Bruce M. Taylor

Biochemical and functional differences between eosinophils from animal species and man.

Eosinophils were isolated from peritoneal lavages of repeated horse serum‐injected guinea pigs or rhesus monkeys and from peripheral blood of normal human donors. Eicosanoid metabolism and chemotaxis by these cells were studied by in vitro techniques. Upon calcium ionophore stimulation guinea pig eosinophils released thromboxane B2 (TXB2) and leukotriene B4 (LTB4) while monkey and human cells produced LTC4, LTB4, and 5‐HETE. Guinea pig cells do not synthesize sulfidopeptide LTs, because they lack the specific LTA4 glutathione S‐transferase. Guinea pig eosinophils exhibit maximal chemotactic responses to LTB4, zymosan activated plasma, and human recombinant C5a, while producing only a negligible response to platelet activating factor (PAF). Monkey and human cells responded maximally to PAF, but exhibit only a weak response to LTB4. These results suggest that the guinea pig eosinophils differ from monkey and human eosinophils in both the synthetic capacity and functional chemotaxis responses to lipid mediators.

Metabolism of prostacyclin. III. Urinary metabolite profile of 6-keto PGF1α in rat

The in vivo metabolism of 6-keto PGF1 alpha was investigated in rats. Following continuous intravenous infusion for 14 days the urinary metabolites were isolated and identified. A substantial amount of unchanged 6-keto PGF1 alpha was recovered in the urine. The metabolic pattern very closely resembles that of PGI2 in rats. Metabolites were found which represented 15-dehydrogenation, beta-oxidation, omega and omega-1-hydroxylation and oxidation. Previous work showed that 6-keto PGF1 alpha is very poorly oxidized by 15-PGDH. We administered 15-[H3]-PGI2 and 15-[H3]-6-keto PGF1 alpha to rats and measured urinary tritiated water as an index for in vivo 15-PGDH activity. The results showed that PGI2 and 6-keto PGF1 alpha were both oxidized to the 15-keto product, although the rate of oxidation of PGI2 was greater than that of 6-keto PGF1 alpha. We concluded that the administered PGI2 was oxidized by 15-PGDH before hydrolysis to 6-keto PGF1 alpha. A portion of the dose is probably hydrolzyed before 15-dehydrogenation.

Metabolism of prostacyclin in Cynomolgus monkey.

The metabolism of prostacyclin (PGI2) in vivo was investigated in Cynomolgus monkey. Following intravenous infusion of 11-[3H]-PGI2 for three days, pooled urine was extracted with Amberlite XAD-2, then chromatographed and purified by Sephadex LH-20, and reverse phase column chromatography. Radioactive fractions were converted to appropriate derivatives for identification by gas chromatography mass spectrometry. Twelve metabolites were characterized, the major of which was 6-keto-PGF1 alpha, accounting for 13% of the urinary radioactivity. The metabolic pathways are similar to those observed earlier in the rat. The excretion of substantial amounts of unchanged 6-keto-PGF1 alpha indicated that the monkey was not able to metabolize PGI2 as avidly as the rat.

Effects of the lipid peroxidation inhibitor tirilazed mesylate (U-74006F) on gerbil brain eicosanoid levels following ischemia and reperfusion

The present study measured the production of eicosanoids in the gerbil brain during early reperfusion after either a 3-h unilateral carotid occlusion (UCO, model of focal ischemia) or a 10-min bilateral carotid occlusion (BCO, model of global ischemia). Arachidonic acid (AA) metabolites were examined to determine if pretreatment with the 21-aminosteroid lipid peroxidation inhibitor U-74006F (tirilazad mesylate) could influence postreperfusion synthesis of brain eicosanoids. In the 3-h UCO focal ischemia model, there was an early (5-min) postreperfusion elevation in brain levels of PGF2 alpha, TXB2 and LTC4 (P < 0.05 vs. sham for all three eicosanoids). LTB4 also rose but not significantly. On the other hand, PGE2 and 6-keto-PGF1 alpha tended to decrease during ischemia and at 5-min postreperfusion (P < 0.05 vs. sham for PGE2). Pretreatment with known neuroprotective doses of U-74006F in this model (10 mg/kg i.p. 10 min before and again immediately upon reperfusion) did not affect the increase in PGF2 alpha or TXB2 but significantly blunted the elevations in LTC4 and LTB4. The postreperfusion decrease in PGE2 was also attenuated. In the 10-min BCO global ischemia model, there was also an increase in each of the measured eicosanoids, except LTB4, at 5 min after reperfusion. Pretreatment with U-74006F (10 mg/kg i.p. 10 min before ischemia) selectively decreased the rise in LTC4 but did not significantly affect the other eicosanoids. In contrast, the antioxidant actually caused a significant enhancement of the postreperfusion increase in PGE2 vs. vehicle-treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Preparation of two dinor-PGI2 metabolites from 6-keto-PGF1α by mycobacterium rhodochrous

Abstract The transformation of 6-keto-PGF1α to two prostacyclin metabolites, 2,3-dinor-6-keto-PGF1α (I) and 2,3-dinor-6,15-diketo-13,14-dihydro-PGF1α (II) by Mycobacterium rhodochrous UC-6176 is described. The finding that the bacterium oxidized 6-keto-PGF1α to the 6,15-diketo metabolite II shows that it contains 15-hydroxy prostaglandin dehydrogenase and Δ13 reductase enzyme systems.

Arachidonic acid metabolism in guinea pig eosinophils: synthesis of thromboxane B2 and leukotriene B4 in response to soluble or particulate activators.

The arachidonic acid metabolism of guinea pig eosinophils isolated from either peritoneal cavity or bronchoalveolar lavages was studied by reverse‐phase high‐performance liquid chromatography. The purified eosinophils (95‐100%) from either source released thromboxane B2 (TxB2), luekotriene B4 (LTB4) and 5‐hydroxy eicosatetraenoic acid (5‐HETE) following calcium lonophore A23187 stimulation. Quantification by radioimmunoassay indicated that maximal mediator output from the stimulated peritoneal cells was reached at 3 min after stimulation. The increase in production of TxB2 and LTB4 was correlated to increasing calcium ionophore A23187 concentration up to 1.0 μg/ml. In addition to calcium ionophore, the guinea pig peritoneal cells were also activated by f‐met‐leu‐phe, phorbol 12‐myristate, 13‐acetate (PMA), and to lesser extent platelet‐activating factor (PAF) to produce TxB2. LTB4 synthesis was stimulated by calcium ionophore, by f‐met‐leu‐phe, as well as by unopsonized glucan, a particulate phagocytotic stimulus. The guinea pig eosinophils do not synthesize sulfidopeptide leukotrienes because of the absence of the specific LTA4 glutathione S‐transferase. These results suggest that the guinea pig eosinophils differ from the human circulating eosinophils in the synthetic capacity of lipid mediators derived from arachidonic acid metabolism. This difference may be important in the understanding of the role of the eosinophils in inflammatory reactions such as that which occurs in the bronchial tissues of asthmatics.

Disappearance and metabolism of leukotriene B4 during carrageenan-induced pleurisy

Leukotriene B4 (LTB4) has been implicated as a mediator in the inflammatory process by virtue of its potent chemotactic activity. At present, very little is known of the stability of this compound in vivo; therefore, the present study was designed to determine the half-life and metabolic fate of radiolabeled LTB4 during a 2-hr intrapleural incubation in rats with acute carrageenan pleurisy. After injection of 0.2 ml of 1% sodium carrageenan (Viscarin), inflammation was allowed to develop for 4 hr. A small polyethylene cannula was then inserted into the chest, and 0.1 microCi of [14C]LTB4 was injected into the chest. Samples for radioactivity determination were taken at 0, 1, 2, 3, 4, 5, 7, 10, 15, 20, 30, 45, 60, 90 and 120 min via the cannula, and at 120 min the entire content of the chest was collected. The half-life for the disappearance of radioactivity from the chest was 45.8 +/- 3.5 min. The 120-min samples were treated with acetone to precipitate protein and extracted with Sep-Paks. The extracts were analyzed by reversed phase high performance liquid chromatography using an ultraviolet detector set at 269 nm and a radioactivity detector. An additional experiment was run using multi-[3H]LTB4, and the only major metabolites detected were omega-hydroxylated compounds. It can be concluded from these results that LTB4 is relatively stable in vivo and could be present for long enough at the inflammatory site to have an influence upon inflammatory cell migration.

Metabolism of leukotriene B4 by guinea pig eosinophils

The metabolism of leukotriene B4 (5(S),12(R)-dihydroxy-6-cis-8,10-trans-14-cis-eicosatetraenoic acid) by isolated guinea pig eosinophils was investigated. Incubation of guinea pig eosinophils with [3H]-leukotriene B4 resulted in the rapid conversion of leukotriene B4 to several more polar metabolites. Two of these metabolites were identified by ultraviolet spectroscopy and gas chromatography-mass spectrometry as the omega oxidation products 5(S),12(R),20-trihydroxy-6,8,10,14-eicosatetraenoic acid (20-hydroxy-leukotriene B4) and 5(S),12(R),19-trihydroxy-6,8,10,14- eicosatetraenoic acid (19-hydroxy-leukotriene B4). Two novel metabolites, 5(S),12(R),18,19-tetrahydroxy-6,8,10,14 eicosatetraenoic acid (18,19-dihydroxy-leukotriene B4) and 5(S),12(R)-dihydroxy-1,18-dicarboxylic-6,8,10,14,16-octadecapentaenoi c acid (delta 16,17-18-carboxy-19,20-dinor-leukotriene B4) were tentatively identified. The identification of these compounds indicates that guinea pig eosinophils are capable of metabolizing leukotriene B4 by both omega and beta oxidation. This catabolic activity may play a role in modulating inflammatory reactions by removing the chemoattractant leukotriene B4 from inflammatory sites.