Enhancement of anaphylactic mediator release from guinea-pig perfused lungs by fatty acid hydroperoxides

Fragments of chopped lung from indomethacin treated guinea-pigs had an anti-aggregating effect when added to human platelet rich plasma (PRP), probably due to the production of prostacyclin (PGI₂) since the effect was inhibited by 15-hydroperoxy arachidonic acid (15-HPAA, 10 μg ml^?1). Both 15-HPAA (1–20 μg ml^?1 min^?1) and 13-hydroperoxy linoleic acid (13-HPLA, 20 μg ml^?1 min^?1) caused a marked enhancement of the anaphylactic release of histamine, slow-reacting substance of anaphylaxis (SRS-A) and rabbit aorta contracting substance (RCS) from guinea-pig isolated perfused lungs. This enhancement was not reversed by the concomitant infusion of either PGI₂ (5 μg ml^?1 min^?1) or 6-oxo-prostaglandin F_1α (6-oxo-PGF_1α, 5 μg ml^?1 min^?1). Anaphylactic release of histamine and SRS-A from guinea-pig perfused lungs was not inhibited by PGI₂ (10 ng - 10 μg ml^?1 min^?1) but was inhibited by PGE₂ (5 and 10 μg ml^?1 min^?1). Antiserum raised to 5,6-dihydro prostacyclin (PGI₁) in rabbits, which also binds PGI₂, had no effect on the release of anaphylactic mediators. The fatty acid hydroperoxides may enhance mediator release either indirectly by augmenting thromboxane production or by a direct effect on sensitized cells. Further experiments to distinguish between these alternatives are described in the accompanying paper (27).     

Effect of bromocriptine on prostacyclin release and cyclic nucleotides on rat aortic and uterine tissues

The effect of bromocriptine mesylate on cyclic nucleotides and PGI₂ release by rat aortic and uterine tissues was investigated. Treatment of rats with bromocriptine (10 mg kg⁻¹ I.P. daily for 14 days) increased PGI₂ release by the thoracic aorta from 0.67 ± 0.02 to 1.4 ± 0.03 ng/mg wet tissue (P < 0.001; n = 6). This increase was antagonized by treatment with sulpiride (15 mg kg⁻¹). Incubation of the arterial tissue with bromocriptive (50 ug ml⁻) in vitro also stimulated PGI₂ release. Mepacrine (160 μg ml⁻) significantly decreased both basal and stimulated PGI₂ release. Incubation of myometrial tissue from pregnant rats with bromocriptine (50 μg ml⁻¹) in vitro significantly decreased PGI₂ release from 1.25 ± 0.07 to 0.60 ± 0.08 ng/mg wet tissue (P < 0.05, n = 6).It also elevated uterine cAMP from 40 ± 2 to 64 ± 3 pmoles/100 mg wet tissue. Both effects were antagonized by sulpiride. Bromocriptine did not affect uterine cGMP or the cyclic nucleotides in the aorta. It is concluded that the increase in aortic PGI₂ was mediated via activation of dopamine D-2 receptors that stimulate phospholipase A₂ enzyme. The decrease in myometrial PGI₂ release may be related to the increase in uterine cAMP resulting from activation of dopamine D-1 receptors. Previous studies suggested a role for PGI₂ in implantation in the rat. The results suggest that the inhibitory effèct on uterine PGI₂ may underlie the reported inhibition of bromocriptine on implantation. On broad basis, the decrease in uterine PGI₂ together with the reported luteolytic effect of bromocriptine point to a potential role for the compound in postcoital contraception.     

Enhancement of anaphylactic mediator release from guinea-pig perfused lungs by fatty acid hydroperoxides.

Fragments of chopped lung from indomethacin treated guinea-pigs had an anti-aggregating effect when added to human platelet rich plasma (PRP), probably due to the production of prostacyclin (PGI2) since the effect was inhibited by 15-hydroperoxy arachidonic acid (15-HPAA, 10 micrograms ml(-1)). Both 15-HPAA (1-20 micrograms ml(-1) min (-1)) and 13-hydroperoxy linoleic acid (13-HPLA, 20 micrograms ml(-1) min(-1)) caused a marked enhancement of the anaphylactic release of histamine, slow-reacting substance of anaphylaxis (SRS-A) and rabbit aorta contracting substance (RCS) from guinea-pig isolated perfused lungs. This enhancement was not reversed by the concomitant infusion of either PGI2 (5 micrograms ml(-1) min (-1)) or 6-oxo-prostaglandin F1alpha (6-oxo-PGF1alpha, 5 micrograms ml(-1) min(-1)). Anaphylactic release of histamine and SRS-A from guinea-pig perfused lungs was not inhibited by PGI2 (10 ng - 10 microgram ml(-1) min(-1)) but was inhibited by PGE2 (5 and 10 micrograms ml(-1) min (-1)). Antiserum raised to 5,6-dihydro prostacyclin (PGI1) in rabbits, which also binds PGI2, had no effect on the release of anaphylactic mediators. The fatty acid hydroperoxides may enhance mediator release either indirectly by augmenting thromboxane production or by a direct effect on sensitized cells. Further experiments to distinguish between these alternatives are described in the accompanying paper (27).     

The effects of a 5-lipoxygenase inhibitor, AA-861, on PGI2-like substance production in guinea-pig lungs

To determine the effects of AA-861 on PGI₂ production in guinea-pig lungs, 3 g of guinea-pig lung was chopped in 4 ml of buffer (control group), in buffer with 4 μg/ml indomethacin (indomethacin group) and in buffer with 2.5 × 10⁻⁵M AA-861 (AA-861 group). The chopped lungs were incubated for 30 min. 250 μl of incubation medium from each group was assessed before and after 3, 5, 10, 15, 20, 25 and 30 min of incubation. The incubation medium was centrifuged and the supernatant was tested for a PGI₂-like substance (PGI₂) by platelet aggregation inhibition. PGI₂ was produced mainly during the initial 3–5 min of incubation and was decreased thereafter. PGI₂ production was almost completely inhibited in the indomethacin group at all of the incubation times and was partially inhibited in the AA-861 group during the initial 3–5 minutes. Endogenous 5-lipoxygenase products generated in the early stages of incubation seem to be involved in PGI₂ production in guinea-pig lungs.     

Role of prostacyclin on steroidogenesis by frog interrenal gland

The role of prostacyclin (PGI₂) on amphibian adrenal steroidogenesis was studied in perifused interrenal fragments from adult male frogs. Exogenous PGI₂ (3×10⁻⁸ M to 3×10⁻⁵ M) and, in a lesser extent, 6-keto-PGF_1α increased both corticosterone and aldosterone production in a dose-related manner. Short pulses (20 min) of 0.88 μM PGI₂ administered at 90 min intervals within the same experiment did not induce any desensitization phenomenon. A prolonged administration (6 h) of PGI₂ gave rise to an important increase in steroid production followed by a decline of corticosteroidogenesis. Indomethacin (IDM, 5 μM) induced a marked reduction of the spontaneous secretion of corticosteroid which confirmed the involvement of endogenous PGs in the process of corticosteroid biosynthesis. The IDM-induced blockade of corticosterone and aldosterone secretion was totally reversed by administration of exogenous PGI₂ in our model. Angiotensin II (AII) induced a massive release of 6-keto-PGF_1α, the stable metabolite of PGI₂. The increase of 6-keto-PGF_1α preceded the stimulation of corticosterone and aldosterone secretions. In contrast, the administration of ACTH did not modify the release of 6-keto-PGF_1α. These results indicate that PGI₂ might be an important mediator of adrenal steroidogenesis in frog. They confirm that the corticosteroidogenic actions of ACTH and AII are mediated by different mechanisms.     

Prostaglandin synthesis by fetal rat bone in vitro: Evidence for a role of prostacyclin

Prostaglandin synthesis by fetal rat bones was examined by thin-layer chromatography of culture media after preincubation with labeled arachidonic acid. Cultures in rabbit complement (non-heat inactivated serum) were compared with cultures in heat-inactivated serum or cultures treated with indomethacin. The major complement-dependent products were PGE₂, PGF_2α and 6-keto-PGF_1α, the metabolite of prostacyclin (PGI₂). Since PGI₂ had not been previously identified in bone its ability to stimulate bone resorption was tested. Repeated addition of PGI₂ stimulated release of previously incorporated ⁴⁵Ca from fetal rat long bones in both short-term and long-term cultures at concentrations of 10⁻⁵ to 10⁻⁹M. Because of the short half life of PGI₂ in solution at neutral pH, we tested a sulfur analog, thiaprostacyclin (S-PGI₂) which was found to be a stimulator of bone resorption at concentrations of 10⁻⁵ to 10⁻⁶M. These studies suggest that endogenous PGI₂ production may play a role in bone metabolism. Since vessels produce PGI₂ it is possible that PGI₂ release may be responsible for the frequent association between vascular invasion and resorption of bone or calcified cartilage in physiologic remodeling and pathologic osteolysis.     

Influence of type of dietary fat on the prostaglandin release from isolated rabbit and rat hearts and from rat aortas

It has previously been found (1) that feeding rats a diet containing a high amount of sunflowerseed oil results in a higher coronary flow and left ventricular work of their isolated hearts as compared to hearts of rats fed hydrogenated coconut oil or lard. It was hypothesized that this phenomenon can be explained by an influence of dietary linoleic acid on prostaglandin synthesis in the heart. To verify this hypothesis rabbits and rats were fed for four weeks sunflowerseed oil (SSO), hydrogenated coconut oil (HCO) or lard (L) to a maximum of 30 to 40 per cent of the total digestable energy, and the prostaglandin release from the isolated perfused hearts and rat aortas was determined by gas chromatography and bio-assay (PGI₂).For the isolated hearts of rabbits fed SSO, the release of PGE₂, PGF_2α and 6-oxo-PGF_1α was 1.7, 0.7 and 3.0 ng min⁻¹ g⁻¹ dry weight respectively; when fed L, these values were 2.9, 1.1 and 5.6 ng min⁻¹ g⁻¹. For the isolated hearts of rats fed SSO, HCO or L, the total release of PGE₂, PGD₂, PGF_2α and thromboxane B₂ (TXB₂) was 5.9, 5.8 and 5.6 ng min⁻¹ g⁻¹ respectively; the release of 6-oxo-PGF_1α was 3.4, 5.7 and 6.4 ng min⁻¹ g⁻¹ respectively. Relatively, 26% PGE₂, 13% PGD₂, 8% PGF_2α, 6% TXB₂ and 47% 6-oxo-PGF_1α were released. For the isolated aortas of rats fed SSO or HCO, the release of PGI₂-like activity was 0.37 ± 0.05 and 0.49 ± 0.05 ng min⁻¹ cm⁻². The release of PGI₂-like activity from hearts of EFA-deficient rats was about 20% of that from control hearts.We conclude that, although feeding sunflowerseed oil, with respect to feeding hydrogenated coconut oil or lard, does increase coronary flow and left ventricular work, it does not increase the basal prostaglandin production in the isolated rat or rabbit heart; instead there is a tendency for a lower PGI₂ synthesis.     

Arachidonic acid stimulates interleukin-6 release from rat peritoneal macrophages in vitro: Evidence for a prostacyclin-dependent mechanism

Interleukin-6 (IL-6) is a cytokine involved in the differentiation of B-cells to antibody secreting plasma cells, the activation of T-cells, and the stimulation of hepatocyte production of acute phase proteins. Because of the pro-inflammatory effects of this cytokine, we investigated the ability of the fatty acid arachidonic acid (AA) to regulate the release of IL-6 from rat resident peritoneal macrophages (Mø) in vitro. AA (0.5–16 μM) stimulated IL-6 release during a 4 h incubation period in a biphasic manner, with 4 μM AA generating a peak of IL-6 release (3-5-fold). AA (0.5–16 μM) also induced an increasing release of the AA metabolite thromboxane B₂ (TXB₂). The AA-induced release of IL-6 occurred within 1–2 h of incubation, whereas TXB₂ concentrations were elevated within 5 min of AA treatment. The TX synthetase inhibitor CGS 12970 (4.0 μM and 40.0 μM) effectively blocked the generation of TXB₂, but increased prostacyclin (PGI₂) generation and potentiated the release of IL-6. In addition, PGI₂, as well as the PGI₂ agonists iloprost and cicaprost, stimulated IL-6 release from Mø by greater than 5-fold over vehicle-treated basal levels. These data suggest that PGI₂ (but not TXA₂) is involved in AA-induced IL-6 release from peritoneal Mø.     

Effect of dipyridamole of prostaglandin generation by human platelets and vessel walls

These experiments were conducted to determine the effects of dipyridemole on human platelet aggregation, platelet thromboxane A₂ (TXA₂) and human vessel wall prostacyclin (PGI₂) generation. Dipyridamole in varying concentrations (5 to 50 μg/ml) had no direct effect on ADP-induced platelet aggregation in vitro, but it potentiated PGI₂-induced platelet aggregation inhibition at these concentrations. Dipyridamole also inhibited arachidonic acid-induced platelet TXA₂ generation at these concentrations. In continuously perfused umbilical vein segments, dipyridamole treatment resulted in stimulation of PGI₂ release determined by bioassay and by measurement of its stable metabolite 6-keto-PGF_1α. Minimum concentration of dipyridamole causing PGI₂ release was 50 μg/ml. These in vitro studies suggest that anti-thrombotic effects of dipyridamole in man are mediated mainly by potentiation of PGI₂ activity and to some extent by TXA₂ suppression. Stimulation of PGI₂ release by human vessels may not be seen in usual therapeutic concentrations.     

Differing mechanisms for leukotriene D4-induced bronchoconstriction in guinea pigs following intravenous and aerosol administration

Leukotriene D₄ (LTD₄) when administered intravenously or by aerosol to guinea pigs produced changes in pulmonary mechanics including a decrease in dynamic compliance and an increase in pulmonary resistance. The effects of intravenous LTD₄ (0.5 μg kg⁻¹) were short lived and abolished by pretreatment of the animal with either cyclooxygenase inhibitors, a thromboxane synthetase inhibitor (OKY 1555) or an SRS-A antagonist (FPL 55712). These findings suggest that bronchoconstriction produced by the intravenous infusion of LTD₄ at 0.5 μg kg⁻¹ is due to the release of thromboxane A₂. However, in animals treated with indomethacin, LTD₄ at higher doses (>0.8 μg kg⁻¹) still elicited a bronchoconstriction which could be blocked by FPL 557112. Nebulization of 0.1 – 1.0 μg of LTD₄ into the lung produced prolonged changes in pulmonary mechanics which were inhibited by FPL 55712 and were potentiated indomethacin. LTD₄, therefore, when administered by aerosol produced effects on the lung which were not mediated by cyclooxygenase products. Responses to nebulized rather than intravenous LTD₄ in the guinea pig may more closely resemble those seen in human tissues.     

The mechanism of action of leukotrienes C4 and D4 in guinea-pig isolated perfused lung and parenchymal strips of guinea pig, rabbit and rat

The biological actions of pure slow-reacting substance of anaphylaxis (SRS-A) from guinea-pig lung, pure slow-reacting substances (SRS) from rat basophilic leukaemia cells (RBL-1) and synthetic leukotrienes C₄ (LTC₄) and D₄ (LTD₄) have been investigated on lung tissue from guinea pig, rabbit and rat. In the guinea pig, the leukotrienes released cyclo-oxygenase products from the perfused lung and contracted strips of parenchyma. The effects of SRS-A, SRS and LTD₄ were indistinguishable. LTC₄ and LTD₄ had similar actions although LTD₄ was more potent than LTC₄. Indo-methacin (1 μg/ml) inhibited the release of cyclo-oxygenase products from perfused guinea-pig lung and caused a marked reduction in contractions of guinea-pig parenchymal strips (GPP) due to LTC₄ and LTD₄. The residual contraction on the GPP was abolished by FPL 55712 (0.5 – 1.0 μg/ml). It appears, therefore, that a major part of the constrictor actions of LTC₄ and LTD₄ in guinea-pig lung are mediated by myotropic cyclo-oxygenase products, i.e. thromboxane A₂ (TxA₂) and prostaglandins (PGs).In rabbit and rat lung, however, SRS-A, SRS and the leukotrienes were much less potent in contracting parenchymal strips and there was little evidence of the release of cyclo-oxygenase products. FPL 55712 at a concentration of 1 μg/ml failed to antagonise leukotriene-induced contractions.     

The effects of microsomal prostaglandin E synthase-1 deletion in acute cardiac ischemia in mice

The goal of the present study was to assess how genetic loss of microsomal prostaglandin E₂ synthase-1 (mPGES-1) affects acute cardiac ischemic damage after coronary occlusion in mice. Wild type (WT), heterozygous (mPGES-1^+/−), and homozygous (mPGES-1^−/−) knockout mice were subjected to left coronary artery occlusion. At 24 h, myocardial infarct (MI) volume was measured histologically. Post-MI survival, plasma levels of creatine phosphokinase (CPK) and cardiac troponin-I, together with MI size, were similar in WT, mPGES-1^+/− and mPGES-1^−/− mice. In contrast, post-MI survival was reduced in mPGES-1^−/− mice pretreated with I prostanoid receptor (IP) antagonist (12/16) compared with vehicle-treated controls (13/13 mPGES-1^−/−) together with increased CPK and cardiac troponin-I release. The deletion of mPGES-1 in mice results in increased prostacyclin I₂ (PGI₂) formation and marginal effects on the circulatory prostaglandin E₂ (PGE₂) level. We conclude that loss of mPGES-1 results in increased PGI₂ formation, and in contrast to inhibition of PGI₂, without worsening acute cardiac ischemic injury.     

Prostacyclin inhibits 5-hydroxytryptamine release but stimulates thromboxane synthesis during cardiopulmonary bypass

The antiaggregating agent prostacyclin (PGI₂) was infused into ten dogs during cardiopulmonary bypass (CPB) to minimize thrombocytopenia and platelet dysfunction. The animals were anesthetized, placed on mechanical ventilation and underwent thoracotomy. After heparinization with 300 u/kg, animals were assigned to control (n=5) or PGI₂ treated groups (n=5). Thoracotomy and then CPB decreased platelet numbers to below 30, 000/mm³ (p < 0.05) and fibrinogen to less than 150 mg/dl (p < 0.05). PGI₂ at 100 ng/kg·min was infused for the 2 h period of CPB. PGI₂ infusion did not prevent these changes, but did prevent platelet serotonin release. In the control group after CPB, platelet serotonin fell from the baseline value of 1.11 μg/10⁹ to 0.35 μg/10⁹ platelets (p < 0.05). In contrast, PGI₂ treatment resulted in a serotonin increase to 2.27 μg/10⁹ platelets (p < 0.05). Thromboxane B₂ concentrations of platelets and plasma rose during CPB (p < 0.05). Surprisingly, PGI₂ infusion accentuated this rise in platelet and plasma thromboxane B₂ (p < 0.05). These data indicate that during CPB, an infusion of PGI₂: 1) does not prevent thrombocytopenia; 2) increases platelet serotonin uptake despite, 3) an associated rise in platelet and plasma thromboxane B₂.     

Comparative study of the pulmonary effects of intravenous leukotrienes and other bronchoconstrictors in anaesthetized guinea pig

Pulmonary responses to intravenous leukotrienes C₄, D₄ and E₄ administered as a bolus injection and by continuous infusion were studied in anesthetized guinea pigs. LTD₄, LTC₄ and LTE₄ (respective ED₅₀ of 0.21 ± .1, 0.64 ± .2 and 2.0 ± .1 μg kg⁻¹) produced dose-dependent increases in insufflation pressure when given as a bolus injection to anesthetized guinea pigs (Konzett-Rössler). Bronchoconstriction was antagonized by FPL-55712 (50–200 μg kg⁻¹), and indomethacin (50–200 μg kg⁻¹) but was not significantly altered by mepyramine (1.0 mg kg⁻¹), methysergide (0.1 mg kg⁻¹), intal (10 mg kg⁻¹) mepacrine (5 mg kg⁻¹) or dexamethasone (10 mg kg⁻¹). The beta adrenoceptor blocker, timolol (5 μg kg⁻¹) produced a significantly greater potentiation of the responses to the leukotrienes than to arachidonic acid, histamine and acetylcholine. Responses to bolus injection of LTE₄ but not LTD₄ or LTC₄ were partially antagonized by atropine (100 μg kg⁻¹) and bilateral vagotomy. In experiments of a different design, continuous infusion of LTD₄ and LTE₄ (2.8–3.2 μg kg⁻¹ min⁻¹) into indomethacin-treated animals produced slowly developing increases in pulmonary resistance and decreases in compliance. The increase in resistance produced by LTE₄ and LTD₄ was partly reversed by intravenous FPL-55712 (1.0 mg kg⁻¹) and atropine (100 μg kg⁻¹) but was almost completely reversed by FPL-55712 (3 – 10 mg kg⁻¹). These findings indicate that leukotrienes can produce bronchoconstriction in guinea pigs through cyclooxygenase-dependent and cyclooxygenase independent mechanisms both of which are blocked by FPL-55712. Cholinergic mechanisms are involved in the mediation of part of the response to bolus injection of LTE₄ as well as a small part of the initial response to continuous infusion of LTD₄ and LTE₄. Intrinsic beta adrenoceptor activation serves to down modulate responses to the leukotrienes to a greater extent than responses to arachidonic acid, histamine and acetylcholine.     

Ammonium excretion by a mutant of the nitrogen-fixing cyanobacterium Anabaena siamensis

Anabaena siamensis isolated from rice fields in Thailand is a fast growing cyanobacterium with a high nitrogen-fixing activity. Mutant strains resistant to the l-glutamate analogue, l-methionine sulfoximine (MSX) were isolated by ethyl methanesulfonate mutagenesis. A stable mutant named A. siamensis SS1, which released ammonium to the medium, was studied further. In batch cultures the rate of ammonium production peaked at the early log phase and gradually decreased until the 4th day of growth when the cultures reached a density of 90 μg chl ml⁻¹. To obtain constant release of ammonium by SS1, continuous culture experiments were performed at a cell density of 5 μg chl ml⁻¹ and the following results were obtained: (1) growth rate as the parent (μ:0·123 h⁻¹) in the presence and absence of 500 μm MSX; (2) 48% GS transferase activity when compared with the parent; (3) ammonium excretion at a rate of 8 μmol (mg chl)⁻¹ h⁻¹ as measured up to 20 generations (120 h); (4) depressed nitrogenase activity; and (5) 30% higher nitrogenase activity than that of the parent. SS1 immobilized in alginate beads (5 μg chl ml⁻¹) exhibited values of glutamine synthetase and nitrogenase activity similar to those of free cells. However, ammonium excretion at the rate of 11·61 μmol (mg chl)⁻¹ h⁻¹ was obtained only up to 20 h after loading in bioreactors, due to the fast growth of SS1 as also occurred in batch cultures.     

Effects of prostacyclin on coronary circulation, heart rate and myocardial contractile force in isolated hearts of guinea PIG and rabbit — Comparison with prostaglandin E2

Infusions of prostacyclin (PGI₂) (3 × 10⁻¹⁰ − 3 × 10⁻⁷M) into the coronary circulation of isolated hearts from guinea pigs or rabbits resulted in a concentration-dependent decrease in the coronary perfusion pressure (CPP). There was a slight decrease in left ventricular systolic pressure in the heart of the rabbit, whereas the heart rate remained unchanged. PGE₂ was without effect on the heart of the rabbit but was as potent as PGI₂ in decreasing the CPP in the guinea pig heart. 6-oxo-PGF_1α (up to 3 × 10⁻⁶ M) did not affect any of the parameters measured.     

High-performance liquid chromatographic analysis of AQ4N, an alkylaminoanthraquinone N-oxide

A simple, highly selective and reproducible reversed-phase high-performance liquid chromatography method has been developed for the analysis of the new anti-cancer pro-drug AQ4N. The sample pre-treatment involves a simple protein precipitation protocol, using methanol. Chromatographic separations were performed using a HiChrom HIRPB (25 cm×4.6 mm I.D.) column, with mobile phase of acetonitrile–ammonium formate buffer (0.05 M) (22:78, v/v), with final pH adjusted to 3.6 with formic acid. The flow-rate was maintained at 1.2 ml min⁻¹. Detection was via photodiode array performed in the UV range at 242 nm and, since the compounds are an intense blue colour, in the visible range at 612 nm. The structurally related compound mitoxantrone was used as internal standard. The validated quantification range of the method was 0.05–10.0 μg ml⁻¹ in mouse plasma. The inter-day relative standard deviations (RSDs) (n=5) ranged from 18.4% and 12.1% at 0.05 μg ml⁻¹ to 2.9% and 3.3% at 10.0 μg ml⁻¹ for AQ4N and AQ4, respectively. The intra-day RSDs for supplemented mouse plasma (n=6) ranged from 8.2% and 14.2% at 0.05 μg ml⁻¹ to 7.6% and 11.5% at 10.0 μg ml⁻¹ for AQ4N and AQ4, respectively. The overall recovery of the procedure for AQ4N was 89.4±1.77% and 76.1±7.26% for AQ4. The limit of detection was 50 ng ml⁻¹ with a 100 μl sample volume. The method described provides a suitable technique for the future analysis of low levels of AQ4N and AQ4 in clinical samples.     

Thromboxane synthase inhibition: Implications for prostaglandin endoperoxide metabolism: I Characterisation of an acute intravenous challenge model to measure prostaglandin endoperoxide metabolism

It has been proposed that thromboxane synthase inhibition (TXSI) may be a useful form of anti-thrombotic therapy and that this is due, in part, to redirection of PGH₂ metabolism in favour of PGI₂, a potent vasodilator and anti-platelet agent. While redirection has been observed there are conflicting reports of its occurrence . We now describe the characterisation of an acute intravenous challenge model using thrombin, collagen, arachidonic acid (AA) and PGH₂ for the study of PGH₂ metabolism. Following challenge, plasma concentrations of TXB₂, 6-oxo-PGF_1α, alleged metabolites of PGI₂ (PGI₂m) and PGE₂ were measured by radioimmunoassay (RIA). Thrombin and collagen challenge resulted in a dose-related increase in plasma TXB₂ while AA and PGH₂, in addition, elevated 6-oxo-PGF_1α and PGI₂m. Injection of PGH₂ elevated 6-oxo-PGF_1α, PGI₂m, TXB₂ and PGE₂ levels. Experimental conditions were defined such that challenge with thrombin (40 NIH units kg⁻¹), collagen (100 kg⁻¹), AA (1mg kg⁻¹) and PGH₂ (5μg kg⁻¹) and measurement of eicosanoids 0.5min following challenge (5μg kg⁻¹) and measurement of eicosanoids 0.5min following challenge were optimal for detection of redirection of PGH₂ metabolism . The identity of immunoreactive TXB₂ and 6-oxo-PGF_1α was further supported by experiments in which the extracted immunoreactive eicosanoids co-eluted with authentic [³H]standards when subject to reverse phase high performance liquid chromatography (RPHPLC). Evidence is also presented that the levels of plasma eicosanoids measured in this model reflect biosynthesis.     

Sulfalene concentrations in plasma and blood cells of Plasmodium falciparum malaria cases after treatment with metakelfin using high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method using acetonitrile–methanol–1 M perchloric acid–water (25:9:0.8:95, v/v/v) at a flow-rate of 1.0 ml min⁻¹ on LiChrospher 100 RP 18 column (250×4 mm; 5 μm) with UV (254 nm) detection has been developed for the determination of sulfalene in plasma and blood cells after oral administration of the antimalarial drug metakelfin. Calibration curves were linear in the range 0.5–100 μg ml⁻¹. The limit of quantification was 50 ng ml⁻¹. Within-day and day-to-day coefficients of variation averaged 3.84 and 5.31%, respectively. Mean extraction recoveries of sulfalene from plasma and blood cells were 87.21 and 84.65%, respectively. Mean concentrations of sulfalene in plasma of P. falciparum cases on days 2, 7 and 15 were 44.58, 14.90 and 1.70 μg ml⁻¹, respectively; in blood cells concentrations of sulfalene were 7.77, 3.25 and 0.75 μg ml⁻¹, respectively, after oral treatment with two tablets (1000 mg) of metakelfin. Significant difference was recorded on day 2 for sulfalene concentration in blood cells of healthy and P. falciparum cases (t=9.49; P<0.001).     

Assay of R-apomorphine, S-apomorphine, apocodeine, isoapocodeine and their glucuronide and sulfate conjugates in plasma and urine of patients with Parkinson's disease

Analytical methods are described for the selective, rapid and sensitive determination of R- and S-apomorphine, apocodeine and isoapocodeine and the glucuronic acid and sulfate conjugates in plasma and urine. The methods involve liquid-liquid extraction followed by high-performance liquid chromatography with electrochemical detection. The glucuronide and sulfate conjugates are determined after enzymatic hydrolysis. For the assay of R- and S-apomorphine a 10 μm Chiralcel OD-R column is used and the voltage of the detector is set at 0.7 V. The mobile phase is a mixture of aqueous phase (pH 4.0)-acetonitrile (65:35, v/v). At a flow-rate of 0.9 ml min⁻¹ the total run time is ca. 15 min. The detection limits are 0.3 and 0.6 ng ml⁻¹ for R- and S- apomorphine, respectively (signal-to-noise ratio 3). The intra- and inter-assay variations are <5% in the concentration range of 2.5-25 ng ml⁻¹ for plasma samples, and <4% in the concentration range of 40-400 ng ml⁻¹ for urine samples. For the assay of apomorphine, apocodeine and isoapocodeine, a 5 μm C₁₈ column was used and the voltage of the detector set at 0.825 V. Ion-pairing chromatography was used. The mobile phase is a mixture of aqueous phase (pH 3.0)-acetonitrile (75:25, v/v). At a flow-rate of 0.8 ml min⁻¹ the total run time is ca. 14 min. The detection limits of this assay are 1.0 ng ml⁻¹ for apomorphine and 2.5 ng ml⁻¹ for both apocodeine and isoapocodeine (signal-to-noise ratio 3). The inter-assay variations are 5% in the concentration range of 5-40 ng ml⁻¹ for plasma samples and 7% in the concentration range of 50-500 ng ml⁻¹ for urine samples. The glucuronic acid and sulfate conjugates of the various compounds are hydrolysed by incubation of the samples with β-glucuronidase and sulfatase type H-1, respectively. Hydrolysis was complete after 5 h of incubation. No measurable degradation of apomorphine, apocodeine and isoapocodeine occurred during the incubation. A pharmacokinetic study of apomorphine, following the intravenous infusion of 30 μg kg⁻¹ for 15 min in a patient with Parkinson's disease, demonstrates the utility of the methods: both the pharmacokinetic parameters of the parent drug and the appearance of apomorphine plus metabolites in urine could be determined.