We recently reported the discovery of a series of bioactive prostaglandin F2-like compounds (F2-isoprostanes) that are produced in vivo by free radical-catalyzed peroxidation of arachidonic acid independent of the cyclooxygenase enzyme. Inasmuch as phospholipids readily undergo peroxidation, we examined the possibility that F2-isoprostanes may be formed in situ on phospholipids. Initial support for this hypothesis was obtained by the rmding that levels of free F2-isoprostanes measured after hydrolysis oflipids extracted from livers of rats treated with CCI4 to induce lipid peroxidation were more than 100-fold higher than levels in untreated animal. Further, increased levels of lipid-associated F2-isoprostanes in livers of CCI4-treated rats preceded the appearance of free compounds in the circulation, suggesting that the free compounds arose from hydrolysis of peroxidized lipids. This concept was supported by demonstrating that free F2-isoprostanes were released after incubation of lipid extracts with bee venom phospholipase A2 in vitro. When these lipid extracts were analyzed by HPLC, fractions that yielded large quantities of free F2-isoprostanes after hydrolysis eluted at a much more polar retention volume than nonoxidized phosphatidylcholine. Analysis of these polar lipids by fast atom bombardment mass spectrometry established that they were F2-isoprostanecontaining species of phosphatidylcholine. Thus, unlike cyclooxygenase-derived prostanoids, F2-isoprostanes are initially formed in situ on phospholipids, from which they are subsequently released preformed, presumably by phospholipases. Molecular modeling of FI-isoprostane-containing phospholipids reveals them to be remarkably distorted molecules. Thus, the formation of these phospholipid species in lipid bilayers may contribute in an important way to alterations in fluidity and integrity of cellular membranes, well-known sequelae of oxidant is ury.

These studies examine the in vivo formation of a unique series of PGF2-like compounds (F2-isoprostanes) derived from free radical-catalyzed nonenzymatic peroxidation of arachidonic acid. We have previously shown that levels of these compounds increase up to 50-fold in rats administered CC14. To understand further the formation of these compounds in vivo, we carried out a series of experiments assessing factors influencing their generation. After CC14 (2 ml/kg) was administered to rats, plasma F2-isoprostanes increased 55-fold by 4 h. Levels declined thereafter, but at 24 h, they were still elevated 21-fold, indicating continued lipid peroxidation. Pretreatment of rats with isonicotinic acid hydrazide and phenobarbital to induce cytochrome P450 enhanced the production of F2-isoprostanes after CC14 administration eightfold and fivefold, respectively, whereas inhibition of the cytochrome P-450 system with SKF-525A and 4-methylpyrazole decreased formation of F2-isoprostanes after CC14 by 55 and $2%, respectively. Further, the glutathione-depleting agents buthionine sulfoximine and phorone augmented the F2-isoprostane response to CC14 by 22-and 11-fold, respectively. F2-isoprostanes are formed in situ esterified to lipids and, in addition to increases in levels of free F2-isoprostanes in the circulation, levels of F2-isoprostanes esterified to lipids in various organs and plasma also increase sharply during CC14 poisoning. The measurement of F2-isoprostanes may facilitate investigation of the role of lipid peroxidation in human diseases. (J. Clin. Invest. 1992. 90:2502-2507

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-a (TGFa) 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 TGFa or TPA treatment. Stimulation of RIE-1 cells with TGFa leads to the increase of a protein (Mr -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 TGFa 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 TGFa 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 TGFa 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. (J. Clin. Invest. 1994. 93:493498.)

The majority of veteran patients are not suitable candidates for HCV treatment because of substance abuse, psychiatric disease, and comorbid medical disease, and many who are candidates decline therapy. Multidisciplinary collaboration is needed to overcome barriers to HCV therapy in this population.

No abstract

A dose of diquat below the amount injurious to selenium-replete animals causes lipid peroxidation and massive liver necrosis in selenium-deficient rats. The current study was undertaken to characterize the lipid peroxidation with respect to the liver injury and to correlate the presence of several selenoproteins with the protective effect of selenium. Lipid peroxidation was assessed by measurement of F2 isoprostanes. Diquat caused an increase in liver and plasma F2 isoprotanes. A gradient of these compounds was detected across the liver in some animals, indicating that this organ was a source of some of the plasma F2 isoprostanes. A time-course experiment showed that liver F2 isoprostane concentration increased before plasma alanine transaminase (ALT) levels rose. Selenium-deficient rats were injected with selenium doses from 2 to 50 micrograms/kg and studied 12 hours later. A dose of 10 micrograms/kg or more prevented diquat-induced lipid peroxidation and liver injury. This dose increased plasma selenoprotein P substantially, and a dose-response was present. Liver cellular and plasma glutathione peroxidase activities remained below 2% of their values in control rats for all selenium doses. In selenium-deficient rats given diquat, hepatic lipid peroxidation precedes hepatic necrosis and could therefore be an important mechanism of the necrosis. Selenoprotein P levels were increased by selenium injections, which protected against diquat injury, but glutathione peroxidase activity was not increased. This is consistent with selenoprotein P being the mediator of the selenium effect.

No abstract

The authors generated and tested a semiautomated carotid artery VWV measurement tool to provide measurements with reduced operator time and interaction, with high Dice coefficients, and with necessary required precision.