Robert W. Egan

Radical scavenging as the mechanism for stimulation of prostaglandin cyclooxygenase and depression of inflammation by lipoic acid and sodium iodide

Certain radical-trapping reducing agents have been shown to stimulate prostaglandin biosynthesis in vitro (1--6) and to depress phorbol myristate acetate-induced mouse ear edema (16). The increased prostaglandin synthesis resulted from influences on the cyclooxygenase. To ascertain whether these alterations were due to direct interaction with the enzyme or to indirect scavenging of the oxidant released during PGG2 reduction, we report the effects of lipoic acid and sodium iodide. Both of these agents stimulated the enzymatic oxygenation of arachidonic acid, increased the reduction of PGG2 to PGH2, quenched the EPR signal induced by arachidonic acid and depressed mouse ear edema. In addition to discovering two unusual antiinflammatory agents, we have confirmed that materials with entirely different structures can have identical effects on the cyclooxygenase, suggesting indirect stimulation of this enzyme due to trapping of the oxidant.

Comparative Biochemistry of Lipoxygenase Inhibitors

This chapter describes some studies on the mechanism of 5-lipoxygenase regulation and inhibition and then presents an overview of lipoxygenase inhibitors that are active against intact-cell, broken-cell, and purifed enzymes from a variety of sources. Although a large number of lipoxygenase inhibitors have been reported, many of these are rather nonselective. Some of the lack of selectivity may result from a common mechanism of lipoxygenase inhibition by antioxidants and redox regulation. It is, therefore, not sufficient to define specificity against broken-cell enzymes. A truly diagnostic agent must be specific in cellular systems and in vivo to be of utility in defining the role of lipoxygenases in pathophysiology.

Biochemical and biological activities of 2,3-dihydro-6-[3-(2-hydroxymethyl)phenyl-2-propenyl]-5-benzofuranol (L-651,896), a novel topical anti-inflammatory agent

The biochemical and biological profile of a topical anti-inflammatory agent, 2,3-dihydro-6-[3-(2-hydroxymethyl)phenyl-2-propenyl]-5-benzofuranol (L-651,896 inhibited the 5-lipoxygenase of rat basophilic leukemia cells with an IC50 of 0.1 microM and leukotriene synthesis by human PMN and mouse macrophages with IC50 values of 0.4 and 0.1 microM respectively. L-651,896 also inhibited prostaglandin E2 synthesis by mouse peritoneal macrophages (IC50 = 1.1 microM). This compound inhibited ram seminal vesicle cyclooxygenase activity at considerably higher concentrations, and this effect was directly related to substrate concentration. When applied topically to the mouse ear, L-651,896 lowered elevated levels of leukotrienes associated with arachidonic acid-induced skin inflammation and delayed hypersensitivity induced by oxazolone. However, while L-651,896 inhibited the increased vascular permeability induced by arachidonic acid, it had no effect on the edema associated with the immune-based response to oxazolone in the same tissue. Thus, it is possible that leukotrienes may play a role in some but not all inflammatory responses.

Acetaminophen as a Cosubstrate and Inhibitor of Prostaglandin H Synthase

Recently, several reports (Marnett et al., 1983; Nordenskjold et al., 1984) have implicated prostaglandin H synthase (PHS) in the bioactivation of xenobiotics to potentially toxic metabolites. Benzidine (Zenser et al., 1983), p-aminophenol (Josephy et al., 1983), and phenacetin (Andersson et al., 1982) are among the compounds known to undergo metabolic activation by PHS. Of particular interest to us is the fact that this enzyme can metabolize acetaminophen (APAP) to a reactive species that can bind to proteins or form a glutathione conjugate (Moldeus and Rahimtula, 1980; Boyd and Eling, 1981; Mohandas et al., 1981; Moldeus et al., 1982). In fact, it has been suggested (Boyd and Eling, 1981; Mohandas et al., 1981) that the nephrotoxicity sometimes associated with APAP overdosage may be due in part to its metabolism by PHS which is present in high levels in the renal inner medulla.

Polarographic assay for phospholipase A2

Abstract A rapid, specific, and quantitative assay for phospholipase A2 from Naja naja venom has been devised, in which phospholipid hydrolysis is measured as soybean lipoxidase-catalyzed oxygen incorporation into the ensuing unsaturated fatty acids. Under conditions where phospholipid was limiting, a linear relationship developed between the extent of oxygen uptake and the amount of egg lecithin metabolized. When phospholipase was rate limiting, the initial rate of oxygen consumption was a linear function of phospholipase concentration over a 14-fold range from 30 to 420 ng/ml. This linear relationship did not exist at higher phospholipase levels, probably due to the micellar nature of the phospholipid. Since this assay can readily detect as little as 17 ng/ml of phospholipase A2 (Naja naja venom) and is insensitive to most potential interfering materials, it should be useful in a variety of applications.

EVIDENCE FOR A PIVOTAL ROLE OF THE ENDOPEROXIDE, PGG 2 , IN INFLAMMATORY PROCESSES

Since the reports by Vane1 as well as Smith and Willis2 that aspirin and indomethacin are potent inhibitors of prostaglandin synthetase, there has been a controversy concerning the role of such non-steroidal anti-inflammatory agents (NSAI) in inflammatory processes. Other studies demonstrated a reasonable correlation between the action of NSAI on PG synthetase and activity both in the rat paw carrageenan assay and the clinic3–5. Nevertheless, the failure of administered PGEs and PGFs to fully mimic the symptoms of inflammation did not foster this interpretation6,7.

A general method for quantitative separation of prostaglandins by paper chromatography.

Abstract Although prostaglandin (PG) mixtures have previously been resolved by chromatography on silica-impregnated paper, drawbacks inherent in each technique have kept them from becoming generally accepted for routine analytical separations. Singh and co-workers (1,2) obtained excellent separation of prostaglandin mixtures on silica-impregnated glass fiber paper. However, this paper was not commercially available and its preparation is tedious. On the other hand, Stamford and Unger (3) separated PGE and PGF on commercially available paper using benzene/chloroform/acetone/methanol/acetic acid as developing solvent. Nevertheless, this solvent does not resolve less polar prostaglandins and fatty acids. More generally acceptable solvent systems cannot be used quantitatively with Stamford and Ungers technique due to irreversible binding of prostaglandins at the origin. Tobias and Paulsrud (11) have separated prostaglandins on commercial silicic acid-impregnated glass fiber sheets, but these are extremely brittle and difficult to accommodate to standard paper radiochromatogram scanners. This communication describes the quantitative chromatographic separation of PGF 1α , PGE 2 , PGA 1 , and arachidonic acid on commercially available Whatman SG-81 silica-impregnated paper using a wide variety of developing solvents. Irreversible binding of prostaglandins at the origin, previously a serious drawback, has been eliminated by applying the sample onto premoistened paper. This method is quantitative, sensitive, reproducible, and applicable to a variety of solvent systems. In addition, it is simple and inexpensive. Although loading capacity is somewhat limited, this is no problem with prostaglandins since they can be readily concentrated in organic solvents.

Free Radical Mechanisms

ABSTRACT The role of the oxidant generated by the reaction of hydroperoxy acids with PG peroxidase is examined with respect to inflammation and other diseases. Data are provided relative to the nature of the oxidant, its similarity to oxygen species elaborated by the neutrophil and its site of action.