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L J Roberts

Publications and source records attributed to L J Roberts.

At least 145 records · Page 8Linked to original sources
PubMed1994

Effect of oxygen tension on the generation of F2-isoprostanes and malondialdehyde in peroxidizing rat liver microsomes.

Although numerous methods have been developed for the detection of lipid peroxidation, it is generally recognized that most of these lack specificity and/or sensitivity, particularly when applied to in vivo situations. We have reported recently that a series of prostaglandin F2-like compounds, termed F2-isoprostanes, are formed in vivo from the free radical catalyzed peroxidation of arachidonic acid and appear to be a useful marker of oxidant stress. Because of formation of other products of lipid peroxidation, such as alkanes and malondialdehyde (MDA), are affected by oxygen tension, which may influence their usefulness as markers of oxidant stress, we carried out a systematic study of the generation of F2-isoprostanes at various oxygen concentrations and compared these changes with the generation of MDA. The disappearance of the F2-isoprostane precursor, arachidonic acid, was used as a reference measure. Rat liver microsomes were peroxidized using an iron-ascorbate system. The incubations were carried out in sealed flasks at 37 degrees under N2 and various concentrations of O2 up to 100%. F2-isoprostanes were quantified by mass spectrometry and MDA by the thiobarbituric acid reaction. Microsomal fatty acids were measured by gas chromatography. Both MDA and F2-isoprostane formation increased in a time-dependent manner up to 15 min. Their formation correlated with a loss of polyunsaturated fatty acid and with an increase in O2 tension up to 21% O2. At oxygen tensions above 21%, MDA generation continued to increase, while F2-isoprostane generation and arachidonic acid loss did not. Levels of MDA and F2-isoprostanes increased a maximum of 65 and 9.4 times baseline values, respectively. These studies, therefore, define factors that influence the formation of F2-isoprostanes in an in vitro model of lipid peroxidation. Further, they demonstrate that higher O2 tensions do not block formation of F2-isoprostanes and validate their usefulness for assessing lipid peroxidation under high, as well as low, oxygen tension.

Animals↗
PubMed1994

Free radical-induced generation of isoprostanes in vivo. Evidence for the formation of D-ring and E-ring isoprostanes.

We recently reported the discovery that a series of novel prostaglandin (PG)F2-like compounds (F2-isoprostanes) are produced in vivo independent of the cyclooxygenase as products of free radical-catalyzed lipid peroxidation. F2-isoprostanes are initially formed in situ from arachidonic acid esterified to phospholipids and then released preformed. We have now investigated whether PGD2/E2-like isoprostanes are also produced by rearrangement of the PGG2-like intermediates involved in isoprostane formation. Using a variety of approaches utilizing mass spectrometry, compelling evidence was obtained for the presence of D2/E2-isoprostane-containing phosphospholipids in the liver (85 +/- 33 ng/g of liver) and free D2/E2-isoprostanes in the circulation (215 +/- 90 pg/ml) of rats treated with CCl4 to induce lipid peroxidation. In untreated rats, levels of D2/E2-isoprostanes esterified in liver phospholipids were much lower (0.90 +/- 0.10 ng/g), and free compounds could not be detected in the circulation (< 5 pg/ml). Interestingly, one of the E2-isoprostanes that would be expected to be formed in abundance, 8-epi-PGE2, was found to be a potent renal vasoconstrictor, and these effects could be abrogated by SQ29548, a thromboxane receptor antagonist. Further understanding of the biological consequences of the formation of these novel compounds and factors that influence their formation may provide valuable insights into the pathophysiology of oxidant injury.

Animals↗
PubMed1994

Evidence that the F2-isoprostane, 8-epi-prostaglandin F2 alpha, is formed in vivo.

F2-isoprostanes are prostaglandin (PG)F2-like compounds that are produced in vivo as non-enzymatic products of free radical catalyzed peroxidation of arachidonic acid. One F2-isoprostane whose formation should be favored is 8-epi-PGF2 alpha. 8-Epi-PGF2 alpha has been shown to exert potent bioactivity but proof that it is formed in vivo is lacking. Evidence is now presented suggesting that 8-epi-PGF2 alpha is, in fact, formed in vivo by demonstrating that an endogenous F2-isoprostane with a retention time on capillary GC identical with that of 8-epi-PGF2 alpha co-chromatographs through four high resolving HPLC purification procedures with authentic radiolabelled 8-epi-PGF2 alpha.

Animals↗
PubMed1994

Actions of the E2-isoprostane, 8-ISO-PGE2, on the platelet thromboxane/endoperoxide receptor in humans and rats: additional evidence for the existence of a unique isoprostane receptor.

D2/E2-isoprostanes, are a recently discovered series of novel prostaglandin-like compounds that are produced in vivo as products of free radical-catalyzed peroxidation of arachidonic acid independent of the cyclooxygenase enzyme. One of the E-ring compounds expected to be produced in abundance by this mechanism, 8-iso-prostaglandin E2 (8-iso-PGE2), is a potent renal vasoconstrictor in the rat, and this effect can be abrogated by the thromboxane/endoperoxide (TxA2/PGH2) receptor antagonist SQ29548, suggesting that 8-iso-PGE2 exerts these effects by interaction with this receptor in the vasculature. Nonetheless, it has recently been suggested that 8-iso-PGE2 induces vasoconstriction by interaction with a unique receptor similar to, but distinct from, the TxA2/PGH2 receptor. Because this issue has not been resolved, we carried out studies to further examine the interaction of this compound with the TxA2/PGH2 receptor on human and rat platelets. Only at concentrations of 10(-5) M or greater did 8-iso-PGE2 induce human platelet aggregation. The aggregation was unaffected by indomethacin but was inhibited by the TxA2/PGH2 receptor antagonist SQ29548. Conversely, 8-iso-PGE2 inhibited the thromboxane receptor agonists U46619 (10(-6) M) and IBOP (3.3 x 10(-7) M) with an IC50 of 5 x 10(-7) M and 5 x 10(-6) M, respectively. 8-iso-PGE2 also inhibited platelet aggregation induced by arachidonic acid but not by ADP. Similarly in rat platelets, 8-iso-PGE2 alone.

Animals↗
PubMed1994

Detection of the major urinary metabolite of prostaglandin D2 in the circulation: demonstration of elevated levels in patients with disorders of systemic mast cell activation.

The symptoms and hemodynamic alterations that accompany episodes of systemic mast cell activation have been largely attributed to excessive prostaglandin (PG)D2 release. Quantification of the major urinary metabolite of PGD2 has been invaluable in elucidating a role for PGD2 in these clinical entities and in the biochemical evaluation of systemic mastocytosis. With the use of a modified mass spectrometric assay for the major urinary metabolite of PGD2, this metabolite was detected in plasma from 10 normal volunteers (3.5 +/- 1.4 pg/ml). Ingestion of niacin, which induces endogenous release of PGD2, increased plasma levels of this metabolite 6.3 to 33 times above the upper limit of normal by 2 hours. Thereafter, levels declined gradually but remained elevated for up to 6 to 8 hours. In contrast, circulating levels of 9 alpha, 11 beta-PGF2, the initial metabolite of PGD2, peaked by 30 minutes and returned to baseline by 2 hours. The clinical utility of measuring the major urinary metabolite in the circulation was demonstrated by detection of markedly increased levels in plasma and serum from patients with systemic mastocytosis and a patient with a severe type I allergic reaction. Thus in the biochemical evaluation of episodes of systemic mast cell activation and endeavors to further elucidate the role of PGD2 in human disease, there are kinetic advantages of measuring the major urinary metabolite of PGD2 in the circulation. One particular advantage is the evaluation of clinical events, which only in retrospect are suspected to be associated with excessive release of PGD2, yet plasma or serum was obtained proximate to the event.

Anaphylaxis↗
PubMed1994

Detection and localization of lipid peroxidation in selenium- and vitamin E-deficient rats using F2-isoprostanes.

Data on the effect of vitamin E and selenium deficiency on lipid peroxidation in vivo have been limited. F2-isoprostanes are novel prostanoids that, free in plasma and esterified to phospholipids in tissues, are markers of lipid peroxidation in vivo. To address the importance of vitamin E and selenium in defense against lipid peroxidation in vivo, we determined F2-isoprostane concentrations in the plasma and organs of rats fed diets deficient in one or both nutrients. Weanling rats were fed a vitamin E- and selenium-deficient diet for 12 wk and then divided into four groups. One group continued to receive the doubly deficient diet, and the other three groups were fed the diet supplemented with vitamin E, selenium or both nutrients (control diet) for 4 wk. Plasma F2-isoprostanes in rats fed the doubly deficient diet were 5.2-fold higher than in animals changed to a control diet. In addition, there were significant differences in liver, lung, kidney, heart and skeletal muscle phospholipid-esterified F2-isoprostanes between these two groups. Lesser increases were noted in the group fed the vitamin E-deficient diet. Selenium deficiency alone was not associated with greater lipid peroxidation. Lipid peroxidation occurs in tissues of rats fed a vitamin E-deficient diet and is increased by concomitant selenium deficiency.

Animals↗
PubMed1994

Regulation of eicosanoid production and mitogenesis in rat intestinal epithelial cells by transforming growth factor-alpha and phorbol ester.

Growth factors and tumor promoters have been shown to play a role in intestinal epithelial growth regulation and transformation. In this study, transforming growth factor-alpha (TGF alpha) and the tumor promoter, tetradecanoyl phorbol acetate (TPA), are shown to stimulate the production of eicosanoids by rat intestinal epithelial (RIE-1) cells in culture. A 4.5-kb mRNA, which hybridizes to the mouse cyclooxygenase-2 cDNA probe, is elevated 18-fold within 30 min after TGF alpha or TPA treatment. Stimulation of RIE-1 cells with TGF alpha leads to the increase of a protein (M(r) approximately 69,000), which binds a monospecific antibody to the mouse cyclooxygenase-2 protein. Dexamethasone markedly inhibits the increase of the 4.5-kb mRNA. Pretreatment of TGF alpha or TPA-stimulated RIE-1 cells with dexamethasone or cyclooxygenase inhibitors prevents the increase in eicosanoid production by these cells. Treatment of quiescent RIE-1 cells with TGF alpha stimulates mitogenesis. This mitogenic activity is blocked by pretreating the cells with dexamethasone or cyclooxygenase inhibitors. A mitogen-inducible cyclooxygenase gene is thus shown to be regulated by TGF alpha and TPA in rat intestinal epithelial cells. We suggest that products of an intestinal growth factor-inducible cyclooxygenase may play a role in the regulation of mitogenesis.

Animals↗
PubMed1994

Formation of non-cyclooxygenase-derived prostanoids (F2-isoprostanes) in plasma and low density lipoprotein exposed to oxidative stress in vitro.

F2-isoprostanes are prostaglandin F2-like compounds that are known to be formed in vivo by free radical oxidation of arachidonyl-containing lipids, and their plasma levels have been suggested as indicators of in vivo oxidative stress. As oxidation of LDL, a likely causal factor in atherosclerosis, involves lipid peroxidation, we investigated whether F2-isoprostanes are formed in plasma and LDL exposed to oxidative stress, and how F2-isoprostane formation is related to endogenous antioxidant status. In plasma exposed to aqueous peroxyl radicals, lipid hydroperoxides and esterified F2-isoprostanes were formed simultaneously after endogenous ascorbate and ubiquinol-10 had been exhausted, despite the continued presence of urate, alpha-tocopherol, beta-carotene, and lycopene. In isolated LDL exposed to aqueous peroxyl radicals or Cu2+, consumption of endogenous ubiquinol-10 and alpha-tocopherol was followed by rapid formation and subsequent breakdown of lipid hydroperoxides and esterified F2-isoprostanes, and a continuous increase in LDL's electronegativity, indicative of atherogenic modification. In Cu(2+)-exposed LDL, the decrease in esterified F2-isoprostane levels was paralleled by the appearance of free F2-isoprostanes, suggesting that hydrolysis by an LDL-associated activity had occurred. Our data suggest that F2-isoprostanes are useful markers of LDL oxidation in vivo. As F2-isoprostanes are potent vasoconstrictors and can modulate platelet aggregation, their formation in LDL demonstrated here may also have important implications for the etiology of cardiovascular disease.

Amidines↗
PubMed1994

Effect of selenium deficiency and glutathione-modulating agents on diquat toxicity and lipid peroxidation in rats.

The dipyridyl herbicide diquat undergoes redox cycling in vivo resulting in superoxide generation. Diquat administration causes hepatic and renal toxicity in rodents. Selenium deficiency worsens this injury and lipid peroxidation is a prominent feature of the toxicity. However, there is limited data regarding the role of lipid peroxidation in diquat-induced toxicity in selenium-adequate animals. In addition, little is known about the effect of glutathione-modulating agents on diquat-induced toxicity and lipid peroxidation in vivo. F2-isoprostanes are novel prostanoids which, both free in plasma and esterified to phospholipids in tissues, are markers of lipid peroxidation in vivo. By using F2-isoprostane quantitation, we examined the effects of selenium deficiency and modulation of glutathione status with 1,3-bis (2-chloroethyl)-1-nitrosourea, phorone or buthionine sulfoximine on diquat-induced toxicity and lipid peroxidation. F2-isoprostanes increased 2- to 9-fold in plasma, liver, kidney and lung in selenium-adequate Fischer 344 rats with liver injury after receiving 100 mumol of diquat per kg. Selenium deficiency or modulation of glutathione status increased diquat toxicity. This was accompanied by 10- to 100-fold increases in plasma and kidney F2-isoprostane levels. Liver F2-isoprostanes were increased 2- to 5-fold. These studies suggest that glutathione, in addition to selenium, is an important defense against diquat-induced toxicity and lipid peroxidation.

Animals↗
PubMed1994

Release of markedly increased quantities of prostaglandin D2 from the skin in vivo in humans following the application of sorbic acid.

BACKGROUND AND DESIGN: Sorbic acid is a preservative that can induce cutaneous vasodilation characterized by erythema, urticaria, and stinging when applied topically to humans. Previous studies have suggested that prostaglandins may mediate the vasodilation, but the prostaglandin responsible has not been established. Recently, we have shown that cutaneous erythema similar to that associated with the application of sorbic acid is induced by topical administration of methylnicotinate and is mediated by the release of prostaglandin D2 (PGD2) from the skin. Therefore, we examined whether the cutaneous vasodilation induced by sorbic acid is also mediated by this prostaglandin in humans. RESULTS: Topical application of 1% sorbic acid to the forearms of four human volunteers resulted in 250- to 620-fold increases in levels of PGD2 and 15- to 58-fold increases in levels of the metabolite of PGD2, 9 alpha,11 beta-PGF2, in blood drawn from the antecubital vein draining the treated sites. There were no increases in the release of the other vasodilatory prostaglandins, PGE2 or prostacyclin (PGI2). The release of PGD2 in response to topically applied sorbic acid occurred in a dose-dependent manner and was not accompanied by a release of histamine, suggesting that the release of PGD2 was not from the mast cell. CONCLUSIONS: The cutaneous vasodilation that occurs following the administration of sorbic acid is primarily due to a release of PGD2 from a cellular source in the skin.

Administration, Topical↗
PubMed1993

Simplification of the mass spectrometric assay for the major urinary metabolite of prostaglandin D2.

The symptoms and hemodynamic alterations that accompany systemic mast cell activation have been attributed in large part to an excessive release of prostaglandin D2 (PGD2). Further, PGD2 has been implicated in the adverse effects of some pharmacologic agents (e.g. nicotinic acid). Quantitation of the major urinary metabolite of PGD2 has been invaluable in elucidating a role for PGD2 in these clinical entities and in the biochemical diagnosis of the disease systemic mastocytosis. However, the stable-isotope mass spectrometric assay originally developed for quantification of this metabolite has been too cumbersome for routine use. We now report improvements in the assay that greatly increase its utility by shortening sample processing and eliminating the need for purification using thin-layer chromatography. The precision and accuracy of the modified assay was evaluated and found to be comparable with the previously described assay. These modifications potentially allow wider use of the assay to explore the role of PGD2 in human disease and in the routine biochemical diagnosis of systemic mastocytosis and other disorders of mast cell activation.

Female↗
PubMed1993

Differentiation of monocytoid THP-1 cells with phorbol ester induces expression of prostaglandin endoperoxide synthase-1 (COX-1).

Prostaglandin endoperoxide synthase (PGH synthase) is responsible for converting arachidonic acid to PGH2, the common precursor of prostaglandins. It has been shown previously that phorbol ester-induced differentiation of the THP-1 human monocytic leukemia cell line is accompanied by induction of PGH synthase enzyme and enhanced capacity to produce prostaglandins. However, the identity of the isoform of PGH synthase that is induced under these conditions has not been established. Northern and Western analyses revealed a dramatic increase in levels of PGH synthase-1 mRNA and protein levels within 24 hr after treatment of THP-1 cells with phorbol ester. No significant increase in PGH synthase-2 mRNA or protein was observed. The increases in PGH synthase-1 were accompanied by enhanced capacity of the cells to produce PGE2. These findings indicate that expression of PGH synthase-1 is greatly enhanced in THP-1 cells by treatment with an agent that induces differentiation.

Cell Differentiation↗
PubMed1993

Identification of non-cyclooxygenase-derived prostanoid (F2-isoprostane) metabolites in human urine and plasma.

Free radicals are thought to play an important role in many types of tissue injury. Recently, we reported that a series of prostaglandin F2-like compounds (F2-isoprostanes) capable of exerting potent biological activity are produced in vivo by free radical-induced lipid peroxidation. Their formation is independent of the cyclooxygenase enzyme and has been shown to increase profoundly in animal models of free radical injury and lipid peroxidation. We now report the identification of F-ring isoprostane metabolites in human urine and plasma utilizing a gas chromatographic/mass spectrometric assay for the major urinary metabolite of prostaglandin D2 (9 alpha,11 beta-dihydroxy-15-oxo-2,3, 18,19-tetranorprost-5-ene-1,20-dioic acid). Evidence confirming these metabolites as tetranor, dicarboxylic acid compounds containing one double bond, cis-cyclopentane ring hydroxyls, and one keto group similar in structure to the major urinary metabolite of prostaglandin D2 was obtained by analysis of human urine by electron ionization mass spectrometry. Levels of these metabolites in normal human urine were determined and found to be unaffected by cyclooxygenase inhibitors. Evidence that these metabolites arise from F2-isoprostanes was obtained by demonstrating that (a) marked increases in plasma levels and urinary excretion of these metabolites, which were unaffected by coadministration of indomethacin, occurred in rats administered CCl4 to induce F2-isoprostane formation and (b) marked increases in levels of these metabolites in plasma and urine resulted from the intravenous infusion of F2-isoprostanes into a rat. Quantification of these isoprostane metabolites in urine and plasma may provide a reliable index of endogenous isoprostane production which could prove to be an important advance in our ability to assess oxidant stress in vivo in humans.

Animals↗
PubMed1993

Prostaglandin D2 and endothelin-1 induce the production of prostaglandin F2 alpha, 9 alpha, 11 beta-prostaglandin F2, prostaglandin E2, and thromboxane in capillary endothelium of human brain.

Endothelial cells derived from human brain capillaries (HBCEC) synthesize prostaglandin D2 (PGD2) which can be stimulated, among other prostanoids, by endothelin 1 (ET-1). Both the PGD2 induced by ET-1 and the exogenously added PGD2 to HBCEC are converted to 9 alpha, 11 beta-prostaglandin F2 (9 alpha, 11 beta-PGF2), a known potent vasoconstrictor. Exogenous PGD2 also dose-dependently enhanced the production of vasoconstrictive PGF2 alpha, thromboxane B2 (TXB2), and the vasodilatory PGE2 as well as cAMP by HBCEC. The PGD2-induced formation of PGF2 alpha, PGE2, and TXB2 was reduced by the cyclooxygenase inhibitors acetylsalicylic acid (ASA) or indomethacin (Indo), indicating for the first time that PGD2 may contribute to the formation of prostanoids in HBCEC. These results strongly suggest that PGD2 may play an important role in the regulation of cerebral capillary function under physiologic and pathologic conditions.

1-Methyl-3-isobutylxanthine↗