Ryszard J. Gryglewski

Flavonoids are scavengers of superoxide anions

Seven flavonoids and three non-flavonoid antioxidants, i.e. butylated hydroxyanisole, chlorpromazine and BW 755 C, were studied as potential scavengers of oxygen free radicals. Superoxide anions were generated enzymatically in a xanthine-xanthine oxidase system and non-enzymatically in a phenazine methosulphate-NADH system, and assayed by reduction of nitro blue tetrazolium. The generation of malonaldehyde (MDA) by the ascorbate-stimulated air-oxidised boiled rat liver microsomes was considered as an index of the non-enzymatic formation of hydroxyl radicals. Flavonoids but not non-flavonoid antioxidants lowered the concentration of detectable superoxide anions in both enzymic and non-enzymic systems which generated these SOD-sensitive radicals. The most effective inhibitors of superoxide anions were quercetin, myricetin and rutin. Four out of seven investigated flavonoids seemed also to suppress the activity of xanthine oxidase as measured by a decrease in uric acid biosynthesis. All ten investigated compounds inhibited the MDA formation by rat liver microsomes. Non-flavonoid antioxidants were more potent MDA inhibitors than flavonoids. It is concluded that antioxidant properties of flavonoids are effected mainly via scavenging of superoxide anions whereas non-flavonoid antioxidants act on further links of free radical chain reactions, most likely by scavenging of hydroxyl radicals.

On the mechanism of antithrombotic action of flavonoids

Flavonols (quercetin and rutin) and flavanes (cyanidol and meciadonol) were studied for their effect on non-enzymatic lipid peroxidation, lipoxygenase and cyclo-oxygenase activities, binding to albumin and platelet membranes. These biochemical properties of four flavonoids were compared with respect to their antithrombotic action in vivo and their efficacy at influencing the platelet-endothelium interaction in vitro. All four flavonoids inhibited the ascorbate-stimulated formation of malondialdehyde by boiled rat liver microsomes (quercetin greater than rutin approximately cyanidol approximately meciadonol) and inhibited platelet lipoxygenase activity (quercetin greater than cyanidol greater than meciadonol greater than rutin) whereas only flavonols, but not flavanes, stimulated cyclo-oxygenase and were bound to platelet membranes. The same two flavonols dispersed platelet thrombi which were adhering to the rabbit aortic endothelium in vitro (EC50 for quercetin was 80 nM and for rutin 500 nM) and prevented platelets from aggregation over blood-superfused collagen strip in vivo (ED50 for quercetin was 5 nmol/kg and for rutin 33 nmol/kg i.v.). Cyanidol and meciadonol were not effective as anti-thrombotic agents. It is concluded that activated platelets adhering to vascular endothelium generate lipid peroxides and oxygen-free radicals which inhibit endothelial biosynthesis of prostacyclin and destroy endothelium-derived relaxing factor (EDRF). Flavonols are anti-thrombotic because they are selectively bound to mural platelet thrombi and owing to their free radical scavenging properties resuscitate biosynthesis and action of endothelial prostacyclin and EDRF. Thus, flavonols release the thrombolytic and vasoprotective endothelial mediators only in these vascular segments which are covered by a carpet of aggregating platelets.

Clinical patterns of hypersensitivity to nonsteroidal anti-inflammatory drugs and their pathogenesis

Abstract Clinical observations coupled with challenge tests using 16 different nonsteroidal anti-inflammatory drugs were carried out in 123 patients with a history of allergy to analgesics. Several different patterns of hypersensitivity could be distinguished. Our clinical and experimental data suggest that in the largest group of patients studied the idiosyncrasy to aspirin-like drugs was not of immunologic type but was due to the suppression of prostaglandin generation in tissues of the patients. The adverse reactions to aspirin-like drugs were manifested as asthmatic attacks or urticaria/angioedema. In another group, the hypersensitivity was limited to pyrazoline drugs and was not related to inhibition of prostaglandin biosynthesis. This type of hypersensitivity, resulting in anaphylactic shock and urticaria, seems to have an immunologic background. In a patient with lupus erythematosus the hypersensitive reactions to analgesics were related neither to their chemical structure nor to their known biochemical activities. In a small group of patients the hypersensitivity to aspirin could possibly be attributed to the minute amounts of impurities present in some commercial products.

Prostacyclin and thromboxane A2 biosynthesis capacities of heart, arteries and platelets at various stages of experimental atherosclerosis in rabbits

Abstract Atherosclerosis was induced by feeding rabbits with 1 g of cholesterol and 3 g of olive oil daily for 1, 3 and 5 months. Arachidonic acid (AA)-induced generation of prostacyclin (PGI 2 ) by perfused hearts and spontaneous generation of PGI 2 by slices of mesenteric arteries and aortas were strongly suppressed after 1 and 3 months of the diet, while after 5 months a tendency for PGI 2 synthesizing capacity to recover was observed. Aggregatability of blood platelets in platelet-rich plasma (PRP) by exogenous AA and endogenous thromboxane A 2 (TXA 2 ) was increased but not earlier than 3 months on the diet. On the other hand, platelet sensitivity to the pro-aggregatory action of adenosine diphosphate (ADP) and to the anti-aggregatory action of PGI 2 was heightened as early as 1 month from the beginning of the experiment. These findings suggest that the first stage of experimental atherosclerosis may be causally related to the strong suppression of PGI 2 generation by arteries, whereas the metabolism of AA in platelets remains unchanged. At this early stage of atherosclerosis increased susceptibility of platelets in PRP to both ADP and PG1 2 may be due to the lowering of cyclic AMP levels in platelets as a consequence of PGI 2 deficiency. A genuine enhancement of AA metabolism in blood platelets occurs only at the second stage of experimental atherosclerosis. This stage is particularly dangerous since an increased generation of TXA 2 by platelets in PRP is combined with a decreased generation of PGI 2 by arteries. At the last stage of atherosclerosis observed, a slow recovery of the enzymic activity occurs in arteries.

Circulatory and anti-platelet effects of intravenous prostacyclin in healthy men

Summary Intravenous infusion of prostacylin (2 - 50 ng/kg/min) into six healthy men caused vasodilation of the skin vessels of head, neck and palms, a lowering of diastolic blood pressure with little effect on systolic blood pressure, an increase in heart rate and a rise in venous oxygen pressure. Prostacyclin infusions also resulted in suppression of platelet aggregability to ADP, as well as a dispersion of circulating platelet aggregates. Template bleeding time became prolonged, while partial thromboplastine time, prothrombine time and euglobulin lysis time remained all unchanged. These effects of prostacyclin were transient and disappeared briefly after termination of the hormone infusion. During infusion of a high dose of prostacyclin (50 ng/kg/min) signs of collapse appeared and waned shortly after withrowal of the prostacyclin infusion. No other serious immediate or remote side effects were observed. It is concluded that vasodilatory and anti-platelet actions of prostacyclin, now proved in men, are of the potential therapeutic interest in thrombo-embolic diseases.

The effect of six prostaglandins, prostacyclin and iloprost on generation of superoxide anions by human polymorphonuclear leukocytes stimulated by zymosan or formyl-methionyl-leucyl-phenylalanine

Prostaglandins (PG) E2,E1,6-keto-E1 and D2 at concentrations of 0.15-0.80 microM inhibited by 25% the generation of superoxide anions (O2-) in human polymorphonuclear leukocytes (PMNs) stimulated with formyl-methionyl-leucyl-phenylalanine (FMLP). The potency of that inhibition by either PGD2 or PGE1 was the same when zymosan was used as a stimulator whereas PGE2 and 6-keto-PGE1 were by 13 and 21 times less potent inhibitors of O2-) in zymosan-stimulated as compared to FMLP-activated PMNs. PGF2 alpha inhibited the generation of O2- by activated PMNs only when used at the highest concentration studied (30 microM). Prostacyclin, 6-keto-PGF1 alpha and Iloprost (a carbacyclin analogue of prostacyclin) at concentrations up to 30 microM showed no significant inhibition of O2- in human PMNs stimulated either with FMLP or with zymosan. It is concluded that PGD2 and PGEs use a common basic mechanism for inhibition of the generation of O2- by PMNs activated with FMLP or zymosan. PGD2 is most generously furnished with these properties. In addition to this basic mechanism PGE2 and 6-keto-PGE1 abrogate the FMLP-induced response by occupation of formyl peptide receptor of PMNs. It is hypothesised that inhibition of the generation of O2- in PMNs and, possibly, in other cells by PGD2, PGE2 and by products of prostacyclin biotransformation might be responsible for their cytoprotective action in myocardial infarction, stroke, liver damage and peripheral vascular disease.

Lungs as a generator of prostacyclin--hypothesis on physiological significance.

SummaryIn vivo anti-platelet de-aggregatory activity of exogenous prostacyclin is enhanced after its passage through the pulmonary circulation of anaesthetized cats, probably because of a concomitant generation of endogenous prostacyclin by the lungs. Evidence is also presented that perfused lungs of guinea pigs and rats spontaneously release considerable amounts of prostacyclin. It is therefore postulated that a continuous biosynthesis of prostacyclin by pulmonary endothelium is a general physiological phenomenon, while the generation of thromboxane A2 by lungs occurs in response to pathological stimuli. Coronary and cerebral arteries are supposed to benefit from this hormonal function of the lungs.

In vivo method for quantitation of anti-platelet potency of drugs

SummaryAortic strips from atherosclerotic rabbits or Achilles tendons from healthy rabbits were superfused with blood (3 ml/min) from anaesthetized and heparinized cats, while blood was returned to the venous system of animals. The superfused tissues gained in weight because of deposition of platelet thrombi on their surface. This gain in weight was continuously monitored and quantified. Forty minutes after intravenous administration of indomethacin (14 mg/kg), aspirin (7 mg/kg) or nictindole (2 mg/kg) the formation of platelet deposits was reduced by half. Three hours after i.v. administration of each drug at a dose of 20 mg/kg the remaining anti-platelet activities were 92% for aspirin, 59% for indomethacin and 18% for nictindole as compared to their antithrombotic action, which was recorded 40 min after their administration. Thrombogenesis was also prevented by a direct infusion of nictindole (50 ng/ml) or indomethacin (2000 ng/ml) into a stream of superfusing blood. Thereby our method enables us to quantify in vivo anti-aggregating potency of drugs, to estimate the duration of this action, and to compare their in vitro and in vivo aggregation-inhibitory activities.

Anti-platelet action of intravenous infusion of prostacyclin in man.

Abstract Sodium salt of prostacyclin in 0.1 M Tris buffer pH 9.0 was infused intravenously at doses of 2, 5, 10, 20 and 50 ng/kg/min into six healthy men, aged 26–46 years. Begining with a dose of 5 ng/kg/min prostacyclin caused a dose-dependent inhibition of ADP-induced platelet aggregation in plasma and dissipation of circulating platelet aggregates in blood. At a dose of 20 ng/kg/min template bleeding time and concomitant blood loss were doubled. Prostacyclin did not influence partial thromboplastin time, prothrombin time and euglobulin lysis time. The anti-platelet action of prostacyclin was accompanied by a moderate fall in diastolic blood pressure, increase in heart rate, vasodilation in the regions of face, neck and palms, as well as by arterialization of venous blood. All of the pharmacological effects of prostacyclin disappeared briefly after termination of the infusion. This is the first report on antiplatelet action of prostacyclin in man and it is concluded that prostacyclin may be a valuble drug in the treatment of thromboembolic diseases.

Steroid hormones, anti-inflammatory steroids and prostaglandins

Abstract Hydrocortisone and synthetic anti-inflammatory steroids inhibit the release of prostaglandins from intact cells, tissues and organs, but do not inhibit conversion of arachidonic acid by microsomal cyclooxygenase to prostaglandins. This type of activity implies the possibility of a direct interaction of corticosteroids with biomembranes, which might either impair the supply of endogenous substrates for prostaglandin biosynthesis or inhibit transmembrane transport of prostaglandins.