Ernst Oliw

Manganese Lipoxygenase DISCOVERY OF A BIS-ALLYLIC HYDROPEROXIDE AS PRODUCT AND INTERMEDIATE IN A LIPOXYGENASE REACTION

Linoleic acid was incubated with manganese lipoxygenase (Mn-LO) from the fungus Gäumannomyces graminis. The product consisted of (13R)-hydroperoxy-(9Z,11E)-octadecadienoic acid ((13R)-HPOD) and a new hydroperoxide, (11S)-hydroperoxy-(9Z,12Z)-octadecadienoic acid ((11S)-HPOD). Incubation of (11R)-[2H]- and (11S)-[2H]linoleic acids with Mn-LO led to the formation of hydroperoxides that largely retained and lost, respectively, the deuterium label. Conversion of the (11S)-deuteriolinoleic acid was accompanied by a primary isotope effect, which manifested itself in a strongly reduced rate of formation of hydroperoxides and in a time-dependent accumulation of deuterium in the unconverted substrate. These experiments indicated that the initial step catalyzed by Mn-LO consisted of abstraction of the pro-S hydrogen of linoleic acid to produce a linoleoyl radical. (11S)-HPOD was converted into (13R)-HPOD upon incubation with Mn-LO. The mechanism of this enzyme-catalyzed hydroperoxide rearrangement was studied in experiments carried out with18O2 gas or18O2-labeled hydroperoxides. Incubation of [11-18O2](11S)-HPOD with Mn-LO led to the formation of (13R)-HPOD, which retained 39–44% of the 18O label, whereas (11S)-HPOD incubated with Mn-LO under 18O2 produced (13R)-HPOD, which had incorporated 57% of 18O. Furthermore, analysis of the isotope content of (11S)-HPOD remaining unconverted in such incubations demonstrated that [11-18O2](11S)-HPOD suffered a time-dependent loss of 18O when exposed to Mn-LO, whereas (11S)-HPOD incorporated 18O when incubated with Mn-LO under 18O2. On the basis of these experiments, it was proposed that the conversion of (11S)-HPOD into (13R)-HPOD occurred in a non-concerted way by deoxygenation into a linoleoyl radical. Subsequent reoxygenation of this intermediate by dioxygen attack at C-13 produced (13R)-HPOD, whereas attack at C-11 regenerated (11S)-HPOD. The hydroperoxide rearrangement occurred by oxygen rebound, although, as demonstrated by the 18O experiments, the oxygen molecule released from (11S)-HPOD exchanged with surrounding molecular oxygen prior to its reincorporation.

Identification of CYP4F8 in Human Seminal Vesicles as a Prominent 19-Hydroxylase of Prostaglandin Endoperoxides

A novel cytochrome P450, CYP4F8, was recently cloned from human seminal vesicles. CYP4F8 was expressed in yeast. Recombinant CYP4F8 oxygenated arachidonic acid to (18R)-hydroxyarachidonate, whereas prostaglandin (PG) D2, PGE1, PGE2, PGF2α, and leukotriene B4 appeared to be poor substrates. Three stable PGH2 analogues, 9,11-epoxymethano-PGH2 (U-44069), 11,9-epoxymethano-PGH2 (U-46619), and 9,11-diazo-15-deoxy-PGH2 (U-51605) were rapidly metabolized by ω2- and ω3-hydroxylation. U-44069 was oxygenated with aV max of ∼260 pmol min− 1 pmol P450− 1 and a K m of ∼7 μm. PGH2decomposes mainly to PGE2 in buffer and to PGF2α by reduction with SnCl2. CYP4F8 metabolized PGH2 to 19-hydroxy-PGH2, which decomposed to 19-hydroxy-PGE2 in buffer and could be reduced to 19-hydroxy-PGF2α with SnCl2. 18-Hydroxy metabolites were also formed (∼17%). PGH1 was metabolized to 19- and 18-hydroxy-PGH1 in the same way. Microsomes of human seminal vesicles oxygenated arachidonate, U-44069, U-46619, U-51605, and PGH2, similar to CYP4F8. (19R)-Hydroxy-PGE1 and (19R)-hydroxy-PGE2 are the main prostaglandins of human seminal fluid. We propose that they are formed by CYP4F8-catalyzed ω2-hydroxylation of PGH1 and PGH2 in the seminal vesicles and isomerization to (19R)-hydroxy-PGE by PGE synthase. CYP4F8 is the first described hydroxylase with specificity and catalytic competence for prostaglandin endoperoxides.

Oxygenation of arachidonic acid by hepatic microsomes of the rabbit: Mechanism of biosynthesis of two vicinal dihydroxyeicosatrienoic acids

[1-14C] Arachidonic (eicosatetraenoic) acid was incubated at 37 degrees C for 15 min with rabbit liver microsomes fortified with NADPH (1 mM). The products were purified by high-pressure liquid chromatography (HPLC) and analyzed by gas chromatography-mass spectrometry. Based on polarity on reversed phase HPLC, the metabolites could be divided into three groups. The major metabolites of lowest polarity were 19- and 20-hydroxyarachidonic acid and 19-oxoarachidonic acid. The major metabolites of medium polarity were two diols, 14,15-dihydroxy-5,-8,11-eicosatrienoic acid and 11,12-dihydroxy-5,8,14-eicosatrienoic acid. Microsomal incubation under atmospheric isotopic oxygen led to incorporation of only one 18O molecule in each diol, indicating that the diols could originate from breakdown of 14(15)-oxido-5,8,11-eicosatrienoic acid and 11(12)-oxido-5,8,14-eicosatrienoic acid, respectively. Major metabolites in the most polar group were 14,15,19- and 14,15,20-trihydroxy-5,8,11-eicosatrienoic acid. 11,12,19- and 11,12,20-trihydroxy-5,8,14-eicosatrienoic acid and 11,12-dihydroxy-19-oxo-5,8,-14-eicosatrienonic acid. About 0.5% of exogenous radioactively labelled arachidonic was covalently bound to microsomal proteins. The metabolites and the protein-bound products were formed in considerably smaller amounts by non-fortified microsomes. Carbon monoxide inhibited this pathway of arachidonic acid metabolism, indicating that these reactions might be catalyzed by the cytochrome P-450-linked monooxygenase systems.

Reduction by indomethacin of furosemide effects in the rabbit

The effects of furosemide on urine flow, sodium and potassium excretion and on plasma renin activity (PRA) were studied in anesthetized rabbits with and without pretreatment with indomethacin 5 mg/kg. Furosemide caused a 10-fold increase in urine flow and in sodium excretion, and a 2-3 fold increase in PRA. Pretreatment with the prostaglandin synthesis inhibitor, indomethacin, reduced the effects of furosemide on diuresis and on electrolyte excretion by over 80% (p less than 0.01) and PRA did not increase over the initial level. The results suggest that the effects of furosemide on PRA and on urinary sodium excretion may be related to the intrarenal activation of the prostaglandin system.

Metabolism of arachidonic acid by isolated rat hepatocytes, renal cells and by some rabbit tissues: Detection of vicinal diols by mass fragmentography

Purified cytochromes P-450 (LM2 and PB-B2) in a reconstituted system and epoxide hydrolase were recently found to metabolize arachidonic (eicosatetraenoic) acid to four vicinal dihydroxyeicosatrienoic acids. These metabolites were chemically synthetized from octadeuterated arachidonic acid and employed as internal standards for mass fragmentography. Isolated rat hepatocytes and renal cells were incubated with arachidonic acid (0.1 mM; 37 degrees C, 15 min) and, following extractive isolation and reversed-phase HPLC, formation of 11,12-dihydroxy-5,8,14-eicosatrienoic acid and 14,15-dihydroxy-5,8,11-eicosatrienoic acid was demonstrated by mass fragmentography using a capillary GC column. Furthermore, these diols were also detected in rabbit liver and renal cortex and they therefore appear to be formed endogenously. Formation of vicinal diols was also studied in cell free systems. Rabbit liver and renal cortical microsomes were incubated with NADPH (1 mM) and arachidonic acid (0.15 mM) for 15 min at 37 degree C and, besides 11,12-dihydroxy- and 14,15-dihydroxyeicosatrienoic acid, small amounts of 8,9-dihydroxy- and 5,6-dihydroxyeicosatrienoic acid could be detected by mass fragmentography. Renal as wall as hepatic monooxygenases can thus epoxidize each of the four double bonds of arachidonic acid. In contrast, rabbit lung microsomes and NADPH metabolized arachidonic acid mainly to prostaglandins and 19-hydroxy- and 20-hydroxyarachidonic acid, while only small amounts of 11,12-dihydroxyeicosatrienoic acid could be found. Monooxygenase metabolism of arachidonic acid by epoxidation might therefore be a significant pathway for the metabolism of this essential fatty acid in isolated rat renal cells and hepatocytes but presumably not in the lung.

Prostaglandins and thromboxanes in amniotic fluid during rivanol-induced abortion and labour

Abortion or delivery were induced by extra-amniotic instillation of Rivanol during the second trimester in twelve patients and during the third trimester in two patients with fetal death and one patient with fetal acrania. Serial sampling of amniotic fluid was performed through a transabdominal catheter and the levels of free arachidonic acid (AA), prostaglandin F2 alpha (PGF2 alpha), prostaglandin E2 (PGE2), 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) and thromboxane B2 (TXB2) were determined. The levels of AA, PGF2 alpha, PGE2, 6-keto-PGF1 alpha and TXB2 in amniotic fluid increased significantly during induction with the exception of AA in fetal death which was high and remained constant during induction. Furthermore, PGF2 alpha, 6-keto-PGF1 alpha and TXB2 were all significantly correlated to AA. These observations suggested that free AA is released during Rivanol-induction of abortion and labour giving an increased synthesis of PGF2 alpha, PGE2 prostacyclin and thromboxane A2 in the fetal membranes and the decidua but not in the fetus. This increase might be relevant for the initiation and progress of abortion and labour in these patients.

Indomethacin and diclofenac sodium increase sodium and water excretion after extracellular volume expansion in the rabbit

The renal effects of an acute extracellular fluid volume expansion (50 ml Ringer/kg body weight/60 min) were studied in aldosterone-treated (100 microgram/kg), anesthetized rabbits with and without pretreatment with either indomethacin (3.0 mg/kg) or diclofenac sodium (3.0 mg/kg), two different inhibitors of renal prostaglandin (PG) biosynthesis. In controls (n = 7), the volume expansion increased urine flow from 1.5 +/- 0.24 to 6.1 +/- 0.5 (S.E.) ml/min/100 g kidney weight and sodium excretion from 0.15 +/- 0.03 to 0.99 +/- 0.10 mmol/min/100 g. PAH and inulin clearance increased by 42 and 58%, respectively, while plasma renin activity and urinary excretion of PGF2 alpha-like immunoreactivity were reduced (P less than 0.05). In animals pretreated with indomethacin (n = 6) or diclofenac sodium (n = 6), the diuresis and the natriuresis following volume expansion were significantly increased about two-fold over controls, whereas PAH and inulin clearance, plasma renin activity and hematocrit did not differ from controls. Both drugs were found to reduce urinary excretion of PGF2 alpha-like immunoreactivity by 75--95% througout the experiment. The results indicate that diclofenac sodium, indomethacin and extracellular volume expansion enhance sodium and water excretion partly by suppression of a PG sensitive reabsorption process in the kidney.

Metabolism of 5(6)-expoxyeicosatrienoic acid by ram seminal vesicles. Formation of novel prostaglandin E1 metabolites.

5(6)-Epoxy-8,11,14-eicosatrienoic acid was incubated with microsomes or ram seminal vesicles in the presence of glutathione (1 mM) for 2 min at 37 degrees C. Following extractive isolation on octadecasilane silica, the products were purified on straight-phase HPLC and separated into three major polar metabolites, which all showed maximal ultraviolet absorbance at 278 nm after treatment with alkali. The least-polar of the three metabolites was identified by capillary column gas chromatography-mass spectrometry as 5(6)- epoxyprostaglandin E1 and the structure was confirmed by comparison with authentic material. The most-polar metabolite was identified as 5,6- dihydroxyprostaglandin E1, while the metabolite of medium polarity was identified as its delta 5-lactone. When glutathione was omitted, 5-hydroxyprostaglandin I 1 alpha and 5-hydroxyprostaglandin I 1 beta were previously identified as the two major metabolites. These results indicate that the postulated epoxyprostaglandin endoperoxide intermediates, 5(6)- epoxyprostaglandin G1 and 5(6)- epoxyprostaglandin H1, might be substrates for the endoperoxide E isomerase enzyme, since this enzyme requires glutathione as a cofactor.

Acute unilateral ureteral occlusion increases plasma renin activity and contralateral urinary prostaglandin excretion in rabbits.

Abstract The effect of an acute, complete unilateral ureteral occlusion was studied on plasma renin activity (PRA), arterial blood pressure and contralateral kidney function in three groups of chloralose-urethane-anesthetized rabbits undergoing a slight mannitol diuresis. Following the acute ureteral occlusion in controls (n =6), PRA increased 3–5 times, mean blood pressure increased 11±5 (S.E.M.) mm Hg and the pressure in the occluded ureter increased to 51 ± 2 mm Hg. In the intact kidney sodium excretion increased from 0.14 ± 0.05 to 0.47 ± 0.12 mmol/min/100 g kidney weight and output of immunoreactive PGE2 (iPGE2) and iPGF2α increased 3–5 fold without significant changes in the clearance of PAH and creatinine. In the second group (n =6), which was pretreated with diclofenac sodium (3 mg/kg i.v.), a PG synthesis inhibitor, no increase in PRA and blood pressure was observed. The pressure of the occluded ureter increased only to 32 ± 2 Hg. The output of iPGE2 and iPGF2α from the intact kidney was depressed by 80–95% and the sodium excretion remained unchanged. The third group (n = 6) was treated by infusion of the angiotensin II antagonist saralasin acetate (6μg/kg/min). In this group the increase in blood pressure was blocked, whereas PRA increased. The pressure of the occluded ureter rose to 40 ± 3 mm Hg. The excretion of iPGE2 from the intact kidney increased 2–4 fold and that of iPGF2α 2–3 fold. These results indicate that in acute unilateral ureteral occlusion angiotensin II might be of importance for the increase in arterial blood pressure but not for PG release from the intact kidney, while on the other hand, the renal PGs could play an important role in the renal release of renin.

Chapter 1 The prostaglandins and essential fatty acids

Publisher Summary This chapter discusses prostaglandins and essential fatty acids. The systematic nomenclature of prostaglandins and prostaglandin metabolites is based on prostanoic acid, a monocarboxylic acid with 20 carbon atoms, arranged as two side chains with 7 and 8 carbons, respectively, linked to a central cyclopentane ring. Prostaglandins have functional groups with oxygen at carbons 9, 11, and 15 of prostanoic acid and one, two or three double bonds in the side chains. Prostaglandins are also classified by the functional groups of the cyclopentane ring. The structures of the cyclopentane ring with functional groups of A, B, C, D, E, F, G, H, and I prostaglandins. Prostaglandins can be metabolized by one or two steps of β-oxidation. These metabolites are systematically named from “2,3-dinorprostanoic acid” and “2,3,4,5-tetranorprostanoic acid,” respectively, but are often referred “C 18 ” and “C 16 ” metabolites. Carbon 20 of prostaglandins may be ω-oxidized to a carboxyl group and this side chain may then also be P-oxidized to shorter compounds. Fatty acids other than the three precursor acids of prostaglandins might be substrates for the prostaglandin synthesizing enzymes in some tissues. Important essential fatty acids in the diet are linoleic (18: 2ω6) and α-linoleic (18: 3ω3) acids, which both occur in plants.