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P Demin

Publications and source records attributed to P Demin.

16 recordsLinked to original sources
PubMed2006

Hepoxilin analogs, potential new therapeutics in disease.

We have chemically synthesized several stable analogs of the naturally occurring hepoxilins, 12-LO products derived from arachidonic acid, which we found to have promising actions in a variety of test systems of disease. The analogs, PBTs, afford chemical and biological stability to the hepoxilin molecule. This article reviews some of our latest observations with the PBTs in the areas of inflammation (inhibition of the bleomycin-evoked lung fibrosis in mice in vivo), platelet aggregation (antagonism of the thromboxane receptor in human platelets in vitro) and thrombosis (inhibitors in vivo), and cancer (apoptosis of the human leukemia cell line, K562 in vitro and in vivo). The demonstration that the PBTs are active in vivo suggests that they can serve as a platform for their further development as novel therapeutics in disease.

8,11,14-Eicosatrienoic Acid↗
PubMed2001

Improved high-performance liquid chromatographic method for the combined analysis of phospholipase, lipoxygenase and cyclooxygenase activities.

We report herein an improved method for the high-performance liquid chromatographic separation and analysis of eicosanoids formed during the stimulation of human platelets in vitro with collagen. Since the products of interest, excepting arachidonic acid, contain hydroxyl groups (one to several), our method involves the conversion of the hydroxyl groups into acetates (pyridine/acetic anhydride) after derivatization with anthryl diazomethane (ADAM) rendering the compounds with much decreased polarity for separation on a reversed-phase column. This procedure is superior to that involving ADAM esters only, i.e. with free hydroxyl groups, as it leads to the excellent separation of the desired compounds from each other and from extraneous peaks observed due to the ADAM reagent and sharpens the peak of thromboxane. We have successfully applied the method to investigate the formation of thromboxane B2 and 12-hydroxyheptadecatrienoic acid (HHT) (products of cyclooxygenase and thromboxane A2 synthase), 12-hydroxyeicosatetraenoic acid (12-HETE, a 12-lipoxygenase product) and arachidonic acid (AA, product of phospholipase A2) formed during the in vitro aggregation of human platelets induced by collagen. A correlation between the inhibition of aggregation by aspirin and thromboxane/HHT formation was observed. All four compounds can be chromatographed in a single run. We employed prostaglandin B1 (PGB1) as internal reference standard to quantify the products. The method is useful to investigate selectivity of drugs which may affect either or all of these enzyme pathways at the same time.

Blood Platelets↗
PubMed2001

Novel platelet antiaggregating substances.

We describe herein a novel action of four stable analogs of the hepoxilins. These analogs inhibit to different degrees, the aggregation of washed human platelets evoked by collagen. One of the analogs, PBT-3, is particularly effective with an IC(50) = 8 x 10(-7) M. The other analogs are about 5-fold less active, but all analogs are about 500-fold more active than the native hepoxilins. The PBT analogs inhibit the collagen-enhanced formation of thromboxane A(2) and HHT but do not affect the formation of 12-HETE or the release of arachidonic acid except at doses higher than those needed to block platelet aggregation. These results demonstrate that these novel compounds may have potential for development into drugs in the treatment of thromboxane-mediated cardiovascular disease.

Arachidonic Acids↗
PubMed1999

Formation of 14,15-hepoxilins of the A(3) and B(3) series through a 15-lipoxygenase and hydroperoxide isomerase present in garlic roots.

We report herein for the first time the formation by freshly grown garlic roots and the structural characterization of 14,15-epoxide positional analogs of the hepoxilins formed via the 15-lipoxygenase-induced oxygenation of arachidonic acid. These compounds are formed through the combined actions of a 15(S)-lipoxygenase and a hydroperoxyeicosatetraenoic acid (HPETE) isomerase. The compounds were formed when either arachidonic acid or 15-HPETE were used as substrates. Both the "A"-type and the "B"-type products are formed although the B-type compounds are formed in greater relative quantities. Chiral phase high performance liquid chromatography analysis confirmed the formation of hepoxilins from 15(S)- but not 15(R)-HPETE, indicating high stereoselectivity of the isomerase. Additionally, the lipoxygenase was of the 15(S)-type as only 15(S)-hydroxyeicosatetraenoic acid was formed when arachidonic acid was used as substrate. The structures of the products were confirmed by gas chromatography-mass spectrometry of the methyl ester trimethylsilyl ether derivatives as well as after characteristic epoxide ring opening catalytically with hydrogen leading to dihydroxy products. That 15(S)-lipoxygenase activity is of functional importance in garlic was shown by the inhibition of root growth by BW 755C, a dual cyclooxygenase/lipoxygenase inhibitor and nordihydroguaiaretic acid, a lipoxygenase inhibitor. Additional biological studies were carried out with the purified intact 14(S), 15(S)-hepoxilins, which were investigated for hepoxilin-like actions in causing the release of intracellular calcium in human neutrophils. The 14,15-hepoxilins dose-dependently caused a rise in cytosolic calcium, but their actions were 5-10-fold less active than 11(S), 12(S)-hepoxilins derived from 12(S)-HPETE. These studies provide evidence that 15(S)-lipoxygenase is functionally important to normal root growth and that HPETE isomerization into the hepoxilin-like structure may be ubiquitous; the hepoxilin-evoked release of calcium in human neutrophils, which is receptor-mediated, is sensitive to the location within the molecule of the hydroxyepoxide functionality.

8,11,14-Eicosatrienoic Acid↗
PubMed1999

The hepoxilins. A review.

This manuscript reviews the literature on hepoxilins to date. It affords a review of the structures, nomenclature, biosynthesis, catabolism and biological actions of the hepoxilins and some of their chemical analogs. Some unpublished data are also presented. The primary biological action of the hepoxilins appears to relate to their ability to release calcium from intracellular stores through a receptor-mediated action. The receptor is intracellular, and appears to be G-protein coupled. The conversion of hepoxilin into its omega-hydroxy catabolite has recently been demonstrated through the action of an omega-hydroxylase. This enzyme is different from that which oxidizes leukotriene B4, as the former activity is lost when the cell is disrupted, while leukotriene B4-catabolic activity is recovered in both the intact and disrupted cell. Additionally, hepoxilin catabolism is inhibited by CCCP, a mitochondrial uncoupler, while leukotriene catabolism is unaffected. As hepoxilins cause the translocation of calcium from intracellular stores in the endoplasmic reticulum to the mitochondria, it is speculated that hepoxilin omega-oxidation takes place in the mitochondria, and the omega-oxidation product facilitates accumulation of the elevated cytosolic calcium by the mitochondria. The biological activity of stable analogs of the hepoxilins is also described which inhibit the calcium-releasing actions of neutrophil inflammatory mediators.

8,11,14-Eicosatrienoic Acid↗
PubMed1996

Hepoxilin A3-specific binding in human neutrophils.

Hepoxilins have been shown to release calcium from intracellular stores in human neutrophils [Dho, Grinstein, Corey, Su and Pace-Asciak (1990) Biochem. J. 266, 63-68; Laneuville, Reynaud, Grinstein, Nigam and Pace-Asciak (1993) Biochem. J. 295, 393-397]. In this paper we report that tritium-labelled hepoxilin A3 (8S) binds to broken neutrophil membranes in a time-, substrate- and temperature-dependent fashion. Specific binding was displaced with unlabelled hepoxilin A3. Specific binding was greatest at 37 degrees C. Competitive binding was best observed with unlabelled hepoxilin A3 (8S); the glutathione conjugate, HxA3-C (8S or 8R), or 12(S)-hydroxyeicosatetraenoic acid was less active. Similarly inactive in displacing the bound radiolabelled hepoxilin A3 was leukotriene B4 as well as a variety of prostaglandins and thromboxane B2. Formylmethionyl-leucylphenylalanine was similarly inactive in competing for the hepoxilin binding sites. Specific binding was inhibited by pretreatment of the broken membranes during 30 min at 37 degrees C with proteinase K, while specific binding of the intact cells was unaffected. Scatchard analysis of binding data revealed a single population of binding sites with apparent KD and Bmax. of 79.3 +/- 9.1 nM and 8.86 +/- 1.4 pmol/ml per 2 x 10(6) cells (+/- S.E.M.) respectively reflecting approx. 2.67 x 10(6) sites/cell. These results demonstrate for the first time that neutrophils contain specific binding sites to hepoxilin A3.

8,11,14-Eicosatrienoic Acid↗
PubMed1995

High-performance liquid chromatographic separation of fluorescent esters of hepoxilin enantiomers on a chiral stationary phase.

Fluorescent anthryl (ADAM) derivatives of hepoxilins have been shown to possess good chromatographic properties affording good sensitivity for the high-performance liquid chromatographic analysis and detection of these compounds and related eicosanoids (12-hydroxyeicosatetraenoic acid) in biological samples. We report herein the separation of all possible stereoisomers of hepoxilins A3 and B3 as their methyl esters as well as their ADAM ester and acetate derivatives on a cellulose trisdimethyphenylcarbamate chiral stationary phase (Chiracel OD) in the normal-phase mode. This methodology is important to address the mechanistic route of biosynthesis of these products.

8,11,14-Eicosatrienoic Acid↗
PubMed1995

Extractive derivatization of the 12-lipoxygenase products, hepoxilins, and related compounds into fluorescent anthryl esters for their complete high-performance liquid chromatography profiling in biological systems.

Facile methods for the detection of intact hepoxilins, monohydroxy-epoxide derivatives of arachidonic acid formed through the 12-lipoxygenase pathway, are unavailable because (i) an absence in these compounds of an appropriate chromophore for sensitive detection by uv exists, (ii) these compounds are sensitive to the acidic workup leading to varying degrees of decomposition, and (iii) they decompose to the derivatization procedures required for their analysis by gas chromatography mass spectrometry. Herein we apply a method which introduces a fluorescent ester chromophore to the carboxylic group of the hepoxilins under conditions which do not require acidification leading to stabilization of the derivative which is extracted into an organic solvent in situ. This procedure quantitatively derivatizes hepoxilins in a biological sample, permitting the detection of hepoxilins after a TLC purification with a limit of 50 pg/sample. This method permits the profiling of 12-HETE, hepoxilins A3 and B3, as well as the corresponding epoxide hydrolase products, trioxilins A3 and B3, in a biological sample by reverse-phase HPLC with fluorescent detection. We also report on the fluorescent and mass spectral properties of these derivatives using a liquid chromatography mass spectrometry LCMS interface with thermospray ionization.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1995

Hepoxilin binding in human neutrophils.

Hepoxilins have previously been shown to release intracellular calcium in human neutrophils. We show herein that tritium-labeled hepoxilin A3 of high specific radioactivity binds to human neutrophils, and this binding is reversed by the addition of unlabeled compound, demonstrating that specific binding for these compounds exists in these cells. Specific binding of both the methyl ester derivative as well as the free acid form of the hepoxilin takes place in broken membrane fragments. In contrast only the methyl ester derivative binds specifically to the intact cells. We also show that intact neutrophils form hepoxilin A3 when incubated in the presence of the hepoxilin precursor, 12(S)-HPETE. These data demonstrate that hepoxilin synthesis can occur in the neutrophil and that hepoxilin binding sites, which appear to be located intracellularly, exist in these cells.

8,11,14-Eicosatrienoic Acid↗
PubMed1994

Hepoxilin A3 formation in the rat pineal gland selectively utilizes (12S)-hydroperoxyeicosatetraenoic acid (HPETE), but not (12R)-HPETE.

Hepoxilins A3 and B3 have previously been shown to be formed from 12-hydroperoxyeicosatetraenoic acid (12-HPETE) through a heat-insensitive ferriheme catalysis as indicated by experiments with hemin or hemoglobin, typical of nonenzymatic rearrangement (Pace-Asciak, C. R. (1984) Biochim. Biophys. Acta 793, 485-488; Pace-Asciak, C. R. (1984) J. Biol. Chem. 259, 8332-8337). In this paper, we demonstrate through use of a mixture of (12S)- and (12R)-HPETE that an enzyme system exists in the rat pineal gland that selectively utilizes (12S)-HPETE for the transformation into hepoxilin A3, while the hemin-catalyzed transformation is not selective, with both (12S)- and (12R)-HPETE being utilized. A new procedure was established to rapidly extract (in the absence of acid) and directly derivatize from the incubation mixture the products of incubation using 9-anthryldiazomethane reagent to form the 9-anthryldiazomethane derivatives of the hydroxyeicosatetraenoic acids (HETEs), hepoxilins and trioxilins, which could be detected by high performance liquid chromatography using a flow-through fluorescence detector. The following observations were made: 1) the pineal experiments showed a high preference for the formation of hepoxilin A3 with minor amounts of hepoxilin B3, while experiments with heme catalysis showed formation of both hepoxilins B3 and A3; 2) chiral phase analysis of the HETEs recovered from the incubation mixture showed the predominance of (12R)-HETE from the pineal experiments, indicating that (12S)-HPETE was selectively used up, whereas both (12S)- and (12R)-HPETE were utilized in the hemin experiments; 3) chiral phase analysis of hepoxilin A3 indicated the presence in the pineal experiments of only hepoxilin with the 11S,12S-configuration formed from (12S)-HPETE, while the hemin experiments contained both the native 11S,12S-configuration as well as the unnative or "bis-epi"-11R,12R-configuration in the epoxide group of hepoxilin A3; 4) reverse phase analysis of the epoxide hydrolase product of hepoxilin A3, i.e. trioxilin A3, showed that the pineal experiments contained only the products of enzymatic hydrolysis derived from the native hepoxilin A3, whose formation was inhibited by the epoxide hydrolase inhibitor, trichloropropene oxide. The pineal system is heat-sensitive, with formation of hepoxilins being abolished by tissue boiling, while the hemin system is insensitive to boiling. These experiments demonstrate that hepoxilin A3 formation in the pineal gland utilizes (12S)-HPETE exclusively, being transformed by a "hepoxilin synthase" primarily into hepoxilin A3.

8,11,14-Eicosatrienoic Acid↗
PubMed1993

Enzymatic formation of hepoxilins A3 and B3.

Available data obtained so far has indicated that hepoxilin formation from 12-HPETE is catalyzed by hemin and hemoglobin and is not affected through heating of these ferri-heme compounds suggestive of nonenzymatic processes. The present paper demonstrates for the first time that 12-HPETE is transformed into the hepoxilins A3 and B3 by intact cells (skin subcutis layer) and slices of several tissues (brain hippocampus and pineal gland) and that this transformation is inhibited by tissue boiling, indicating an enzymatic catalysis. The tissues employed are pharmacologically responsive to hepoxilins and hence the present data offer biochemical support of a potential biological role for the hepoxilins in these tissues.

8,11,14-Eicosatrienoic Acid↗