Development of a radioimmunoassay for prostaglandin D2 using an antiserum against 11-methoxime prostaglandin D2

A sensitive and specific radioimmunoassay for prostaglandin D₂ has been developed using its stabilized 11-methoxime derivative, which was obtained after treatment of prostaglandin D₂ with methoxamine-HCl. The antiserum was obtained after injection of prostaglandin D₂-methoxamine coupled to bovine serum albumin. A (¹²⁵I)-Histamide prostaglandin D₂-methoxamine tracer was prepared by iodination of the corresponding histamide, followed by thin layer chromatography purification. The sensitivity of the assay was 280 femtomoles per ml at 50% displacement. The cross reactivities were 15% with prostaglandin D₁-methoxamine and less than 0.20% with other prostaglandins. Determination of the half-life of prostaglandin D₂ in a solution containing albumin was also carried out, since it has been shown to catalyze prostaglandin D₂ destruction. The unstability of this prostaglandin is due to the presence of a β-hydroxy ketone group, and all prostaglandins possessing this labile moiety could be stabilized by such a derivatization before developing a radioimmunoassay.     

Isomerization of prostaglandin H2 into prostaglandin D2 in the presence of serum albumin.

The prostaglandin endoperoxide, prostaglandin H2, decomposes in aqueous media mainly into prostaglandin E2. This paper shows that in the presence of serum albumin from a number of species prostaglandin H2 decomposes mainly into prostaglandin D2, an isomer of prostaglandin E2. The effect on endoperoxide decomposition exerted by serum albumin may well have physiological significance since intace endoperoxides can be released from tissues and since the biological properties of prostaglandins E2 and D2 are quite different.     

Methyl ester of prostaglandin D2 as a delivery system of prostaglandin D2 into brain

We examined the permeability of the blood-brain barrier to a methyl ester of prostaglandin D2 and the brain uptake was assessed by radioactivity measurements and radioimmunoassay. When the methyl ester (1 mg/kg) was administered intravenously into mice, it was rapidly taken up by the brain (189 ng/g brain at 30 s) and disappeared from the brain with a half-life of 9 s, whereas it was hardly detectable in the blood. The methyl ester transported into the brain was hydrolyzed to prostaglandin D2 and the time course of prostaglandin D2 levels showed an accumulation phase with a peak at 30 s. The total amount of prostaglandin D2 and its methyl ester was 279 ng/g brain at 30 s after injection, corresponding to 0.5% of the administered dose and being 6-times higher than that after prostaglandin D2 injection. The advantage of the methyl ester over prostaglandin D2 for brain uptake was observed at doses higher than 0.2 mg/kg where the methyl ester which escaped from hydrolysis in the blood was taken up more effectively than prostaglandin D2. In in vitro experiments, the esterase activity on the methyl ester was shown to be 20-times greater in the plasma than in the brain homogenate. These results indicate that the esterification of prostaglandin D2 may serve as a good system for the delivery of prostaglandin D2 into the brain.     

Characterization of the biosynthetic pathway of prostaglandin D2 in human platelet-rich plasma

The biosynthetic mechanism of prostaglandin D2 in human platelet-rich plasma has been investigated. Platelet-rich plasma was separated into washed platelets and platelet-poor plasma, and [1-14C]prostaglandin H2 was incubated with each fraction. The enzymatic conversion of the endoperoxide to prostaglandin D2 was found only in platelet-poor plasma and not in washed platelets or platelet lysate. This prostaglandin D synthetase activity was purified to homogeneity and identified as serum albumin by sodium dodecyl sulfate polyacrylamide gel electrophoresis, isoelectric focusing, and immunoelectrophoresis. The optimal pH and Km value for prostaglandin H2 were 9.0 and 6 microM, respectively. Glutathione was not required for the activity. Although prostaglandin H2 ws converted to prostaglandin D2 and E2 in the reaction, only the prostaglandin D2 formation was dependent on the protein amount and abolished by prior boiling. The action of this activity under physiological conditions was examined in a model system constituted of serum albumin and washed platelets. Prostaglandin D2 formation was observed in association with thrombin-evoked platelet aggregation in this system and was proportional to the number of platelets and the concentration of serum albumin, suggesting that thrombin-stimulated platelets released prostaglandin H2, and the latter compound was then converted to prostaglandin D2 by the action of serum albumin. Consistent with this interpretation, prostaglandin H2 added to platelet-rich plasma was converted in part to prostaglandin D2, and the aggregation caused by this endoperoxide was greatly enhanced by neutralizing the action of prostaglandin D2 with anti-prostaglandin D2 antiserum.     

Metabolism of prostaglandin D2 in isolated rat lung: the stereospecific formation of 9 alpha,11 beta-prostaglandin F2 from prostaglandin D2

The metabolic transformation of exogenous prostaglandin D2 was investigated in isolated perfused rat lung. Dose-dependent formation (2-150 ng) of 9 alpha,11 beta-prostaglandin F2, corresponding to about 0.1% of the perfused dose of prostaglandin D2, was observed by specific radioimmunoassay both in the perfusate and in lung tissue after a 5-min perfusion. To investigate the reason for this low conversion ratio, we analyzed the metabolites of tritium-labeled 9 alpha,11 beta-prostaglandin F2 and prostaglandin D2 by boric acid-impregnated TLC and HPLC. By 5 min after the start of perfusion, 9 alpha,11 beta-prostaglandin F2 disappeared completely from the perfusate and the major product formed remained unchanged during the remainder of the 30-min perfusion. The major product was separated by TLC and identified as 13,14-dihydro-15-keto-9 alpha,11 beta-prostaglandin F2 by GC/MS. In contrast, pulmonary breakdown of prostaglandin D2 was slow and two major metabolites in the perfusate increased with time, each representing 56% and 11% of the total radioactivity at the end of the perfusion. The major product (56%) was identified as 13,14-dihydro-15-ketoprostaglandin D2 and the minor one (11%) was tentatively identified as 13,14-dihydro-15-keto-9 alpha,11 beta-prostaglandin F2 based on the results from radioimmunoassays, TLC, HPLC, and the time course of pulmonary breakdown. These results demonstrate that the metabolism of prostaglandin D2 in rat lung involves at least two pathways, one by 15-hydroxyprostaglandin dehydrogenase and the other by 11-ketoreductase, and that the 9 alpha,11 beta-prostaglandin F2 formed is rapidly metabolized to 13,14-dihydro-15-keto-9 alpha,11 beta-prostaglandin F2.     

LC-MS/MS-analysis of prostaglandin E2 and D2 in microdialysis samples of rats

For the determination of prostaglandins in microdialysis samples, usually immunoassays are used. However, these assays may show cross-reactivity among various prostaglandins. To overcome this problem a specific method for the determination of prostaglandin E2 and D2 in rat microdialysis samples by using liquid chromatography-electrospay ionization-tandem mass spectrometry (LC-ESI-MS/MS) is described. Prostaglandin E2 and D2 were extracted from microdialysis samples with liquid-liquid extraction using deuterated prostaglandin D2, [2H4]-PGD2, as internal standard. Subsequently, prostaglandins were separated with a phenomenex Synergi Hydro-RP column and determined with a PE Sciex API 3000 mass spectrometer equipped with a turbo ion spray interface operating in negative ionization mode. The method showed a LLOQ of 25 pg/ml for prostaglandin E2 and 50 pg/ml for prostaglandin D2. The applicability of the method is shown in rat spinal cord microdialysis samples following peripheral nociceptive stimulation.     

Stimulation of prostaglandin D2 receptors on human platelets by analogs of prostacyclin.

RS-93427, a novel analog of prostacyclin, increased adenylate cyclase activity in human platelet membranes (EC50 = 42 nM) to approximately the same maximum level as that produced by prostacyclin (EC50 = 87 nM). The concentration-response curve for RS-93427 appeared to be monophasic. However, a selective prostaglandin D2 antagonist (BW A868C) significantly reduced the stimulation of adenylate cyclase produced by low concentrations of RS-93427 (3.2 to 32 nM). RS-93520, a stereoisomer of RS-93427, also stimulated adenylate cyclase activity but in a biphasic pattern. BW A868C reduced the activation produced by low concentrations of RS-93520 with a 100-fold shift in the response curve. Maximum stimulation by RS-93520 (4.5-fold) was less than that obtained with prostaglandin D2 (7.3-fold). Thus, the stimulation of adenylate cyclase activity by low concentrations of RS-93520 is due to an interaction with prostaglandin D2 receptors while the activation by RS-93427 is mediated by both prostacyclin and prostaglandin D2 receptors. Additional data in support of these conclusions was obtained when these prostaglandins were tested as inhibitors of ADP-induced platelet aggregation in the presence or absence of BW A868C. The potent stimulation of prostaglandin receptors with chimeric molecules provides some insight into the structural features required for receptor activation.     

Cyclooxygenase-2-derived prostaglandin D(2) is an early anti-inflammatory signal in experimental colitis

The ability of nonsteroidal anti-inflammatory drugs and cyclooxygenase-2 inhibitors to exacerbate inflammatory bowel disease suggests that prostaglandins are important anti-inflammatory mediators in this context. Prostaglandin D(2) has been suggested to exert anti-inflammatory effects. We investigated the possibility that prostaglandin D(2) derived from cyclooxygenase-2 plays an important role in downregulating colonic inflammation in rats. Colitis was induced by intracolonic administration of trinitrobenzene sulfonic acid. At various times thereafter (from 1 h to 7 days), colonic prostaglandin synthesis and myeloperoxidase activity (index of granulocyte infiltration) were measured. Prostaglandin D(2) synthesis was elevated >4-fold above controls within 1-3 h of induction of colitis, preceding significant granulocyte infiltration. Treatment with a selective cyclooxygenase-2 inhibitor abolished the increase in prostaglandin D(2) synthesis and caused a doubling of granulocyte infiltration. Colonic granulocyte infiltration was significantly reduced by administration of prostaglandin D(2) or a DP receptor agonist (BW-245C). These results demonstrate that induction of colitis results in a rapid increase in prostaglandin D(2) synthesis via cyclooxygenase-2. Prostaglandin D(2) downregulates granulocyte infiltration into the colonic mucosa, probably through the DP receptor.     

Properties of prostaglandin synthetase of rabbit kidney medulla.

The formation in vitro of prostaglandins E2, D2, and F2alpha from arachidonic acid by rabbit kidney medulla homogenate or microsomal fraction is markedly affected by the composition of the incubation medium employed. Optimal biosynthesis is obtained in 0.1 M potassium phosphate buffer, with the optimum pH being 8.0--8.8. Under these conditions prostaglandin formation is linear up to arachidonic acid concentration of 30 muM. The initial rate of formation of prostaglandin E2 + prostaglandin D2 is 3--4 times higher than that of prostaglandin F2alpha. Reduced glutathione (1 mM) did not affect the biosynthesis by medulla homogenate and produced only small stimulation of the biosynthesis by microsomal powder. Hydroquinone produced a small stimulation at a low concentration of 0.005 mM, and a strong inhibition at concentrations of 0.1 mM or higher. Addition of bovine serum albumin (0.1%) reduced the microsomal biosynthesis of prostaglandins by approximately 80%. Addition of boiled homogenate or boiled 140 000 X g supernatant produced small stimulation of microsomal biosynthesis while 140 000 X g supernatant (not boiled) caused small inhibition which was not dose-related. It appears that rabbit kidney prostaglandin-synthetase converts arachidonic acid to prostaglandins E2 and F2alpha in comparable amounts, without apparent need for a cytoplasmic soluble cofactor or specific reducing agents.     

Prostaglandin D2 in rat brain,spinal cord and pituitary: Basal level and regional distribution

A highly sensitive and specific radioimmunoassay for prostaglandin D₂ has been developed and used to determine the basal level and regional distribution of this prostaglandin in rat brain, spinal cord and pituitary. The assay can detect as little as 20 pg of prostaglandin D₂, and the antiserum used shows 20% cross-reactivity to prostaglandin D₁, 0.1% cross-reactivity to 13,14-dihydro-15-ketoprostaglandin D₂ and even lower cross-reactivity to other prostaglandins. Prostaglandin D₂-like immunoreactivity was extracted with ethanol from the rat tissues. The immunoreactivity comigrated with authentic prostaglandin D₂ on silica gel thin layer chromatography, showed the dilution curve parallel to that of the authentic compound, and decreased in amounts by the pretreatment of animals with indomethacin, suggesting that it was prostaglandin D₂ itself. To avoid a postmortem formation of prostaglandins, we sacrificed animals by microwave irradiation at 4.5 kW for 1.2 sec under which conditions both prostaglandin D synthetase and prostaglandin D dehydrogenase were completely inactivated. The amount of prostaglandin D₂ in whole brain measured under these conditions was 3.42±0.59 ng (mean+S.E.M.), and those of prostaglandin E₂ and F_2α measured by the respective radioimmunoassays were 1.32±0.24 and 0.96±0.20 ng, respectively. Prostaglandin D₂ was widely distributed in rat brain, spinal cord and pituitary. The highest concentrations were seen in pineal gland and neurointermediate pituitary followed by anterior pituitary. Lower but significant concentrations were observed in other parts of brain, among which hypothalamus and septum showed the relatively high concentrations.     

The low molecular weight fraction of human serum albumin upregulates COX2, prostaglandin E2, and prostaglandin D2 under inflammatory conditions in osteoarthritic knee synovial fibroblasts

BackgroundThe ability to decrease inflammation and promote healing is important in the intervention and management of a variety of disease states, including osteoarthritis of the knee (OAK). Even though cyclooxygenase 2 (COX2) has an established pro-inflammatory role, evidence suggests it is also critical to the resolution that occurs after the initial activation phase of the immune response. In this study, we investigated the effects of the low molecular weight fraction of 5% human serum albumin (LMWF-5A), an agent that has proven to decrease pain and improve function in OAK patients after intra-articular injection, on the expression of COX2 and its downstream products, prostaglandins (PGs).MethodsFibroblast-like synoviocytes from the synovial membrane of OAK patients were treated with LMWF-5A or saline as a control with or without the addition of interleukin-1β (IL-1β) or tumor necrosis factor α (TNFα) to elicit an inflammatory response. Cells were harvested for RNA and protein at 2, 4, 8, 12, and 24 h, and media was collected at 24 h for analysis of secreted products. COX2 mRNA expression was determined by qPCR, and COX2 protein expression was determined by western blot analysis. Levels of prostaglandin E2 (PGE2) and prostaglandin D2 (PGD2) in the media were quantified by competitive ELISA.ResultsIn the presence of either IL-1β or TNFα, LMWF-5A increased the expression of both COX2 mRNA and protein, and this increase was significant compared to that observed with IL-1β- or TNFα-stimulated, saline-treated cells. Downstream of COX2, the levels of PGE2 were increased only in TNFα-stimulated, LMWF-5A-treated cells; however, in both IL-1β- and TNFα-stimulated cells, LMWF-5A increased the release of the anti-inflammatory prostaglandin PGD2.ConclusionLMWF-5A appears to trigger increased anti-inflammatory PG signaling, and this may be a primary component of its therapeutic mode of action in the treatment of OAK.     

Cholesterol arachidonate as a prostaglandin precursor in adrenocortical cells.

Rat adrenocortical cells were incubated with labeled arachidonate, and the radioactivity in unesterified fatty acids was reduced by washing with 2% albumin solutions. These cells were then incubated for two hours in the absence and presence of 7.1 x 10(-10)M ACTH. During subsequent incubation of prelabeled cells with ACTH, both the mass and radioactivity of arachidonate in adrenocortical cholesteryl esters was depleted to the same extent (30--32%). The released arachidonate was in part incorporated into phospholipids, and there was also a significant increase in unesterified arachidonic acid. During this period, there was also increased incorporation of arachidonate into labeled prostaglandins. Of this increase, 92% by isotope analysis, and 88% by radioimmunoassay techniques was attributable to prostaglandins of the E pathway. These data demonstrate that prostaglandin E synthesis is specifically increased during ACTH stimulation of rat adrenocortical cells and suggest that a major source of the arachidonate substrate for this synthesis is derived from hormone-dependent hydrolysis of cortical cholesteryl esters.     

Effect of prostaglandin I2 and related compounds on vascular permeability response in granuloma tissues

The effects of prostaglandin I2, 6-ketoprostaglandin F1alpha, prostaglandin E1 and thromboxane B2 on the vascular permeability response in rat carrageenin granuloma were studied with the aid of 131I- and 125I-human serum albumin as indicators for the measurement of local vascular permeability. A single injection of 5 microgram of prostaglandin I2 methyl ester or I2 sodium salt into the locus of the granulomatous inflammation elevated local vascular permeability 2.0-2.5 times over the control within 30 min. The potency was equal to that of the positive control prostaglandin E1 which has been known to be the most potent mediator in this index among several candidate prostaglandins for chemical mediator of inflammation. The other prostaglandin and thromboxane B2 tested were essentially inactive.     

Cholesterol arachidonate as a prostaglandin precursor in adrenocortical cells

Rat adrenocortical cells were incubated with labeled arachidonate, and the radioactivity in unesterified fatty acids was reduced by washing with 2% albumin solutions. These cells were then incubated for two hours in the absence and presence of 7.1 × 10⁻¹⁰M ACTH. During subsequent incubation of prelabeled cells with ACTH, both the mass and radioactivity of arachidonate in adrenocortical cholesteryl esters was depleted to the same extent (30–32%). The released arachidonate was in part incorporated into phospholipids, and there was also a significant increase in unesterified arachidonic acid. During this period, there was also increased incorporation of arachidonate into labeled prostaglandins. Of this increase, 92% by isotope analysis, and 88% by radioimmunoassay techniques was attributable to prostaglandins of the E pathway. These data demonstrate that prostaglandin E synthesis is specifically increased during ACTH stimulation of rat adrenocortical cells and suggest that a major source of the arachidonate substrate for this synthesis is derived from hormone-dependent hydrolysis of cortical cholesteryl esters.     

Biosynthesis of prostaglandins in rabbit kidney medulla. Properties of prostaglandin synthase.

A simple radioactive-substrate assay for prostaglandin synthase (EC 1.14.99.1), which uses t.l.c. to measure simultaneously different prostaglandins synthesized from one precursor substrate, was developed. Rabbit kidney-medulla prostaglandin synthase catalyses the formation of prostaglandin E2, prostaglandin F2alpha and prostaglandin D2 from arachidonic acid. Fractionation of crude homogenates indicated that the microsomal fraction possessed the highest specific activity of prostaglandin synthase, whereas the soluble fraction exhibited little enzyme activity but rather contained a heat-labile inhibitory macromolecular factor(s), which might be attributed to the serum albumin present in this fraction. The microsomal fraction possessed low intrinsic enzyme activity, but the actvity could be fully stimulated by the presence of both GSH (reduced glutathione) and a phenolic cofactor. Only cysteine could partially replace GSH, whereas other thiols were inactive and some were even inhibitory. A variety of phenolic compounds, including catecholamines, dopamine (3,4-dihydroxyphenethylamine), 5-hydroxytryptamine and quinol, were active in stimulating prostaglandin synthase. In all cases, the stimulation was reflected in the synthesis of all three prostaglandins with ratios not significantly altered by different phenolic cofactors. The synthesis of each of the different prostaglandins appeared to have similar pH optima. The enzyme system was not inhibited by thiol-group inhibitors or a variety of metal chelators except for cyanide and 8-hydroxyquinoline. Characterization of the kidney-medulla prostaglandin synthase system indicated that it exhibited properties similar to those of the enzyme system present in seminal vesicles.     

Basal level of prostaglandin D2 in rat brain by a solid-phase enzyme immunoassay

A solid-phase enzyme immunoassay for prostaglandin D2 (PGD2) was developed in which PGD2 was labeled with horseradish peroxidase. After competitive binding to the immobilized antibody between enzyme-labeled and free PGD2, the activity of the enzyme bound to the antibody was assayed fluorometrically using 3-(p-hydroxyphenyl)-propionic acid and hydrogen peroxide as substrates. The procedure allowed determinations of 3-100 pg for PGD2. The IC50 value for PGD2 in the solid-phase enzyme immunoassay was about 25 pg and the sensitivity was improved about 10 times compared to those in radioimmunoassay and in solution-phase enzyme immunoassay. The solid-phase enzyme immunoassay was applied to the measurement of PGD2 content in rat brain and thereby an octadecylsilyl silica cartridge and a reversed-phase HPLC were sequentially used for sample preparations. Heads were immediately frozen in liquid nitrogen after decapitation to avoid a postmortem formation of PGD2. PGD2 contents measured by solid-phase enzyme immunoassay correlated well with the values obtained by radioimmunoassay (r = 0.966) after raising its contents by intravenous administration of PGD2. The in vivo level of PGD2 in rat brain was extremely low but determined to be 0.11 +/- 0.03 ng/g tissue (mean +/- S.E.M.) with this enzyme immunoassay. The result was equal to the value extrapolated to zero time from the postmortem change.     

The content of prostaglandin E and prostaglandin F2alpha in the exudate of carrageenin granuloma of rats.

1.Granuloma was made by the subcutaneous injection of 2% carrageenin solution on the dorsum of male rats. Eight, 16, 24 and 72 h after the injection. the exudate from each rat granuloma was withdrawn and extracted for rpstaglandins. 2.Extracted prostaglandins were separated prostaglandin E and prostaglandin F group by silicic acid mini-column chromatography. Then the amount of prostaglandin E and prostaglandin F2alpha were determined by the radioimmunoassay method. 3.The levels of prostaglandin E in the granuloma exudates were 4.6 ng/ml at 8 h after the carrageenin injection, then decreased 3.6 ng/ml and to 1.1 ng/ml at 16 h and 24 h, respectively. Seventy-two h after the injection, prostaglandin E level was increased to 8.1 ng/ml. 4.The levels of prostaglandin F2alpha in the exudate were as follows: At 8 h after the carrageenin injection, the level was 9.4 ng/ml, then decreased to 1.3 ng/ml and to 0.8 ng/ml at 16 h and 24 h, respectively. Seventy-two h after the carrageenin injection, it was again elevated to 4.7 ng/ml. 5.The exudate of granuloma, 24 and 72 h after the carrageenin injection, was incubated with [3H]prostaglandin E1 at 37 degrees C for 30 min. Then the acidic ether extract was subjected to reversed phase partition chromatography. It was found that the exudate of 24 h and 72 h granuloma had little activity of prostaglandin 15alpha-hydroxy dehydrogenase.     

Inhibition of brain prostaglandin D synthetase and prostaglandin D2 dehydrogenase by some saturated and unsaturated fatty acids

The activities of rat brain prostaglandin D synthetase and swine brain prostaglandin D2 dehydrogenase were inhibited by some saturated and unsaturated fatty acids. Myristic acid was most potent among saturated straight-chain fatty acids so far tested. The IC50 values of this acid were 80 microM for prostaglandin D synthetase and 7 microM for prostaglandin D2 dehydrogenase, respectively. Little inhibition was found with methyl myristate and myristyl alcohol. The IC50 values of these derivatives were more than 200 microM for both enzymes, suggesting that the free carboxyl group was essential for the inhibition. The effects of cis double bond structure of fatty acids on the inhibition potency were examined by the use of the carbon 18 and 20 fatty acids. The inhibition potencies for both enzymes increased with the number of cis double bonds; the IC50 values of stearic, oleic, linoleic and linolenic acid were, respectively, more than 200, 60, 30 and 30 microM for prostaglandin D synthetase, and 20, 10, 8.5 and 7 microM for prostaglandin D2 dehydrogenase. Arachidonic acid also inhibited the activities of both enzymes with respective IC50 values of 40 microM for prostaglandin D synthetase and 3.9 microM for prostaglandin D2 dehydrogenase, while arachidic acid showed little inhibition. The kinetic studies with myristic acid and arachidonic acid demonstrated that the inhibition by these fatty acids was competitive and reversible for both enzymes. Myristic acid and other fatty acids also inhibited the activities of several enzymes in prostaglandin metabolism, although to a lesser extent. The IC50 values of myristic acid for prostaglandin E isomerase, thromboxane synthetase and NAD-linked prostaglandin dehydrogenase (type I) were 200, 700 and 100 microM, respectively. However, this fatty acid showed little inhibition on fatty acid cyclooxygenase (20% at 800 microM), glutathione-requiring prostaglandin D synthetase from rat spleen (20% at 800 microM), and NADP-linked prostaglandin dehydrogenase (type II) (no inhibition at 200 microM).     

Metabolic fate of radiolabeled prostaglandin D2 in a normal human male volunteer

50 microCi of [3H]prostaglandin D2 tracer (100 Ci/mmol) was infused intravenously into a normal human male volunteer. 75% of the infused radioactivity was excreted into the urine within 5 h. This urine was added to urine obtained from two mastocytosis patients with marked overproduction of prostaglandin D2. Radiolabeled prostaglandin D2 urinary metabolites were chromatographically isolated and purified and subsequently identified by gas chromatography-mass spectrometry. 25 metabolites were identified. 23 of these compounds comprising 37% of the recovered radioactivity had prostaglandin F-ring structures, and only two metabolites comprising 2.7% of the recovered radioactivity retained the prostaglandin D-ring structure. The single most abundant metabolite identified was 9,11-dihydroxy-15-oxo-2,3,18,19-tetranorprost-5-ene-1,20-dioic acid which was isolated in a tricyclic form as a result of formation of a lower side chain hemiketal followed by lactonization of the terminal carboxyl and the hemiketal hydroxyl. Different isomeric forms of several prostaglandin F-ring metabolites were identified. An isomer of prostaglandin F2 alpha was also excreted intact into the urine as a metabolite of prostaglandin D2. 15 PGF-ring compounds were treated with n-butylboronic acid and 13 failed to form a boronate derivative, suggesting that the orientation of the hydroxyl group at C-11 in these 13 metabolites is beta. This study documents that prostaglandin D2 is metabolized to prostaglandin F-ring metabolites in vivo in humans. These results also bring into question the accuracy of quantifying prostaglandin F2 alpha metabolites as a specific index of endogenous prostaglandin F2 alpha biosynthesis, as well as quantifying urinary prostaglandin F2 alpha as an accurate index of renal production of prostaglandin F2 alpha.     

Purification and properties of prostaglandin D synthetase from rat brain.

The prostaglandin D synthetase system was isolated from rat brain. Prostaglandin endoperoxide synthetase solubilized from a microsomal fraction catalyzed the conversion of arachidonic acid to prostaglandin H2 in the presence of heme and tryptophan. Prostaglandin D synthetase (prostaglandin endoperoxidase-D isomerase) catalyzing the isomerization of prostaglandin H2 to prostaglandin D2 was found predominantly in a cytosol fraction and was purified to apparent homogeneity with a specific activity of 1.7 mumol/min/mg of protein at 24 degrees C. The enzyme also acted upon prostaglandin G2 and produced a compound presumed to be 15-hydroperoxy-prostaglandin D2. Glutathione was not required for the enzyme reaction, but the enzyme was stabilized by thiol compounds including glutathione. The enzyme was inhibited by p-chloromercuribenzoic acid in a reversible manner. The purified enzyme was essentially free of the glutathione S-transferase activity which was found in the cytosol of brain.