Thromboxane B2 uptake and metabolism in isolated perfused lung. Identification and comparison with prostaglandin F2 alpha.

Thromboxane B2 was metabolised in isolated perfused guinea-pig lungs to a product identified by negative ion chemical ionisation mass spectrometry as 13,14-dihydro-15-ketothromboxane B2. Conversion was measured by radio TLC and was greater in guinea-pig than rat lungs (29.1 vs. 13.8% at 10 ng/ml), but similar in lungs from normal and sensitized guinea-pigs. Thromboxane B2 metabolism was less than that of prostaglandin F2 alpha but, like it, was prevented at 5 degrees C and reduced by cycloheximide pretreatment. Tissue to medium ratio in perfused guinea-pig lungs was 3.4 for thromboxane B2, but 0.2 for insulin (showing that thromboxane B2 is accumulated within the lung) and was altered after experimental manipulations. Neither lung slices, crude homogenates, cytosolic and microsomal fractions nor purified prostaglandin 15-hydroxydehydrogenase metabolised thromboxane B2 in vitro, although prostaglandin F2 alpha was extensively inactivated. Quantitative partition coefficient and albumin-binding data confirm that thromboxane B2 lacks prominent lipophilicity, implying that cellular uptake in lung must be carrier-mediated. We conclude that thromboxane B2 is a substrate for pulmonary degradation which may form a route for the biological inactivation of thromboxane A2. Its resistance to prostaglandin 15-hydroxydehydrogenase as conventionally tested remains paradoxical and is discussed.

Prostaglandins D2 and E2 are not regulators of amino acid release from rat cortical synaptosomes.

Rat cerebral cortex synaptosomes synthesise prostaglandins, and analysis by gas chromatography-mass spectrometry revealed that of the prostaglandins quantified PGD2 (10.9 ng/mg protein) was produced in highest concentration. Treatment with high potassium or veratrine caused release of putative amino acid transmitters, but not of prostaglandins, and prostaglandins D2 and E2 were unable to stimulate release of amino acids. The release of putative amino acid transmitters, evoked by high potassium levels, was not inhibited by these prostaglandins. Prostacyclin receptors could not be identified on synaptosomes by radioligand binding techniques.

Total profiling by GC/NICIMS of the major cyclo-oxygenase products from antigen and leukotriene-challenged guinea-pig lung.

Separation and quantitation of all the major cyclo-oxygenase products in perfused guinea-pig lungs challenged with antigen or leukotrienes C4 and D4 were achieved using a novel combined capillary column gas chromatography/negative ion chemical ionization mass spectrometric (GC/NICIMS) method. In descending order of magnitude, unchallenged lungs released thromboxane B2 (TXB2) plus its pulmonary metabolite (TXDK) greater than 6-keto-PGF1 alpha plus its 13,14-dihydro-15-keto metabolite (K2H1F1 alpha) greater than PGE2 plus PGF2 alpha greater than PGD2; after ovalbumin anaphylaxis there were increases of X 26 in TXB2 plus TXDK, X 28 in PGD2 and histamine (measured fluorometrically) but of only X 3 in 6-keto-PGF1 alpha plus K2H2F1 alpha and PGE2 plus PGF2 alpha. FPL55712 treatment greatly reduced the release of TXB2 and 6-keto-PGF1 alpha and their metabolites (showing this to be a secondary effect mediated by leukotriene action) but did not affect PGD2 output. LTC4 and LTD4 themselves induced the release of TXB2 and TXDK, as did bradykinin, but neither substance caused appreciable PGD2 release. Aside from illustrating the great value of the GC/NICIMS method for simultaneously determining all cyclooxygenase products, the main conclusions are: (i) PGD2 may be an in vitro marker for activation of lung inflammatory cells; (ii) prostacyclin and thromboxanes are actively metabolized in situ in the lung; and (iii) 'pathological subversion' of pulmonary function by anaphylaxis, leukotrienes or bradykinin principally causes thromboxane release from unknown target cells, with a smaller release of prostacyclin which may be compensatory in nature.

Prostacyclin binding to guinea pig pulmonary receptors.

[3H] Prostacyclin bound to membranes of guinea pig lung. Specific binding was saturable, and the results revealed two receptor classes (Kd =16 nM and 258 nM). The binding capacity of the high affinity site was 105 fmol [3H]prostacyclin per mg of membrane protein, and that of the low affinity site was 1257 fmol per mg membrane protein. A comparison of selected prostaglandins as inhibitors of [3H] prostacyclin binding revealed some of the structural requriements of the ligand for occupation of the high affinity receptor.

Thromboxane synthase inhibition causes re-direction of prostaglandin endoperoxides to prostaglandin D2 during collagen stimulated aggregation of human platelet rich plasma.

Prostanoid synthesis and release during collagen-induced aggregation of human platelet rich plasma (PRP) was studied using a novel gas chromatography/mass spectrometry assay technique. Aggregation was associated with the production of mainly thromboxane A2 (TXA2), measured as TXB2, and smaller amounts of the prostaglandins (PGs) D2, E2 and F2 alpha. UK 37,248 inhibited TXB2 formation by greater than 95% and increased the production of PGD2, PGE2 and PGF2 alpha twenty-fold. The relative amounts of these three prostanoids were not changed by UK 37,248. Even though high concentrations of PGD2 were formed, aggregation was not inhibited. In contrast, flurbiprofen inhibited aggregation, demonstrating that platelet aggregation produced by this concentration of collagen is cyclooxygenase dependent. These results support the proposal that the prostaglandin endoperoxides can induce aggregation alone, irrespective of the amount of PGD2 that is produced.

Plasma levels and urinary excretion of orally administered propantheline bromide in man.

After a single oral dose of 30 or 60 mg of propantheline bromide peak plasma levels of the drug were reached within 2 h in six healthy men. Mean peak plasma concentrations were 20.6 and 53.1 ng/ml after 30 mg and 60 mg respectively. The mean apparent absorption and elimination half-lives after 30 mg dose were 0.22 and 1.57 h respectively, and similar half-lives were found at the higher dose level. There was a dose related change in plasma levels and AUCinfinity of the drug, and some 3% to 4% of the administered dose of propantheline bromide was excreted unchanged in urine at each dose level. Comparison of the plasma levels and urinary excretion of the drug with those seen after i.v. administration in an earlier study indicated an apparently low systemic availability of orally administered propantheline bromide. There was tentative evidence of a qualitative relationship between the oral dose administered, plasma concentrations and the effects of propantheline bromide on salivary excretion.

The measurement of clonidine in human plasma and urine by combined gas chromatography mass spectrometry with ammonia chemical ionization.

The dimethyl derivatives of clonidine and [2H4]clonidine give good chemical ionization mass spectra when ammonia is used as reagent gas. A gas chromatographic mass spectrometric selected ion monitoring assay for the estimation of clonidine in plasma and urine using ammonia chemical ionization is described.

A "carrier effect" observed in assay of antidiarrhoeal drug compounds.

A carrier effect has been shown to exist in a stable isotope dilution assay for diphenoxylate. When a tetradeuterated analogue is used as a carrier and internal standard a sevenfold increase in sensitivity is observed for the unlabelled compound. Examination of a related pharmacologically active compound (SC-27166) showed a much smaller effect.

A stable isotope dilution assay for disopyramide and its [13C, 15N]labelled analogue in biological fluids.

The mass spectra of disopyramide phosphate and two stable isotopically labelled analogues have been obtained using electron impact and chemical ionization. The low isotopic purity of [13C, 15N)disopyramide phosphate was shown to be due to the low isotopic purity of the 15N label. A stable isotope dilution assay for disopyramide and [13C, 15N]disopyramide in biological fluids has been developed using [2H14]disopyramide phosphate as the internal standard. This assay will be used to analyse samples obtained after the co-administration of disopyramide phosphate intravenously and [13C, 15N]disopyramide phosphate orally to several animal species.

Analysis of picomolar concentrations of 6-oxo-prostaglandin F1 alpha in biological fluids.

A highly sensitive and specific assay for the quantitation of 6-oxo-prostaglandin F1 alpha, the stable hydrolysis product of prostacyclin, is described. The method involves the addition of [3,3',4,4'-2H4]-6-oxo-prostaglandin F1 alpha as internal standard, extraction from biological fluids using muBondapak C18 reversed-phase Sep-Paks, and preliminary purification by normal-phase chromatography. Following conversion to the methoxime, tris-trimethylsilyl, pentafluorobenzyl derivative, samples were analysed using combined capillary column gas chromatography negative ion chemical ionisation mass spectrometry. Fragments ions at m/z 614 (1H) and 618 (2H) [M-C7H2F5]- were monitored for quantitation. This method was used for the measurement of endogenous levels of 6-oxo-prostaglandin F1 alpha in human urine and for the determination of prostacyclin release from rat peritoneal mast cells and from rat aortic rings incubated in human plasma.

Quantitative analysis of prostanoids in biological fluids by combined capillary column gas chromatography negative ion chemical ionization mass spectrometry.

An assay for the quantitative analysis of six biologically important prostanoids based on combined gas chromatography negative ion chemical ionization mass spectrometry has been developed. Prostanoids were extracted from biological fluids by liquid chromatography on Sep-Pak cartridges and converted to pentafluorobenzyl ester derivatives. Samples were injected on capillary column by the splitless technique and injections were made in a high boiling hydrocarbon solvent (n-dodecane) in order to minimize chromatographic run times. Quantification was carried out using selected ion monitoring of the appropriate [M-pentafluorobenzyl]- anion. The assay has been used for profiling cyclooxygenase metabolites of arachidonic acid in guinea pig lung perfusate after induction of anaphylaxis and platelet rich plasma after collagen-stimulated aggregation.

Correlation of prostacyclin synthesis by human umbilical artery with status of essential fatty acid.

Prostacyclin production by human umbilical artery rings was measured in a variety of incubation media and related to gestational age, mode of delivery, and birth weight. Prostacyclin production was directly correlated with the status of essential free fatty acid in the tissue as assessed by the ratio of tissue concentrations of arachidonic acid and Mead's acid. The results indicate a possible association of the essential fatty acid status both with intrauterine growth retardation and with fetal vascular prostacyclin production.

Prostacyclin is not a circulating hormone in man.

A highly specific stable isotope dilution assay for plasma 6-oxo-prostaglandin F1 alpha has been developed. The method employs capillary column gas chromatography coupled with negative ion chemical ionisation mass spectrometry. The limit of sensitivity of the assay is 0.5 pg.m1(-1). Concentrations of 6-oxo-prostaglandin F1 alpha in the plasma of 20 healthy volunteers determined by this assay were all below 3 pg.m1(-1). The levels were much lower than any previously reported and confirms that prostacyclin is not a circulating hormone in man under normal physiological conditions.

The absorption of disopyramide in animals determined using a stable isotope co-administration technique.

The stable isotope co-administration technique for estimating the bioavailability of drugs has been investigated in a series of experiments using rhesus monkeys. The compound chosen for study was disopyramide phosphate. A cross-over study was designed whereby the animals received disopyramide phosphate (administered intravenously at 5 mg kg-1) and [13C, 15N]disopyramide phosphate (administered orally at 5 mg kg-1), with a wash-out period between doses. A co-administration study was carried out whereby both the oral and intravenous doses were administered together. The co-administration study was repeated. The results from the cross-over study showed [13C, 15N]disopyramide to have an oral availability of 4.9 +/- 0.9% (by comparing areas under the plasma concentration versus time curves). The bioavailability was estimated to be 5.7 +/- 0.3% comparing totals excreted in urine over 48 h. The bioavailability of the oral dose was calculated as 8.2 +/- 2.5% (comparing areas under plasma concentration versus time curves) and 9.3 +/- 3.0% (comparing totals excreted in urine) after co-administration. The differences between these results and the cross-over results were examined in a further study, using oral administration only. The animals were dosed orally with a solution containing both disopyramide phosphate (5 mg kg-1) and [13C, 15N]disopyramide phosphate (5 mg kg-1). No differences were observed between the plasma concentration versus time curves or urinary excretion for either isotope. It is unlikely that the discrepancy in bioavailability is due to absorption, metabolism or exretion of the oral dose. It is probable that the high concentration of disopyramide obtained after the intravenous dosage affects the disposition of the oral dose, and this gives the higher figure.

Synthesis of leukotriene B4, and prostanoids by human alveolar macrophages: analysis by gas chromatography/mass spectrometry.

Human alveolar macrophages, obtained during diagnostic bronchoscopy, were maintained in monolayer culture. Challenge of these cells (greater than 95% purity) with 1.2 mg/ml zymosan A particles (opsonized with human serum) was followed by a rapid release of leukotriene B4 into the medium, 7.28 +/- 5.99 ng/mg cell protein at 2 h (mean +/- S.D.4, n = 4). Leukotriene B4 was identified and measured by a novel technique employing capillary column gas chromatography coupled to negative ion chemical ionization mass spectrometry. The release of thromboxane B2, prostaglandins D2, E2, F2 alpha and the lysosomal enzyme N-acetyl-beta-D-glucosaminidase was also measured. Thromboxane B2 was the most abundant metabolite of arachidonic acid released into the culture medium (65.2 +/- 14.8 ng/mg cell protein 2 h after the addition of zymosan A, n = 4), and the synthesis of thromboxane B2 was inhibited by greater than 90% in 1 microM Na flurbiprofen. Inhibition of cyclooxygenase activity was accompanied by a 2-fold increase in leukotriene B4 synthesis.