Frank F. Sun

The chemical structure of prostaglandin X (prostacyclin)

The chemical structure of prostaglandin X, the anti-aggregatory substance derived from prostaglandin endoperoxides, is 9-deoxy-6, 6alpha-epoxy-delta5-PGF1alpha. The stable compound formed when prostaglandin X undergoes a chemical transformation in biological systems in 6-keto-PGF1alpha. Prostaglandin X is stabilized in aqueous preparations by raising the pH to 8.5 or higher. The trivial name prostacyclin is proposed for 9-deoxy-6, 9alpha-epoxy-delta5-PGF1alpha.

Calcium stimulation of a novel lipoxygenase.

Abstract Homogenates of rat basophilic leukemia (RBL-1) cells have a novel lipoxygenase which was stimulated by calcium in a concentration dependent fashion and inhibited by epinephrine. The major compounds formed from [14C]-arachidonic acid were identified by gas chromatography — mass spectrometry to be 5-hydroxyeicosatetraenoic acid and 5,12-dihydroxyeicosatetraenoic acid. Other compounds present in small amounts were 12-hydroxy- and 5,6-dihydroxyeicosatetraenoic acid. The stimulation by calcium of this pathway in basophils links it closely to the release reaction which is calcium dependent.

Hepatic metabolism of prostacyclin (PGI2) in the rabbit: formation of a potent novel inhibitor of platelet aggregation.

Abstract Metabolism of [9-3H]-PGI2 was studied in the isolated Tyrodes perfused rabbit liver. Five products, four radioactive and one non-radioactive, were identified in the perfusate: 19-hydroxy-6-keto-PGF1α, 6-keto-PGF1α, dinor-6-keto-PGF1α, pentanor PGF1α and a 6-keto-PGE1-like substance. The first two, 19-hydroxy-6-keto-PGF1α and 6-keto-PGF1α, represented 5% and 45% respectively, of the total radioactivity; the last two accounted for 39%. The presence of dinor and pentanor derivatives of 6-keto-PGF1α indicated that β -oxidation and oxidative-decarboxylation occurs in the liver as the major metabolic pathway of PGI2. One non-radioactive metabolite which co-migrated with authentic 6-keto-PGE1 was found to inhibit platelet aggregation, having a potency similar to authentic 6-keto-PGE1, and its effect can be eliminated by boiling and by alkali treatment. This metabolite, having similar Rf value on TLC and biological behavior as 6-keto-PGE1, may arise from oxidation of 6-keto-PGF1α via the 9-hydroxyprostaglandin dehydrogenase pathway, as suggested by recovery of tritiated water in the aqueous phase of the perfusate. This material, a potent inhibitor of platelet aggregation, may arise from PGI2 or its hydrolysis product, 6-keto-PGF1α.

Biosynthesis of thromboxanes in human platelets. I. Characterization and assay of thromboxane systhetase

Abstract An enzyme system which catalyzes the rapid conversion of prostaglandin endoperoxide to thromboxane B2 was found in the microsomal fraction of human platelet homogenate. The products of the reaction were identified by gas chromatography-mass spectrometry as thromboxane B2 and the C-17 hydroxy fatty acid HHT. A simple radiometric TLC method was developed for the determination of the enzyme activity. Various parameters affecting the enzyme activity have been defined. Thromboxane synthetase was strongly inhibited by its substrate analogs. The activity was completely abolished when low amounts (5 × 10−5 M ) of the 9,11 (epoxymethano) prostanoic acid was included in the assay mixture. The enzyme reaction was not affected by nonsteroidal antiinflammatory agents.

Prostaglandin 15-hydroxy dehydrogenase and Δ13 reductase levels in the lungs of maternal, fetal and neonatal rabbits

Abstract The levels of prostaglandin 15-hydroxy dehydrogenase and reductase have been studied in the lungs of maternal, fetal and neonatal rabbits. Fetal lungs obtained at gestational age of 28–30 days (full term 31 days) had the same levels of prostaglandin dehydrogenase as the adults, while the reductase levels in the fetal lungs were only one fourth that in the adults. The lungs of maternal rabbits at near term possessed very high levels of prostaglandin dehydrogenase — approximately twenty-fold higher than in the adult non-pregnant female controls. The Δ13 reductase appeared slightly elevated during pregnancy. Neonatal animals at different ages showed the same levels of both enzymes as the near term fetus and/or the non-pregnant adults, which suggests that the development of the ability for prostaglandin metabolism is completed at least several days before birth. The high dehydrogenase levels in the near term maternal lungs indicated the requirement for extra protection against prostaglandin release during late pregnancy.

Metabolism of arachidonic acid by human neutrophils: Characterization of the enzymatic reactions that lead to the synthesis of leukotriene B4

Human neutrophils stimulated with calcium ionophore A23187 synthesized 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B4. Time-course studies showed that the concentrations of both products reached a maximum after 2 min after which the products were rapidly removed. In longer incubations, 5-HETE was esterified into membrane lipids, and leukotriene B4 was converted to 20- hydroxyleukotriene B4 and/or 20- carboxyleukotriene B4. The reaction is apparently self-limiting. After the maximum was reached, addition of fresh ionophore, Ca2+ or oxygen had little effect. Fresh arachidonic acid increases the yields of 5-HETE and delta 6-trans-leukotriene B4 but not additional leukotriene B4. Only the addition of fresh neutrophils gave additional leukotriene B4. This finding suggests that leukotriene B4 synthesis is limited by both substrate availability and enzyme inhibition by hydroperoxide intermediate. Exogenous arachidonic acid added with ionophore had different effects on the syntheses of leukotriene B4, delta 6-trans-leukotriene B4, and 5-HETE. As the arachidonic acid concentration increases, product formation increases in the following order: 5-HETE greater than delta 6-trans-leukotriene B4 greater than leukotriene B4. At a high concentration (more than 10 microM) of arachidonic acid, the synthesis of delta 6-trans-leukotriene B4 was greater than leukotriene B4 itself. Since delta 6-trans-leukotriene B4 represents the nonenzymatic decomposition of leukotriene A4, we suggest that one of the rate-limiting steps in the synthesis of leukotriene B4 is the leukotriene A4 hydrolase. Our data suggest the synthesis of leukotriene B4 is under the control of three factors: (1) substrate availability; (2) limited capacity of the leukotriene A4 hydrolase, and (3) enzyme inactivators generated during the reaction, such as hydroperoxide intermediate. The tightly controlled system assures only a finite amount of this powerful bioactive substance will be produced under most conditions.

Prostaglandins H1 and H2. Convenient biochemical synthesis and isolation. Further biological and spectroscopic characterization

An easy biochemical preparation of the prostaglandin endoperoxides, PGH1 and PGH2, is described. Both of the endoperoxides are potent contractors of isolated gerbil colon smooth muscle. Contracture with PGH2 is about equal to that caused by the standard, PGE1, while contracture with PGH1 is about half of that caused by PGE1. PGH1 was found to inhibit platelet aggregation induced by PGH2 and is about 1/10 as potent a stimulator of cAMP accumulation as is PGE1. The mass spectra of the methyl esters of both PGH1 and PGH2 were obtained, as were the infrared spectra of the two compounds. The nuclear magnetic resonance spectrum of PGH2 is characterized by signals at 4.58 delta and 4.47 delta for the C-9 and C-11 protons, respectively.

6-keto-prostaglandin E1 inhibits the aggregation of human platelets

6-Keto-prostaglandin E1 (6-keto-PGE1) was found to have a similar potency to prostacyclin (PGI2) as an inhibitor of platelet aggregation. It caused a time and concentration dependent inhibition of ADP, collagen and epinephrine induced platelet aggregation, the dose ratios for 70% inhibition by 6-keto-PGE1, PGI2 and PGE1 being approximately 1 : 1 : 13. In doses similar to those of PGI2, 6-keto-PGE1 partially inhibited the release of [3H]-serotonin from platelet-rich plasma induced by collagen.

Generation and Detection of Hydroxyl Radical Following Experimental Head Injury

Oxygen-free radicals have been postulated to play a role in the acute pathophysiology of blunt head injury.14. Much of this work points to the cerebral microvasculature as a major target of oxygen radical-mediated damage. Moreover, free-radical scavengers such as superoxide dismutase and lipid antioxidants, including methylprednisolone,h U-72099E, U-74006F,X and U-78517F: which block free radical damage to membrane polyunsaturated fatty acids (i.e., lipid peroxidation), have been reported to attenuate posttraumatic pathophysiology and/or to promote survival and recovery in experimental head injury. However, a firm association of oxygen radicals and lipid peroxidation with pathophysiological events has been hindered by the lack of analytical methodology that will directly measure cerebral tissue levels of specific radicals or lipid hydroperoxides. In recent studies in both the mouse concussive and the rat controlled cortical impact head injury models, we have directly measured brain hydroxyl radical (.OH) levels via the salicylate trapping method in which the production of 2,3and/or 2,5-dihydroxybenzoic acid (DHBA) in brain, 15 min after salicylate administration was used as an index of .Ou formation. In the mouse model, we have also examined the brain levels of enzymatically derived eicosanoids (e.g., PGFZa, PGE2, TXB2, 6-keto PGFI,, LTC.,). Additionally, in the rat model, we have employed the highpressure liquid chromatographic-chemiluminescence (HPLC-CL) method to measure the lipid peroxidation product, phosphatidylcholine hydroperoxide (PCOOH), and its time course in relation to that of .OH generation. Finally, to provide a pathophysiological correlation, we have further compared the time course of bloodbrain barrier (BBB) disruption in both models as an index of microvascular damage by oxygen radical-induced lipid peroxidation. These studies have shown a clear association between posttraumatic -OH generation, membrane lipid peroxidation, and subsequent increases in cerebral microvascular permeability.

Metabolism of prostaglandin F2α in the rat

Abstract Tritium-labeled prostaglandin F 2α (Tris salt) was given intravenously to female rats at a dose level of 1.2 mg/kg body wt. Sequential urine and feces specimens were collected and analyzed for radioactivity. It was found that average recovery of total radioactivity was 92.7% with 65.1% in the urine and 15.3% in the feces. The amount of tritiated water produced metabolically was estimated to be 12.32% of the dose. The metabolic transformation of tritium-labeled prostaglandin F 2α was investigated in rats following chronic intravenous infusion. The following six urinary metabolites were isolated and identified: 5,7-dihydroxy-II-ketotetranorprostanoic acid; 5,7,15-trihydroxy-II-ketotetranorprostanoic acid; 5,7,16-trihydroxy-II-ketotetranorprostanoic acid; 5,7-dihydroxy-II-ketotetranorprosta-1,16-dioic acid; 5,7-dihydroxy-II-ketotetranor-(ω-dinor)-prosta-1,14-dioic acid and 5,7,11-trihydroxy-tetranorprosta-1,16-dioic acid. The six metabolites were also present as the corresponding δ-lactones. The major metabolite was 5,7-dihydroxy-11-keto-tetranorprosta-1, 16-dioic acid which accounted for slightly less than half of the radioactivity extracted from the urine.