Jorge Capdevila

The reaction of arachidonic acid epoxides (epoxyeicosatrienoic acids) with a cytosolic epoxide hydrolase

Epoxyeicosatrienoic acids, formed during the cytochrome P-450-catalyzed oxidation of arachidonic acid, react with a liver cytosolic epoxide hydrolase to form vicinal diols of eicosatrienoic acid. The role of this cytosolic enzyme, rather than a microsomal bound type, explains previous results illustrating the ability to accumulate epoxides during the in vitro aerobic steady state of oxidative metabolism of arachidonic acid by liver microsomes. The inability of the 5,6-epoxyeicosatrienoic acid to serve as a suitable substrate for this enzyme is discussed in light of recent studies concerning possible unique physiological functions for this metabolite.

Intestinal vasodilation by epoxyeicosatrienoic acids: arachidonic acid metabolites produced by a cytochrome P450 monooxygenase.

Purified synthetic products from the cytochrome P450 pathway of arachidonate metabolism were applied to the intestinal serosa. Arteriolar blood flow was calculated using video microscopy. After a steady-state baseline, a bolus containing 10-60 micrograms 14,15-epoxyeicosatrienoic acid/ml (14,15-EET) had no detectable effect on blood flow. However, 25 +/- 3 micrograms 11,12-EET/ml and 36 +/- 2 micrograms 8,9-EET/ml caused increases (134 +/- 8% and 127 +/- 6%) that were similar to those elicited by 8 +/- 2 micrograms adenosine/ml (138 +/- 12%). Furthermore, the increases (275 +/- 38%) produced by 32 +/- 6 micrograms 5,6-EET/ml exceeded those elicited (160 +/- 10%) by a similar concentration (27 +/- 3 micrograms/ml) of adenosine. Thus, a structure-activity relationship is suggested. Nevertheless, these values probably underestimate the potency of the EETs because the vasoactivity was reduced by contact with water. The activity of the cyclooxygenase pathway seemed to limit the formation of vasoactive quantities of EETs, or other nonprostanoids, from exogenous arachidonate in the serosa but not the mucosa. A bolus (1.3 +/- 0.2 mg/ml) or continuous application (122 +/- 45 micrograms/ml) of arachidonate caused blood flow increases (236 +/- 14% or 229 +/- 27%) that were almost eliminated (129 +/- 5% or 121 +/- 9%) by a cyclooxygenase inhibitor; the residual response was abolished by a cytochrome P450 inhibitor. However, cytochrome P450 inhibitors alone did not attenuate the arachidonate response. In contrast, a continuous application of 194 micrograms arachidonate/ml to the mucosa caused a markedly smaller blood flow increase (119 +/- 8%) and cyclooxygenase inhibitors potentiated (132 +/- 8%), rather than reduced, this response. We conclude that EETs are a labile class of vasodilators with a potency comparable to adenosine in the intestinal microcirculation. Indirect evidence suggests regional differences in the formation of vasoactive quantities of arachidonate metabolites within the intestinal wall.

The oxidative metabolism of arachidonic acid by purified cytochromes P-450☆

Abstract Arachidonic acid is catalytically oxidized using either of two types of purified cytochrome P-450 reconstituted with the purified flavo-protein, NADPH-cytochrome P-450 reductase. The reaction is dependent on the presence of cytochrome P-450, NADPH, and oxygen. The patterns of products formed are unique for the type of cytochrome P-450 used. This suggests an enzyme-directed specificity of the site of attack on the unsaturated fatty acid by the hemeprotein. Additional experiments show a possible role for cytochrome b5 since the addition of purified cytochrome b5 enhances the rate of metabolism of arachidonic acid 2 to 3 fold.

Renal Microsomal Cytochrome P-450 and the Oxidative Metabolism of Arachidonic Acid*

Arachidonic acid is oxidized by the microsomal cytochrome P-450 monooxygenases of rabbit kidney cortex and medulla to products that include W and W-1 alcohols and several epoxyacids. In vivo administration of deoxycorticosterone acetate results in a time dependent and selective induction of the arachidonic acid W/W-1 oxygenase activity of cortex and medulla microsomal fractions (145% and 220% of controls, respectively). Initial segmental analysis shows the presence of an active epoxygenase activity in the isolated rabbit pars recta and its in vitro activation by p-aminohippuric acid.

Novel glutathione conjugates formed from epoxyeicosatrienoic acids (EETs)

The catalysis of glutathione (GSH) conjugation to epoxyeicosatrienoic acids (EETs) by various purified isozymes of glutathione S-transferase was studied. A GSH conjugate of 14,15-EET was isolated by HPLC and TLC; this metabolite contained one molecule of EET and one molecule of GSH. Fast atom bombardment mass spectrometry of the isolated metabolite confirmed the structure as a GSH conjugate of 14,15-EET. Studies designed to determine the isozyme specificity of this reaction demonstrated that two isozymes, 3-3, and 5-5, efficiently catalyzed this conjugation reaction. The Km values for 14,15-EET were approximately 10 microM and the Vmax values ranged from 25 to 60 nmol conjugate formed min-1 mg-1 purified transferase 3-3 and 5-5. The 5,6-, 8,9-, and 11,12-EETs were also substrates for the reaction, albeit at lower rates. These results demonstrate that the EETs can serve as substrates for the cytosolic glutathione S-transferases.

Synthesis of arachidonic acid metabolites produced by purified kidney cortex microsomal cytochrome P-450

Abstract Reported are the synthesis and structure confirmation of the major metabolites produced during the NADPH dependent oxidation of arachidonic acid by a reconstituted enzyme system containing purified kidney cortex microsomal cytochrome P-450.

Synthesis of epoxyeicosatrienoic acids and heteroatom analogs.

Publisher Summary The chapter presents a study on synthesis of epoxyeicosatrienoic acids and heteroatom analogs. The chapter mentions the need for a comprehensive review of the current status of the epoxygenase pathway with a critical discussion of its implications. The most characteristic and extensively studied epoxygenase metabolites are the four regioisomeric cis -epoxyeicosatrienoic acids (EETs). They are relatively stable to hydrolysis at physiological pH [for example, leukotriene A 4 (LTA 4 ) and hepoxilin A 3 ]. The exception is 5,6-EET which, in its free-acid form, readily decomposes to 5,6-dihydroxyeicosatrienoic acid and/or the corresponding δ -1actone. The absolute configuration of the EETs has been determined using material obtained from in vitro incubation of arachidonic acid with the major phenobarbital-inducible form of rat liver microsomal cytochrome P -450. Recent results suggest that the enantiomeric composition is highly dependent on the identity of the cytochrome P -450 isozyme. This may be significant because in some instances biological activity is sensitive to EET stereochemistry. Chiral syntheses of all the EET enantiomers have been achieved. Most investigators have relied on racemic EETs prepared by Coreys site-specific oxidation methodology or, more conveniently, by nonselective peracid epoxidation of arachidonic acid. This chapter describes procedures based on the latter reaction that have proved reliable in laboratories for the production and purification of nanomole-millimole amounts of EETs and their methyl esters. In light of the increasing interest in EET heteroatom analogs, protocols for converting EETs to cis -thiiranes and cis -aziridines are also included.

Effects of newly arachidonic acid metabolites on microsomal Ca++ binding, uptake and release

The 5,6-; 8,9-; 11,12- and 14,15-epoxyeicosatrienoic acids and their respective hydration products, the vic-diols, recently reported as metabolites of arachidonic acid in rat liver microsomes, were examined for effect on release of 45Ca from canine aortic smooth muscle microsomes. At 10(-6) M, the diols had no effect, but the 5,6-; 11,12- and 14,15-epoxyacids increased the loss of 45Ca. Further studies with the 14,15-epoxyacid demonstrated a dose-dependent decrease of Ca++ uptake (ATP present) in canine aortic microsomes in 0.03 mM Ca++, whereas Ca++ binding (ATP absent) was not affected. Ca++ uptake, binding and release in rat liver microsomes was similarly affected by the 14,15-epoxyacid, the major epoxyeicosatrienoic acid derivative produced by rat liver microsomal incubations. It is suggested that alterations in Ca++ metabolism might be a possible mechanism of action for these derivatives of arachidonic acid.

Palladium mediated allylic Mitsunobu displacement : stereocontrolled synthesis of hepoxilin A3 and trioxilin A3 methyl esters

A regio- and stereoselective palladium mediated allylic displacement under Mitsunobu conditions was exploited for the preparation of several hepoxilin A3 and trioxilin A3 stereoisomers.

The mechanistic plurality of cytochrome P-450 and its biological ramifications.

The mechanistic plurality of the microsomal cytochrome P-450 enzyme system is illustrated by studies of the oxidative metabolism of benzo[a]pyrene, 3-hydroxybenzo[a]pyrene and arachidonic acid. Rat liver microsomal metabolism of benzo[a]pyrene or 3-hydroxy-benzo[a]pyrene, supported by cumene hydroperoxide, generates benzo[a]pyrene quinones via molecular oxygen-dependent and -independent pathways. Arachidonic acid is metabolized by rat liver microsomal fractions to a variety of oxygenated products, including cis-trans diene conjugated monohydroxy-acids, epoxy-acids as well as omega- and omega-1-oxidation products. The chemistry of the different reaction products is discussed in terms of the possible mechanisms responsible for their formation and the role of the haemoprotein during catalysis. An integrated view for the reaction cycle of cytochrome P-450 is presented.