Metabolic fate of endogenously synthesized prostaglandin D2 in a human female with mastocytosis

Increased production of prostaglandin D₂ was recently demonstrated in patients with systemic mastocytosis. One female patient investigated with mastocytosis was found to have overproduction of prostaglandin D₂ of such magnitude (150-fold above normal) that it provided the unique opportunity to delineate the metabolic fate of endogenously synthesized prostaglandin D₂. A five percent aliquot of a twenty-four hour urine collection from the patient was extracted, purified by silicic acid chromatography, methylated, and finally subjected to high pressure liquid chromatography. Column fractions collected were derivatized and analyzed by gas chromatography-mass spectrometry. Increased quantities of sixteen urinary metabolites were identified and included a series of metabolites retaining the PGD-ring as well as series of metabolites with a PGF-ring. PGF-ring metabolites were excreted in approximately 4-fold greater relative abundance than PGD-ring metabolites. More than one apparent isomeric form of some PGF-ring metabolites were found. The predominant urinary metabolite was 2,3-dinor-prostaglandin F₂. This study provides evidence that endogenously synthesized prostaglandin D₂ is converted in substantial part to prostaglandin F₂ metabolites in humans.     

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.     

Metabolism of prostaglandin D2 in the monkey.

[3H7]Prostaglandin D2 was biosynthesized and infused into an unanesthetized monkey. The urinary metabolites were isolated and subsequently identified by gas chromatography-mass spectrometry. Two pathways of prostaglandin D2 metabolism were identified and resulted in metabolites with prostaglandin D (3-hydroxycyclopentanone) and prostaglandin F (cyclopentane-1,3-diol) ring structures. The major prostaglandin D ring metabolite was identified as 9,20-dihydroxy-11,15-dioxo-2,3-dinorprost-5-en-1-oic acid. Nine other prostaglandin D ring metabolites were identified reflecting various combinations of metabolism by beta and omega oxidation, 15 dehydrogenation, and 13-14 reduction. In greater abundance were those prostaglandin D2 metabolites which had the prostaglandin F ring structure. The major prostaglandin D2 metabolite which had the prostaglandin F ring structure was identified as 9,11,15-trihydroxy-2,3-dinorprosta-5,13-dien-1-oic acid (dinor prostaglandin F2 alpha). Nine other metabolites with the prostaglandin F ring structure were identified, including prostaglandin F2 alpha itself. These, for the most part, were the structural counterparts of the metabolites with the prostaglandin D ring. Since many prostaglandin D2 metabolites were found to be identical with the metabolites of prostaglandin F2 alpha, quantitative determinations of prostaglandin F ring metabolites may not be a specific indicator of prostaglandin F2 alpha biosynthesis. Likewise, data involving the measurement of a biological effect of prostaglandin D2 must be re-examined to account for the possible contribution of prostaglandin F2 alpha, a metabolite of prostaglandin D2, to the biological response.     

Radioimmunologic identification of prostaglandins produced by serum-stimulated mouse embryo palate mesenchyme cells

High-performance liquid chromatography and radioimmunoassay were used to identify the prostaglandins synthesized by mouse embryo palate mesenchyme cells. Serum stimulated the release of several different metabolites of arachidonic acid including 6-ketoprostaglandin F1 alpha (the stable product of prostacyclin, prostaglandin I2), prostaglandin E2 and prostaglandin F2 alpha. Compared to control cells, the serum-stimulated cells produce elevated levels of prostaglandin E2 (36-fold), 6-ketoprostaglandin F1 alpha (15-fold) and prostaglandin F2 alpha (7-fold). The acetylenic analogue of arachidonic acid, 5,8,11,14-eicosatetraynoic acid prevented this accelerated synthesis.     

Metabolism of prostaglandins D2 and F2 alpha in primary cultures of rat hepatocytes

3H-Labeled prostaglandins D2 and F2 alpha rapidly degraded to more-polar metabolites in primary cultured rat hepatocytes. The metabolites of prostaglandins D2 and F2 alpha accumulated in the culture medium. The metabolites extracted by ethyl acetate at pH 3 were purified by silicic acid column and thin-layer chromatography of silica gel, and were analysed by gas chromatography-mass spectrometry. The major metabolites from prostaglandin D2 were identified as dinor-prostaglandin D1 (7 alpha,13-dihydroxy-9-ketodinorprost-11-enoic acid) and tetranor-prostaglandin D1 (5 alpha,11- dihydroxy-7-ketotetranorprost-9-enoic acid). Those from prostaglandin F2 alpha were identified as dinor-prostaglandin F1 alpha (7 alpha,9 alpha,13-trihydroxydinorprost-11-enoic acid), tetranor-prostaglandin F1 alpha (5 alpha,7 alpha,11-trihydroxytetranorprost-9-enoic acid) and 9 alpha,11 alpha,15-trihydroxyprost-13-ene-1,20-dioic acid. These data indicate that prostaglandins D2 and F2 alpha mainly degraded by beta-oxidation, which is the same process as reported earlier for prostaglandins E1 and E2, and that prostaglandin F2 alpha was also subjected to omega-oxidation.     

Enzymatic formation of prostaglandin F2 alpha from prostaglandin H2 and D2. Purification and properties of prostaglandin F synthetase from bovine lung

Prostaglandin F synthetase from bovine lung was purified 540-fold to apparent homogeneity, as assessed by polyacrylamide gel electrophoreses and ultracentrifugation. The purified enzyme proved to be a monomeric protein with a molecular weight of about 30,500. The enzyme catalyzed not only the reduction of the 11-keto group of prostaglandin D2 but also the reduction of 9,11-endoperoxide of prostaglandin H2 and various carbonyl compounds (e.g. phenanthrenequinone). Experiments using column chromatography, polyacrylamide gel electrophoreses, immunotitration using antibody against the purified enzyme, and heat treatment indicated that three enzyme activities resided in a single protein. Although phenanthrenequinone and prostaglandin D2 competitively inhibited the prostaglandin D2 and phenanthrenequinone reductase activities, respectively, these two substrates were all but ineffective on the prostaglandin H2 (at the Km value) reductase activity up to 14-fold of those Km values. These results suggest that a single enzyme protein purified from the bovine lung catalyzes the reduction of prostaglandin D2, prostaglandin H2, and various carbonyl compounds and that prostaglandin D2 and prostaglandin H2 are metabolized at two different active sites, yielding prostaglandin F2 alpha as the reaction product.     

Comparison of the production of eicosanoids by human and rat peritoneal macrophages and rat Kupffer cells

Human and rat peritoneal macrophages and rat Kupffer cells were labelled with [1-14C] arachidonic acid and stimulated with the calcium ionophore A23187. The metabolites formed were separated by high pressure liquid chromatography (HPLC). Human peritoneal macrophages formed especially leukotriene B4, 5-hydroxy-6,8,11,14 eicosatetraenoic acid and small amounts of leukotriene C4 and thromboxane B2, 12-hydroxy-5,8,10 heptadecatrienoic acid and 6-keto-prostaglandin F1 alpha, whereas rat peritoneal macrophages mainly produced cyclooxygenase products and in particular thromboxane B2 and 12-hydroxy-5,8,10 heptadecatrienoic acid. Rat Kupffer cells synthesized mainly cyclooxygenase products such as prostaglandin F2 alpha, prostaglandin D2 and prostaglandin E2. These results indicate that the profile of eicosanoids production by macrophages is dependent both on the species and on the tissue from which the macrophage is derived.     

Cellular confluence determines injury-induced prostaglandin E2 synthesis by human keratinocyte cultures

When keratinocyte cultures become confluent, their prostaglandin E2 synthesis is suppressed. To determine whether the injury response is characterized by increased prostaglandin E2 synthesis, an in vitro injury model was developed. When confluent keratinocyte cultures were focally lethally irradiated using ultraviolet light B, a dose-dependent increase in prostaglandin E2 synthesis was induced by the injury. After irradiation, confluent cultures' prostaglandin E2 synthesis increased for 2 days to 8-fold more than controls, then decreased to control values by day 6. Increased prostaglandin E2 synthesis was first detected 8 h after injury. Focal irradiation of non-confluent cultures (killing isolated colonies) caused no change in prostaglandin E2 synthesis, indicating that culture continuity must be disrupted before synthesis increases. In addition, partial irradiations of petri dishes demonstrated that enhanced metabolism was confined to cells adjacent to the injury site and was not mediated by a soluble factor. When confluent and injured cultures were incubated with [14C]arachidonic acid, and the products formed analyzed by thin layer chromatography, 10-fold more prostaglandin E2 microgram protein was seen in irradiated cultures relative to confluent controls. The products formed by each group were the same, and no consistent increases in metabolites other than prostaglandin E2 were observed. The increased synthesis of prostaglandin E2 by injured cultures was apparently due to an increase in cyclooxygenase activity as determined by kinetic experiments. These data indicate that the pattern of metabolism of arachidonic acid seen in non-confluent cultures is similar to that seen in injury, and that cell-cell contact modulates enhanced prostaglandin E2 synthesis.     

Streamlined F2-isoprostane analysis in plasma and urine with high-performance liquid chromatography and gas chromatography/mass spectroscopy

F2-Isoprostanes in plasma and urine are generally determined by labor-intensive methods requiring sample purification by solid-phase extraction and thin-layer chromatography (TLC). A streamlined and more sensitive method for the measurement of esterified plasma F2-isoprostanes was developed by replacing these steps with high-performance liquid chromatography (HPLC) using an amino column with a hexane/2-propanol gradient. Pentafluorobenzyl esters of F2-isoprostanes were prepared and purified by HPLC, silylated, and then analyzed by gas chromatography (GC) with negative chemical ionization mass spectroscopy (NCI-MS). This method permits analysis with lower plasma volumes (100 microL) and greater sensitivity (to 10 pg; allowing detection to 50 pg/mL) than provided by other methods. Urinary F2-isoprostanes can also be efficiently quantified by this method, with 8-iso-PGF2alpha being identified as a major isomer. With this procedure, esterified plasma F2-isoprostanes were found to be 8.3-fold higher in an end-stage renal failure patient on hemodialysis and urinary 8-iso-PGF2alpha was 7.1-fold higher in a cigarette smoker than respective control subjects. This method, particularly the substitution of the TLC step common to other methods with HPLC, results in a more sensitive and reproducible assay.     

Identification of the major urinary metabolite of thromboxane B2 in the monkey.

Thromboxane B2 (TxB2) was biosynthesized from prostaglandin endoperoxides (PGG2, PGH2) using guinea pig lung microsomes and infused into an unanesthetized monkey. Urine was collected and TxB2 metabolites were isolated by reversed phase partition chromatography and high performance liquid chromatography. A major metabolite (TxB2-M) was found to be excreted in greater than two-fold abundance relative to other metabolites. Its structure was determined by gas chromatography-mass spectrometry to be dinor-thromboxane B2. In vitro incubation of TxB2 with rat liver mitochondria yielded a C18 derivative with a mass spectrum identical to that of TxB2-M, substantiating that the major urinary metabolite of TxB2 in the monkey is a product of a single step of beta-oxidation.     

Quantification of F-ring isoprostane-like compounds (F4-neuroprostanes) derived from docosahexaenoic acid in vivo in humans by a stable isotope dilution mass spectrometric assay

Lipid peroxidation has been implicated in the pathophysiological sequelae of human neurodegenerative disorders. It is recognized that quantification of lipid peroxidation is best assessed in vivo by measuring a series of prostaglandin (PG) F2-like compounds termed F2-isoprostanes (IsoPs) in tissues in which arachidonic acid is abundant. Unlike other organs, the major polyunsaturated fatty acid (PUFA) in the brain is docosahexaenoic acid (DHA, C22:6 omega-6), and this fatty acid is particularly enriched in neurons. We have previously reported that DHA undergoes oxidation in vitro and in vivo resulting in the formation of a series of F2-IsoP-like compounds termed F4-neuroprostanes (F4-NPs). We recently chemically synthesized one F4-NP, 17-F4c-NP, converted it to an 18O-labeled derivative, and utilized it as an internal standard to develop an assay to quantify endogenous production of F4-NPs by gas chromatography (GC)/negative ion chemical ionization (NICI) mass spectrometry (MS). The assay is highly precise and accurate. The lower limit of sensitivity is approximately 10 pg. Levels of F4-NPs in brain tissue from rodents were 8.7 +/- 2.0 ng/g wet weight (mean +/- S.D.). Levels of the F4-NPs in brains from normal humans were found to be 4.9 +/- 0.6 ng/g (mean +/- S.D.) and were 2.1-fold higher in affected regions of brains from humans with Alzheimer's disease (P = 0.02). Thus, this assay provides a sensitive and accurate method to assess oxidation of DHA in animal and human tissues and will allow for the further elucidation of the role of oxidative injury to the central nervous system in association with human neurodegenerative disorders.     

Estradiol is responsible for reduced renal prostaglandin dehydrogenase activity in female rats

The contribution of sex steroids to sex-related differences in renal prostaglandin dehydrogenase activity and urinary prostaglandin excretion was examined in 7-8-week-old male and female rats subjected to sham-operation or gonadectomy at 3 weeks of age. Rats were injected subcutaneously twice over a 6-day interval with vehicle (peanut oil, 0.5 mg/kg) or with depot forms of testosterone (10 mg/kg), estradiol (0.1 mg/kg), progesterone (5 mg/kg), or with estradiol and progesterone combined (0.1 and 5 mg/kg). After the second injection, 24-h urine samples were collected for prostaglandin measurement by radioimmunoassay; the rats were killed, and renal and pulmonary prostaglandin dehydrogenase activities were determined by radiochemical assay. Renal prostaglandin dehydrogenase activity was 10-times higher in intact male rats than in intact females. Gonadectomy increased renal prostaglandin dehydrogenase activity 4-fold in females, but had no effect in males; estradiol, alone or combined with progesterone, markedly suppressed renal prostaglandin dehydrogenase activity in both sexes, while testosterone or progesterone alone had no effect. Pulmonary prostaglandin dehydrogenase did not differ between the sexes and was unaffected by gonadectomy or sex-steroid treatment. Intact female sham-operated rats excreted 70-100% more prostaglandin E2, prostaglandin F2 alpha, and 6-keto-prostaglandin F1 alpha in urine than did males; gonadectomy abolished the difference in urinary prostaglandin E2 excretion. Estradiol decreased urinary prostaglandin E2 in females but not in males; treatment with other sex steroids did not alter urinary prostaglandin excretion.     

Arachidonic acid metabolism by murine peritoneal macrophages infected with Leishmania donovani: in vitro evidence for parasite-induced alterations in cyclooxygenase and lipoxygenase pathways

Leishmania donovani is an obligate intracellular protozoan that resides within mononuclear phagocytes of infected mammals. Affected human and rodent hosts commonly show abnormalities of T cell function, which may be related to altered macrophage physiology resulting from intracellular parasitism. To examine this possibility, we studied the metabolism of endogenous arachidonyl-phospholipids and [3H]-arachidonyl-phospholipids by murine peritoneal exudate macrophages infected with amastigotes of L. donovani. Our results indicated that infected cells synthesized increased amounts of both cyclooxygenase and lipoxygenase metabolites of arachidonic acid. Increased synthesis of immunoreactive prostaglandin (PG)E2 was evident as early as 1 to 4 hr after infection, was correlated with the fraction of cells infected, and was inhibited by sodium meclofenamate (0.2 and 20 microM) but not nordihydroguaiaretic acid (3 microM). As determined by thin-layer chromatography, infected cells also produced markedly increased amounts of prostaglandin F2 alpha (also inhibited by sodium meclofenamate) with insignificant increases in thromboxane B2 and the stable metabolite of prostacyclin, 6-oxo-PGF1 alpha. In contrast, stimulation of cells with opsonized zymosan resulted in significantly increased synthesis of all four eicosanoids. L. donovani infection was also found to induce marked increases in synthesis of lipoxygenase metabolites of arachidonic acid by infected cells. This was evidenced by increased amounts of [3H]-labeled material in cell extracts that co-migrated with authentic standards of 5 and 12/15-hydroxy-eicosate-traenoic acids in thin-layer chromatograms. Increased synthesis of these products was largely inhibited by both NDGA (3 microM) and sodium meclofenamate (20 and 0.2 microM). Additional evidence for augmentation of 5-lipoxygenase by Leishmania was provided by the demonstration of increased leukotriene-C4 in conditioned medium from infected cells. These results indicate that macrophages infected with L. donovani produce increased amounts of arachidonic acid metabolites with the potential for influencing cellular immune function and the inflammatory response to infection.     

Quantification of dinor, dihydro metabolites of F2-isoprostanes in urine by liquid chromatography/tandem mass spectrometry

The F2-isoprostanes (F2-IsoP) are a series of prostaglandin (PG)-F2-like compounds that are produced by free-radical-mediated oxidation of arachidonic acid. One F2-IsoP with potent biological activity is 15-F2t-IsoP and increased levels of 15-F(2t)-IsoP have been measured in several diseases. The major urinary metabolite of 15-F2t-IsoP (8-iso-PGF(2alpha)) is 2,3-dinor-5,6-dihydro-15-F2t-IsoP (15-F2t-IsoP-M). Previously, we developed a stable isotope dilution gas chromatography/negative chemical ionization/mass spectrometry (MS) assay for 15-F2t-IsoP-M, which, while highly sensitive, required time-consuming derivatization and thin-layer chromatography purification. We now report the development of a more rapid high-performance liquid chromatography method coupled to electrospray ionization-tandem mass spectrometry (LC/MS/MS) to analyze all of the dinor,dihydro metabolites of the F2-IsoP isomers (F2-IsoP-M). The precision of this assay was +/-5.0% and the accuracy 80%. The assay remained linear over a range of 1-100 ng injected onto the LC column. Levels of F2-IsoP-M determined by the LC/MS/MS assay method significantly correlated with levels of 15-F2t-IsoP-M determined by the GC/MS assay (R = 0.77y = 67.2x-0.5). The levels of F2-IsoP-M detected in spot urines from 40 normal subjects were 38.1+/-19.1 ng/mg creatinine (mean+/-SD). This method provides an accurate and rapid assay to assess oxidative status in vivo.     

Prostacyclin and prostaglandin synthesis in isolated brain capillaries

The synthesis of prostacyclin and prostaglandins was examined in isolated blood-free brain capillaries of guinea-pigs and rats using 1-14C-arachidonic acid as a precursor. The main prostaglandins synthesized by guinea-pig microvessels were prostaglandin D2 and prostaglandin E2. Substantially less prostaglandin F2 alpha or the prostacyclin stable metabolite, 6-oxo-prostaglandin F1 alpha was synthesized. Rat capillary prostaglandin distribution differed substantially from that of the guinea-pigs although the principle prostaglandin was also PGD2. Total prostaglandin conversion was greater in guinea-pig capillaries than in the rat. Norepinephrine stimulated the prostaglandin forming capacity of blood free cerebral microvasculature of guinea-pigs. Prostacyclin and prostaglandins could be involved in the activity dependent regulation of regional cerebral blood flow and permeability.     

Prostaglandins as endogenous mediators of interleukin 1 production

We examined the role of cyclooxygenase (CO)-derived metabolites of arachidonic acid (AA) in the regulation of interleukin 1 (IL 1) production by lipopolysaccharide (LPS)-stimulated murine resident peritoneal macrophages. The use of LPS proved to be an efficacious probe, because it stimulated both IL 1 production and AA metabolism via only the CO pathway. The production of the CO metabolites prostaglandin E2 (PGE2) and prostaglandin I2 (PGI2; measured as its stable metabolite 6-Keto prostaglandin F1 alpha) by LPS-stimulated macrophages was demonstrated by high pressure liquid chromatography and radioimmunoassay. The addition of exogenous PGE2 or PGI2 resulted in a dose-dependent suppression of macrophage IL 1 production. Inhibitors of the CO pathway (indomethacin, piroxicam, and ibuprofen) caused a dose-dependent augmentation in the LPS-induced IL 1 response. This augmentation directly correlated with the efficacy of the compounds as CO inhibitors. Similar results were found when macrophage-derived fibroblast growth factor was assessed. The addition of exogenous IL 1 to macrophage cultures caused an increase in the levels of PGE2, over a narrow dose range (0.05 to 0.6 IL 1 units). These studies provide detailed evidence that AA metabolites synthesized via the CO pathway can modulate the production of growth factors by LPS-stimulated macrophages. In addition, our data support the concept that IL 1, as with classical hormones, can regulate its own production through a self-induced inhibitor, PGE2.     

Regulation of prostaglandin synthesis by thyrotropin, insulin or insulin-like growth factor-I, and serum in FRTL-5 rat thyroid cells.

The present report shows that thyrotropin (TSH) regulates all three steps involved in prostaglandin synthesis in FRTL-5 rat thyroid cells, i.e. arachidonic acid release from membrane phospholipids, cyclooxygenase (prostaglandin H synthase) action, and individual prostaglandin formation; however, its action at specific steps may require the presence of, or can be duplicated by, insulin, insulin-like growth factor-I (IGF-I), and/or a serum factor. Thus, TSH releases free arachidonic acid from rat FRTL-5 thyroid cells whose phospholipid fraction is radiolabeled with [3H]arachidonic acid; this action involves a pertussis toxin-sensitive G protein, is not cAMP mediated, and does not require insulin or 5% serum. To quantitate TSH effects on cyclooxygenase activity and on individual prostaglandin formation, a homogenate system and a rapid reversed-phase high pressure liquid chromatography procedure have been developed to measure cyclooxygenase metabolites. TSH increased cyclooxygenase activity in homogenates only if the cells were also exposed to insulin, IGF-I, and/or 5% calf serum; TSH alone had no apparent effect on the activity. Maximal activation, 4-fold over basal/micrograms of DNA, took 36 h to achieve and reflected, at least in part, an increase in cyclooxygenase gene expression. Like cyclooxygenase activity, induction of prostaglandin E2 production required 2 or more factors, i.e. TSH plus insulin/IGF-I or TSH plus insulin/IGF-I plus serum. Increased production of prostaglandin D2, could, however, be detected if cells were treated with TSH alone and the TSH activity could be duplicated by insulin, IGF-I, or calf serum alone.     

Characterization of arachidonic acid metabolic profiles in animal tissues by high-resolution gas chromatography-mass spectrometry

A standardized, highly specific routine method was developed for the quantitative profiling of cyclooxygenase metabolites of arachidonic acid in animal tissues. Whole homogenates were used to assess the potential capacity of tissues to metabolize endogenous arachidonic acid. Samples were analyzed by high-resolution gas chromatography-mass spectrometry in the selected ion monitoring mode. The screening of several rat tissues by this method revealed marked tissue-specificity in both the synthesis capacity and prostaglandin profile. The major products detected were: 6-ketoprostaglandin F1alpha for lung, stomach, muscle and heart; prostaglandin D2 for spleen, brain and liver; prostaglandin F2alpha for kidney and prostaglandin E2 for seminal vesicles. Marked species differences were found when guinea pig tissues were analyzed.     

Survey of aspirin administration in systemic mastocytosis

BackgroundPrevious recommended doses for aspirin use in systemic mastocytosis have been 3.9–5.2 g/d. Here, the aspirin doses and biochemical responses of patients with systemic mastocytosis given aspirin to decrease prostaglandin D₂ levels and prevent symptoms were reviewed.MethodsTwenty patients with systemic mastocytosis who had been given aspirin were identified, and their clinical and laboratory records were reviewed including changes in the excretion of the prostaglandin D₂ metabolite 11β-prostaglandin F_2α in response to aspirin.ResultsTwo of 20 patients developed either a delayed reaction or flushing during outpatient updosing with aspirin. In 20 of 20 patients with elevated baseline urinary excretion of 11β-prostaglandin F_2α, aspirin therapy caused a reduction to normal levels of excretion. Doses of aspirin required ranged from 81 mg twice daily to 500 mg twice daily.ConclusionsControl of elevated prostaglandin D₂ levels in systemic mastocytosis can be achieved with lower doses of aspirin than previously reported as necessary in this disorder.     

Quantification of the major urinary metabolite of prostaglandin D2 by a stable isotope dilution mass spectrometric assay

Prostaglandin D2 (PGD2) has been found to be an important pathophysiological mediator in a number of human disorders. Thus a means to assess the endogenous production of PGD2 is of considerable clinical value. To accomplish this goal, we developed a method for the quantification of the major urinary metabolite of PGD2, 9 alpha, 11 beta-dihydroxy-15-oxo-2,3,18,19-tetranorprost-5-ene-1,20-dioic acid, by gas chromatography/negative ion chemical ionization mass spectrometry. This metabolite was chemically synthesized and converted to an 18O4-labeled derivative for use as an internal standard. Novel derivatization and purification procedures were incorporated in the assay taking advantage of the ability of the lower side chain of this molecule to undergo cyclization at acidic pH to form a hemiketal, gamma-lactone, and uncyclization with methoximation. Precision of the assay is +/- 7% and accuracy is 96%. The lower limit of sensitivity is approximately 50 pg. Normal levels for the urinary excretion of this metabolite in 18 normal adults was found to be 1.08 +/- 0.72 ng/mg creatinine (mean +/- 2SD). Substantial elevations in the urinary excretion of the metabolite were found in clinical situations in which prostaglandin D2 has been shown to be released in increased quantities. Thus, this assay provides a sensitive and accurate method to assess endogenous production of prostaglandin D2 as a means to explore the pathophysiological role of prostaglandin D2 in human disease.