 10 mM ammonium acetate, and 1 M acetic acid (70:30:0.1, v:v:v) at a flow rate of 1.0 mL/min. The UV detection wavelength was set at 240 nm. The calibration curve was linear within the concentration range from 10 to 1000 ng/mL (r(2) > 0.999, n = 7), the limit of detection was 1.0 ng/mL and the limit of quantitation was 10.0 ng/mL for a 100 microL injection. The relative error (%) for intra-day accuracy was from 93.14 to 112.50% and the RSD (%) for intra-day precision was from 0.21 to 2.60% over the concentration range 10-1000 ng/mL. By applying this method, amounts of 5-HETE were quantitated in human lung cancer tissues from 27 human subjects. The established HPLC method was validated to be a simple, reliable and cost-effective procedure that can be applied to conduct translational characterization of 5-HETE in human lung cancer tissues.

Ascospore release from bottle-shaped asci in Dipodascus albidus.

Yeasts utilize different mechanisms to release ascospores of different lengths from bottle-shaped asci. Using electron microscopy, confocal laser scanning microscopy, gas chromatography-mass spectrometry and digital live imaging, the individual release of oval ascospores from tight-fitting narrow bottle-necks, is reported in the yeast Dipodascus albidus. These ascospores are surrounded by compressible, oxylipin-coated sheaths enabling ascospores to slide past each other when forced by turgor pressure and by possible sheath contractions towards the narrowing ascus-neck. In this paper, the release mechanisms of ascospores of various lengths from bottle-shaped asci and produced by different yeasts are compared. We suggest that different release mechanisms, utilizing compressible sheaths or geared-alignment, have possibly evolved to compensate for variation in ascospore length. Alternatively, sheaths and ridges might be two evolutionary solutions to the same biomechanical problem, i.e. to release ascospores irrespective of length from bottle-shaped asci.

Urinary excretion of lipoxin A(4) and related compounds: development of new extraction techniques for lipoxins.

LX are tetraene-containing eicosanoids generated by lipoxygenase (LO) transformation of arachidonic acid (Serhan and Romano, 1995). LX possess potent anti-inflammatory activity in vivo, and temporal biosynthesis of LX, concurrent with spontaneous resolution, has been observed during exudate formation (Levy et al, 2001). Limited results are currently available on the involvement of LX in clinical settings. Recently, a rabbit anti-LXA(4) antiserum has been raised to produce an enzyme-linked immunosorbent assay (ELISA) kit for LXA(4) (Levy et al, 1993). Although specific and accurate with isolated cells, this kit has not been tested with complex biological matrix such as urine. Initial attempts to determine urinary excretion of LXA(4) using the LXA(4) ELISA kit were unsuccessful because of high unspecific absorbance readings. In this report, we show that the LXA(4) extraction procedure indicated in the ELISA kit is inadequate for urinary measurements of immunoreactive (i)LXA(4). We present the development of a new extraction technique, more selective for LX, that abolishes background contamination and minimizes the unspecific readings. Using this method, we show for the first time that urine from healthy subjects contain (i)LXA(4) material and identify a urinary tetraene with the physical properties of a LXA(4) metabolite. Although reliable methods have been previously established to quantitate LXA(4) from whole blood (Brezinski et al, 1992), the present extraction technique, which optimizes for LXA(4) recovery from human urine, represents a substantial achievement for LX investigation and may open a new avenue of clinical studies on LXA(4).

16(R)-hydroxy-5,8,11,14-eicosatetraenoic acid, a new arachidonate metabolite in human polymorphonuclear leukocytes.

Intact human polymorphonuclear leukocytes (PMNL) incubated with substimulatory amounts of arachidonic acid in the absence of a calcium ionophore formed four metabolites that were isolated by reverse-phase HPLC and characterized structurally by GC/MS. A major metabolite eluting as the most abundant peak of radioactivity lacked UV chromophores above 215 nm, and its formation was sensitive to 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride (SKF525A) but not 3-amino-1-[m(trifluoromethyl)phenyl]-2-pyrazoline (BW755C), suggesting that it was likely to be a product of cytochrome P450. The GC/MS analysis revealed the presence of two components: 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) and 16-hydroxy-5,8,11,14-eicosatetraenoic acid (16-HETE) in an approximate ratio of 4:1. The minor metabolites were identified as 15-HETE and 5-HETE. Although 20-HETE has been observed previously as a product of arachidonic acid metabolism in PMNL, the occurrence of 16-HETE was a novel finding. The stereochemistry of the hydroxyl group in PMNL-derived 16-HETE was established by analysis of 1-pentafluorobenzyl-16-naphthoyl derivatives on a chiral-phase chromatographic column and comparison with authentic synthetic stereoisomers. The PMNL-derived radioactive metabolite co-eluted with the synthetic 16(R)-HETE stereoisomer. Analysis of the total lipid extracts from intact PMNL followed by mild alkaline hydrolysis resulted in detectable amounts of 16-HETE (108+/-26 pg/10(8) cells) and 20-HETE (341+/-69 pg/10(8) cells), which suggested that these HETEs were formed from endogenous arachidonic acid and esterified within PMNL lipids. Thus, in contrast to calcium ionophore-stimulated neutrophils that generate large amounts of 5-lipoxygenase products, the intact PMNL generate 20-HETE and 16(R)-HETE via a cytochrome P450 omega- and omega-4 oxygenase(s).

Metabolism of arachidonic acid to 20-hydroxy-5,8,11, 14-eicosatetraenoic acid by P450 enzymes in human liver: involvement of CYP4F2 and CYP4A11.

20-Hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) is a principal arachidonic acid (AA) metabolite formed via P450-dependent oxidation in hepatic and renal microsomes. Although 20-HETE plays an important role in the regulation of cell and/or organ physiology, the P450 enzyme(s) catalyzing its formation in humans remain undefined. In this study, we have characterized AA omega-hydroxylation to 20-HETE by human hepatic microsomes and identified the underlying P450s. Analysis of microsomal AA omega-hydroxylation revealed biphasic kinetics (KM1 and VMAX1 = 23 microM and 5.5 min-1; KM2 and VMAX2 = 144 microM and 18.8 min-1) consistent with catalysis by at least two enzymes. Of the human P450s examined, CYP4A11 and CYP4F2 were both potent AA omega-hydroxylases, exhibiting rates of 15.6 and 6.8 nmol 20-HETE formed/min/nmol P450, respectively. Kinetic parameters of 20-HETE formation by CYP4F2 (KM = 24 microM; VMAX = 7.4 min-1) and CYP4A11 (KM = 228 microM; VMAX = 49.1 min-1) resembled the low and high KM components, respectively, found in liver microsomes. Antibodies to CYP4F2 markedly inhibited (93.4 +/- 6%; n = 5) formation of 20-HETE by hepatic microsomes, whereas antibodies to CYP4A11 were much less inhibitory (13.0 +/- 9%; n = 5). Moreover, a strong correlation (r = 0.78; P < .02) was found between microsomal CYP4F2 content and AA omega-hydroxylation among nine subjects. The correlation (r = 0.76; P < .02) also noted between CYP4A11 content and 20-HETE formation stemmed from the relationship (r = 0.83; P < . 02) between hepatic CYP4A11 and CYP4F2 levels in the subjects. Finally, immunoblot analysis revealed that in addition to liver, both P450s also were expressed in human kidney. Our results indicate that AA omega-hydroxylation in human liver is catalyzed by two enzymes of the CYP4 gene family, namely CYP4F2 and CYP4A11, and that CYP4F2 underlies most 20-HETE formation occurring at relevant AA concentrations.

High-pressure liquid chromatography of oxo-eicosanoids derived from arachidonic acid.

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.

Lipoxygenase activity in heart cells.

Arachidonic acid (AA) metabolism via the lipoxygenase (LOX) pathway in rat hearts and in cultured rat cardiomyocytes was investigated using 1-[14C]AA. LOX activity was detected in the microsomal fraction, in the high speed supernatant prepared from rat hearts and in rat cardiomyocyte supernatant. LOX products from all fractions comigrated in thin layer chromatography as 12-hydroxyeicosatetraenoic acid (12-HETE) and 15-HETE. Enzyme linked immunosorbent assay for 12-HETE showed its formation by the microsomal fraction, the ammonium sulfate (AS) pellet, and by rat cardiomyocyte supernatant, while radioimmunoassay for 15-HETE showed its formation only by the AS pellet. The properties of LOX in each fraction are reported here.

Synthesis of hydroxyeicosatetraenoic (HETEs) and epoxyeicosatrienoic acids (EETs) by cultured bovine coronary artery endothelial cells.

Endothelial cells release several factors which influence vascular tone, leukocyte function and platelet aggregation. Some of these factors are metabolites of arachidonic acid, most notably prostacyclin. However, many of the endothelial metabolites of arachidonic acid have not been positively identified. The purpose of these studies is to identify the arachidonic acid metabolites synthesized by bovine coronary endothelial cells. Cultured bovine coronary artery endothelial cells were incubated with [14C]arachidonic acid. The incubation media was extracted and the radioactive metabolites resolved by a combination of reverse phase- and normal phase-high pressure liquid chromatography (HPLC). The cells synthesized 6-keto prostaglandin (PG)F1 alpha, PGE2, 12-hydroxyheptadecatrienoic acid (HHT), 12-, 15-, and 11-hydroxyeicosatetraenoic acids (HETE), and 14,15-, 11,12-, 8,9-, and 5,6-epoxyeicosatrienoic acids (EET). Several of the HETEs were further analyzed by chiral-phase HPLC. The cells synthesized predominately 12(S)-, 15(S)-, and 11(R)-HETE. The synthesis of the S optical isomers of 12- and 15-HETE suggested that the 12- and 15-lipoxygenases were present in these cells. 11(R)-HETE is probably derived from cyclooxygenase. They also synthesized smaller amounts of 9-, 8- and 5-HETEs. The structures of the HETEs and EETs were confirmed by mass spectrometry. The release of 6-keto PGF1 alpha and 15-HETE was measured by specific radioimmunoassays. Melittin, thrombin, arachidonic acid and A23187 stimulated the release of both eicosanoids in a concentration-related matter. Under all conditions, the release of 6-keto PGF1 alpha exceed the release of 15-HETE. Therefore, cultured bovine coronary artery endothelial cells synthesize cyclooxygenase, lipoxygenase and cytochrome P-450 metabolites of arachidonic acid.

5-15-diHETE and lipoxins generated by neutrophils from endogenous arachidonic acid as asthma biomarkers.

5-Lipoxygenase (5-LO) activation of human blood polymorphonuclear cells (PMN) from healthy subjects (HS) and from asthmatic patients (AP) was investigated comparing their respective capacities to produce lipoxins, 5,15-dihydroxyeicosatetraenoic acid (5,15-diHETE) and leukotrienes, under in vitro stimulation by ionophore A23187. PMN from AP were able to generate higher leukotriene levels from endogenous sources than PMN from HS. Moreover they produced 5,15-diHETE (from 50 to 280ng/10(7) cells) and lipoxins (from 1 to 30ng/10(7) cells), in a linear manner, whereas in the same experimental conditions no detectable amounts of these compounds appeared in PMN from HS. The enhanced 5-LO activation of blood PMN may reflect transcellular signalisation priming indicating that lipoxins and 5,15-diHETE could be much more specific inflammatory state biomarkers than leukotriene B4.

Method for simultaneous isolation and quantitation of platelet activating factor and multiple arachidonate metabolites from small samples: analysis of effects of Staphylococcus aureus enterotoxin B in mice.

A novel method has been developed for the extraction and simultaneous separation and quantitation of key arachidonate metabolites and platelet activating factor (PAF) from plasma samples of limited size. Aqueous solutions of these metabolites were added onto a solid phase C-18 cartridge and arachidonate metabolites and PAF were eluted, successively, with acetonitrile-methanol (85:15, v/v), followed by 100% methanol. Arachidonate metabolites (first eluate) were fractionated by C-18 reverse-phase high-performance liquid chromatography using a program designed for the resolution of 31 arachidonate metabolites (3-min separation). The fractions were collected and assayed by radioimmunoassay or radiography, if radioactively labeled. Two internal standards were added to each sample, 15-hydroxyeicosadienoic acid (detected at 235 nm) to determine gradient shifts and [1-14C]eicosatrienoic acid to estimate the recoveries of arachidonate metabolites at any stage of the process. This method was developed to answer specific questions concerning the mode of action of Staphylococcus aureus enterotoxin B (SEB). Plasma samples from mice challenged with SEB were analyzed and major differences were seen in 5-lipoxygenase metabolites. Low doses of SEB vs saline stimulated 5-hydroxyeicosatetraenoic acid production, while high doses of SEB stimulated leukotriene D4 production.

Egg hatching activity of trihydroxylated eicosanoids in the barnacle Balanus balanoides.

Previous work has shown that eicosanoid hatching factors can be isolated from tissues of a variety of barnacle species. To further investigate the nature of barnacle hatching factor an eicosanoid fraction isolated from homogenates of the barnacle Balanus balanoides was purified on RP-HPLC and trihydroxy fatty acid fractions were bioassayed for hatching activity and analysed by GC-MS. The identified eicosanoids were 8,11,12-trihydroxy-5,9,14-eicosatrienoic acid, 8,11,12-trihydroxy-5,9,14,17-eicosatetraenoic acid, 10,11,12-trihydroxy-5,8,14,17-eicosatetraenoic acid and 10,13, 14-trihydroxy-4,7,11,16,19-docosapentaenoic acid. These studies suggest that barnacles possess a potent n-9 lipoxygenase which can metabolise either arachidonic acid, eicosapentaenoic acid or docosahexaenoic acid. The predominant eicosanoids common to all active fractions were either 8,11,12-trihydroxyeicosatrienoic acid or an isomer of 8,11-12-trihydroxyeicosatetraenoic acid, which suggests that a particular configuration of an 8,11,12 triol is barnacle hatching factor.

Formation of 19(S)-, 19(R)-, and 18(R)-hydroxyeicosatetraenoic acids by alcohol-inducible cytochrome P450 2E1.

When reconstituted with cytochrome b5 and NADPH cytochrome P450 oxidoreductase, cytochrome P450 2E1 metabolized lauric, stearic, oleic, linoleic, linolenic, and arachidonic acid to multiple metabolites. Two major metabolites, accounting for 78% of the total metabolism, were produced with arachidonic acid. The Vmax for total metabolite formation from arachidonic acid was 5 nmol/min/nmol P450 with an apparent Km of 62 microM. Gas chromatography-mass spectrometry analysis identified the two major metabolites as monohydroxylated eicosatetraenoic acids (HETEs). The major HETE was 19-hydroxyeicosatetraenoic acid (19-HETE) and comprised 46% of the total metabolite produced. The second metabolite was the omega-2 hydroxylated metabolite (18-HETE) and comprised 32% of the total product formed. Chiral analysis demonstrated that 19-HETE was 70% 19(S)-HETE and 30% 19(R)-HETE. In contrast, 18-HETE was essentially 100% R isomer. Approximately 18% of the total metabolite produced from arachidonic acid coeluted with epoxyeicosatrienoic acid (EET) standards. The EET metabolites were 56.4% 14,15-EET and 43.6% as a mixture of 11,12-EET and 8,9-EET. 5,6-EET was not detected. Anti-P450 2E1 IgG inhibited arachidonic acid metabolism by renal and hepatic microsomes prepared from acetone-treated rabbits. With renal cortex microsomes, the formation of 18-HETE and 19-HETE was inhibited 67 and 25%, respectively, by the antibody. Liver microsomal formation of 18-HETE was inhibited by 87% and 19-HETE by 70%. Thus, under conditions where cytochrome P450 2E1 is induced, the enzyme could contribute significantly to the formation of the omega-1 and omega-2 hydroxylated metabolites of arachidonic acid.

12-Lipoxygenase from rat basophilic leukemia cells, an oxygenase with leukotriene A4-synthase activity.

Rat basophilic leukemia cells exhibit 12-lipoxygenase activity only upon cell disruption. 12-Lipoxygenase may also possess 15-lipoxygenase activity, as is indicated by the formation of low amounts of 15(S)-HETE, in addition to the predominant product 12(S)-HETE, upon incubation of partially purified 12-lipoxygenase with arachidonic acid. With 5(S)-HPETE as substrate not only 5(S), 12(S)-diHETE and 5(S), 15(S)-diHETE are formed, but also LTA4, as was indicated by the presence of LTA4-derived LTB4-isomers. 12-Lipoxygenase from rat basophilic leukemia cells has many features in common with 12-lipoxygenase from bovine leukocytes. As was suggested for the latter enzyme, 12-lipoxygenase from rat basophilic leukemia cells may represent the remaining LTA4-synthase activity of 5-lipoxygenase, of which the 5-dioxygenase activity has disappeared upon cell disruption. Such a possible shift from 5-lipoxygenase activity to 12-lipoxygenase activity could not simply be induced by interaction of cytosolic 5-lipoxygenase with a membrane fraction after cell disruption, but may involve release of membrane-associated 5-lipoxygenase upon disruption of activated rat basophilic leukemia cells.

A unique pool of free arachidonate serves as substrate for both cyclooxygenase and lipoxygenase in platelets.

Stimulation of platelets induces a rapid release of arachidonate from specific phospholipids and subsequent remodeling of arachidonate-containing phospholipids. This process is accompanied by transformation of released arachidonate by cyclooxygenase and lipoxygenase enzymes. We addressed the question of whether the cyclooxygenase and the lipoxygenase products originated from the same arachidonate-containing phospholipids. [14C]Arachidonate prelabeled platelets were stimulated by thrombin or by ionophore A 23187. We monitored the cyclooxygenase pathway by following 12-hydroxy-5,8,10-heptadecatrienoic acid [12(S)-HHT] formation and the lipoxygenase pathway by following 12-hydroxy-5,8,10,14-eicosatetraenoic acid [12(S)-HETE] formation and compared specific activities. The data showed that the same pool of released arachidonate can be utilized by either cyclooxygenase or by lipoxygenase. Indeed, the specific activity of both products was identical when both enzymes were acting. Since cyclooxygenase was rapidly deactivated while lipoxygenase continued to be active, the specific activity of 12(S)-HETE became lower than the specific activity of 12(S)-HHT when large amounts of 12(S)-HETE were synthesized. Based on comparison of specific activity between phospholipids and oxygenated products, the pools of arachidonate-containing phospholipids involved in the synthesis of oxygenated products are dependent on the amount of arachidonate released.