Effects of dietary fish oil on malondialdehyde production and glutathione peroxidase activity in hyperlipidaemic patients.

In 20 adult patients suffering from hyperlipidaemia we measured the lipid composition of erythrocyte membrane, the glutathione peroxidase activity in both erythrocytes and platelets, the production of malondialdehyde by platelets stimulated with thrombin, as well as the level of plasma selenium, retinol and alpha-tocopherol, before and after 8 weeks of fish oil supplementation (20 ml daily). We noted a remarkable reduction in plasma triglycerides which was associated with a significant decrease in blood pressure; moreover, we noted a reduction in the amount of arachidonic acid compensated by an increment of omega-3-fatty acid (particularly eicosapentaenoic and docosahexaenoic acids). The dietary supplementation with fish oil was associated with a significant increase in glutathione peroxidase activity in both erythrocytes and platelets. On the contrary, the production of malondialdehyde, which was originally higher than normal in hyperlipidaemics, was inhibited significantly after fish oil (p less than 0.001). Whereas no changes were observed in the concentration of plasma selenium and alpha-tocopherol, an increment of plasma retinol occurred. These data indicate that in hyperlipidaemics there is a proaggregant status; this situation may be normalized by using a dietary supplementation of fish oil; the increase of polyunsaturated fatty acids on the cell membrane, with a possible increment of the formation of lipoperoxides, induced by fish oil, is compensated by an increased activity of the scavenger enzyme glutathione peroxidase.

Inhibitory effects of trimetazidine dihydrochloride on aggregation, serotonin release and malondialdehyde production in rabbit platelets.

Aggregation, serotonin release and malondialdehyde (MDA) production via cyclooxygenase and thromboxane A2 synthetase were investigated in rabbit platelets. Trimetazidine dihydrochloride (TMZ) attenuated the collagen-induced aggregation more strongly than the arachidonic acid (AA)-, thromboxane A2 agonist (U-46619)-, Ca2+-ionophore (A-23187)- and ADP-induced aggregation: IC50 values were 1.0 +/- 0.1, 4.4 +/- 0.3, 4.3 +/- 0.4, 4.1 +/- 0.7 and 3.3 +/- 0.2 mM, respectively. TMZ decreased dose-dependently the serotonin release induced by collagen and A-23187, but did not decrease that induced by AA. TMZ also decreased the MDA production induced by collagen and A-23187 (IC50: 0.3 +/- 0.03 and 1.0 +/- 0.1 mM, respectively), but did not decrease the production induced by AA. Furthermore, TMZ decreased dose-dependently the MDA production induced by exogenous phospholipase A2. On the other hand, indomethacin (10 microM) attenuated the aggregation induced by collagen and AA, but not by the other agents, and decreased the serotonin release and the MDA production induced by collagen, A-23187 and AA. The present results suggest that TMZ may inhibit the process preceding the cyclooxygenase pathway in the AA cascade, and subsequently may attenuate the aggregation and the serotonin release via thromboxane A2 production from endogenous AA.

Iron chelation as a possible mechanism for aspirin-induced malondialdehyde production by mouse liver microsomes and mitochondria.

To investigate the possibility that lipid peroxidation is the mechanism responsible for aspirin-induced liver damage, pure neutralized acetylsalicylic acid (ASA), 0.6-90.9 mM, was added to calcium-aggregated mouse liver microsomes followed by incubation in NADPH buffer at 37 degrees C for 60 min and subsequent measurement of malondialdehyde (MDA). MDA production at ASA concentrations from 1.2 to 4.6 mM was greater than control (P less than 0.004). Peak MDA values were observed with 4.6 mM ASA, 39.58 +/- 6.73 nmol MDA/mg protein vs. 16.16 +/- 2.85 (P less than 0.004). Higher concentrations of ASA were inhibitory compared with the value at 4.6 mM (P less than 0.001). Aspirin had similar effects on MDA production by mouse liver mitochondria. MDA production with either ASA or buffer was completely suppressed by the potent iron-chelating agents desferrioxamine and alpha,alpha' dipyridyl when these were added to the microsomal preparations. Since MDA production in this system is known to be affected by iron-chelating agents (enhanced at low concentration, inhibited at higher concentration), the iron-chelating properties of ASA were investigated. Conductivity titration curves of Fe(OH)3 added to water or ASA suggested that the ASA was complexing with iron. The presence of an iron-ASA complex was established by high pressure liquid chromatographic analysis of the solution from this study. We conclude that aspirin enhances MDA production by hepatic microsomes and mitochondria via an aspirin-iron chelate and that this represents at least one mechanism by which aspirin may produce liver damage.

Malondialdehyde production from spermine by homogenates of normal and transformed cells.

The oxidation of spermine in vitro by a mixture of polyamine oxidase and diamine oxidase from pig kidney gives rise to malondialdehyde via 3-aminopropanol as the intermediate. Conversely, with spermidine, under similar experimental conditions, no evidence could be obtained for malondialdehyde formation within the limits of sensitivity of the assay (2.0 nmol). The activities of both these enzymes show about a 2-fold increase in normal rat kidney cells (LA31 NRK) transformed by the temperature sensitive mutant of Rous sarcoma virus (LA31) and incubated at the non permissive temperature (39 degrees C) compared to the activities in LA31 NRK at the permissive temperature (33 degrees C). These same enzymatic activities show no temperature dependent changes in normal rat kidney cells (NRK) or in these same cells infected by the wild type virus (NRK B77). In extracts derived from Friend erythroleukemic cells induced to differentiate by dimethyl sulfoxide or hexamethylene bis acetamide, spermine oxidation takes place more efficiently than in non induced cells. A rise in diamine oxidase activity is seen in LA31 NRK (39 degrees C) 12 h after the temperature shift, whereas morphological manifestations of normalcy are seen only at 48 h. The Km of diamine oxidase is 10(-6) M for putrescine and 10(-3) M for 3-aminopropanol. A possible mechanism involving the well documented acetylation of putrescine [23,26] is proposed for diverting intracellular putrescine away from cytosolic diamine oxidase and towards intramitochondrial monoamine oxidase.

In vitro study of paraquat effects on malondialdehyde formation in thymus cells.

The present experiments have shown that paraquat enhanced both O2- production and oxidation of exogenous NADPH in thymus cells by NADPH-dependent enzyme system. The effects of paraquat on malondialdehyde formation were also NADPH-dependent and opposite in the absence and in the presence of ferrous ions: Paraquat inhibited the NADPH-dependent malondialdehyde formation in the absence of Fe2+; it activated this formation in the presence of Fe2+ and had no effect on the nonenzymic Fe2+- and Fe2+-ascorbate-stimulated lipid peroxidation. It is suggested that the activating effect of paraquat on the enzymic NADPH-dependent MDA formation in the presence of Fe2+ is due to the formation of a powerful oxidant product (FeO2)+ or to some Fe2+-paraquat radical complex, acting similarly to the ADP-chelated ferrous ions.

Platelet activity in mitral valve prolapse: a study of platelet aggregation, malondialdehyde production, and plasma beta-thromboglobulin.

Platelet aggregation, malondialdehyde (MDA) production, and recovery after aspirin (ASA) administration and plasma levels of beta-thromboglobulin (BTG) were determined in 40 asymptomatic patients with mitral valve prolapse (MVP) and 17 normal subjects. Platelet aggregation was similar in patients and controls, although two patients presented spontaneous aggregation. Production of MDA and plasma levels of BTG were higher in MVP than in controls; however, recovery after ASA was similar in both groups. The results further indicate that platelet hyperactivity is present in a significant number of asymptomatic patients with MVP.

Effect of SAS 650 (Furofenac) on malondialydehyde production by platelets.

The activity of SAS 650, a new anti-inflammatory drug, on ex vivo and in vitro MDA production by platelets was compared to that of aspirin. The drug induced dose-dependent inhibition of in vitro MDA production by rat and guinea-pig platelets and also had good activity after 30 second of incubation in rat platelets, quicker than aspirin. SAS 650 preincubation reduced the in vitro inhibitory effect of ASA, as shown also by ex vivo experiments. The results of the present study support the involvement of SAS 650 in the platelet cyclooxygenase pathway.

A novel phospholipid in irreversibly sickled cells: evidence for in vivo peroxidative membrane damage in sickle cell disease.

In individuals with sickle cell disease, a variable number of irreversibly sickled cells (ISC) is present that may contribute to the pathophysiology of sickle cell anemia. The present study was undertaken to determine the possible role of membrane lipid peroxidation in the genesis of ISC. After 24 hr of simple aerobic incubation, sickle cells accumulated 2-3 times more malonyldialdehyde (MDA), an end product of lipid peroxidation, than normal cells. To assess the possibility of peroxidative damage in ISC in vivo, ISC were separated from sickle blood using Stractan density gradients. Lipid extracts of the untreated ISC-enriched fraction of sickle blood showed significant fluorescence and contained a novel phospholipid:MDA adduct that was not seen in control cells. Taken together, these observations suggest that ISC have previously undergone lipid peroxidative damage and the accumulation of MDA in vivo.

Levels of malondialdehyde production in rat liver following loading and unloading with iron.

Rats were given daily injections of an iron sorbitol citric acid complex in a total dose of 50 mg Fe3+/100 g of body weight and either killed immediately after iron loading, or investigated 2 months later. Among the latter animals, one group was subjected to weekly phlebotomies in order to mobilize iron from the stores, while another group was not further treated. Quantitation of iron and malondialdehyde production was performed on homogenates of liver, kidney and spleen from controls and rats in the different experimental groups, and the distribution of iron in granular form was studied in the livers by means of electron microscopy. The results showed substantially increased amounts of iron in the organs studied after iron-loading and also augmented malondialdehyde production in the liver and kidney (but not in the spleen). A decreased malondialdehyde production was recorded two months after iron-loading in the kidney and spleen of non-bled animals; this decrease was exaggerated in the same organs from bled animals. The production of malondialdehyde as well as the iron content in the livers of both bled and non-bled rats 2 months after iron loading was higher than in the controls. The evidence obtained suggested that the accumulation of iron in the liver was causally related to increased lipid peroxidation. Judging from the morphological appearances this change did not result in cell damage, the only pertinent morphologic alteration being the occurrence of iron particles in the lysosomal vacuome and the cell sap.

The effects of hypolipidemic agents derived from procetofenic acid on the activity of superoxide dismutase and glutathione peroxidase and on malonyl dialdehyde production of rat liver.

Male and female rats were treated with hypolipidemic agents derived from procetofenic acid, namely fenofibrate ( procetofene ), cholestyrammonium alpha-(4-p-chlorobenzoyl-phenoxy)-isobutyrate (Alfa-1081) and alpha-(4-p-chlorobenzoyl-phenoxy)-isobutyryl-taurine (35/ipo). A marked decrease of liver superoxide dismutase and glutathione peroxidase was observed in the male rats treated with fenofibrate. In this sample a significant increase of malonyl dialdehyde was also detected when the liver homogenates were subjected to a test of lipid peroxidation induced by active oxygen species. Alfa-1081 and 35/ipo produced no substantial changes of superoxide dismutase and glutathione peroxidase and no significant increase of peroxidation products. These results, while providing an in vivo evidence for the role of superoxide dismutase and glutathione peroxidase as antioxidant enzymes, indicate that hypolipidemic agents can affect also enzymes not bound to membranes as they do with peroxisomal enzymes. The side effect presented here may be harmful, since it can lead to an augmented risk of lipid peroxidation in tissues. It is, however, clear that also within the same class of hypolipidemic drugs the effect is not always present. Peroxidative damage should therefore be considered as a key parameter in the characterization of hypolipidemic agents.

Low density lipoprotein (LDL) from male volunteers stimulated the thromboxane formation by human platelets.

LDL (0.5 - 2.0 mg LDL-cholesterol/ml) isolated from venous blood of healthy male volunteers stimulated dose-dependently the malondialdehyde (MDA) formation by frozen human platelets, used as a marker for the activity of the thromboxane synthetase. HDL (0.25 - 1.0 mg HDL-cholesterol/ml) and human serum albumin (1 - 10 mg/ml) had no concentration-dependent influence on the MDA formation. If these results can be extended to in vivo they suggested the strong connection between the prostaglandin and lipoprotein hypothesis of pathogenesis of atherosclerosis.

Modulation of platelet thromboxane and malonaldehyde by dietary vitamin E and linoleate.

Thromboxane (TXB2) and malonaldehyde (MDA) production by thrombin-stimulated washed platelet were evaluated in rats fed 6 combinations of dietary vitamin E (0, 100, 1000 ppm) and linoleate (6.5 and 17.0 en%) for 23 weeks. The molar ratio of MDA:TXB2 was consistently near 3 in all groups studied. In animals receiving the lower linoleate diets, TXB2 and MDA synthesis were inversely related to the dietary vitamin E concentrations and the levels of MDA and TXB2 were positively correlated (r = 0.99) with decreasing vitamin E in the diet. High dietary linoleate (17.0 en%), independent of vitamin E status, reduces TXB2 and MDA synthesis. The importance of dietary antioxidant on platelet prostanoid synthesis is discussed.

Glutathione peroxidase blockage inhibits prostaglandin biosynthesis in rat platelets and aorta.

In the present study we demonstrated that the compound 3-amino-1,2,4-triazole (AMT) is a strong inhibitor of erythrocyte glutathione peroxidase (GSH-Px) activity. Moreover, AMT inhibits arachidonic-induced malondialdehyde formation in platelet-rich plasma and prostacyclin-like activity generation in aorta rings. These results give new lines of evidence on the connection between GSH-Px activity and prostaglandin synthesis in rat platelets and arterial vessel wall.

Platelet malondialdehyde production kinetics after cyclooxygenase block. A study performed with a modified high sensitivity assay.

A modification of the original thiobarbituric acid (TBA) method for malondialdehyde (MDA) assay is described. The improvement is essentially based on the clearing effect of KClO4 that makes measurements more simple and sensitive. MDA values obtained in normal subjects were almost eightfold higher than those obtainable with Stuart's original assay method, so that after cyclooxygenase block it was possible to assess platelet regeneration time even in thrombocytopenic patients with at least 60,000 platelets/microliter and MDA production early after aspirin intake. To challenge this modification, platelet regeneration time was studied in normal subjects as well as in thrombocytopenic patients, either hypoplastic or idiopathic, and in hypoxemic patients with increased platelet consumption. The initial disappearance kinetics of platelet MDA and thromboxane B2 production after aspirin suggests that TBA-reactive material is synthesized through the lipoxygenase pathway. The reversible block determined by acetylsalicylic acid and salicylate on hydroperoxi-eicosatetraenoic acid peroxidase can be responsible for the initial increase of this TBA-reactive material.

[The effect of triphenyltin fluoride on aggregation, ATP secretion and malondialdehyde formation of rabbit platelets in vitro].

Recent studies have demonstrated that triphenyltin fluoride (TPTF), widely used as an agricultural chemical and a marine antifoulant, inhibits collagen-induced platelet aggregation and ATP secretion in rabbits ex vivo. The aim of the present investigation was to elucidate the mechanism of the inhibitory action of TPTF by investigating platelet malondialdehyde (MDA) formation, aggregation and ATP secretion following the stimulation by various stimuli of rabbit platelets treated in vitro with TPTF, other triphenyl metals and aspirin. Although no inhibitory effect of TPTF was found on sodium arachidonate-induced platelet aggregation and ATP secretion, TPTF inhibited dose-dependently both platelet aggregation and ATP secretion induced by collagen. The antiaggregating (IC50) concentration of TPTF was 6.0 X 10(-6) M against collagen. In addition, TPTF prevented the collagen-, and thrombin-induced formation of MDA, but had little inhibitory effect on the conversion of exogenous arachidonic acid to MDA in platelets. In contrast, aspirin (10(-3) M) inhibited platelet aggregation, ATP secretion and MDA formation induced by all the stimuli tested. Other triphenyl metals did not any inhibitory effect on collagen-, and sodium arachidonate-induced platelet aggregation and ATP secretion even at a final concentration at 10(-3) M. These results suggest that TPTF has a specific inhibitory effect on platelet aggregation and ATP secretion by acting at some step(s) of platelet membrane between the binding site of collagen and thrombin and the release of arachidonic acid.

Effect of heparin on platelet monolayer adhesion, aggregation and production of malondialdehyde.

Heparin inhibited monolayer adhesion of washed human and rabbit platelets to collagen-coated glass at 2.5 and 20 units/ml concentration, in the absence of red cells. Adhesion of rabbit platelets to de-endothelialized rabbit aorta, under similar conditions, was less strongly inhibited but no inhibition was seen at 40% haematocrit. Addition of plasma reduced, rather than enhanced heparin activity and hirudin 0.5 units/ml had no significant effect. Heparin also inhibited platelet aggregation, release of (14C) 5-HT and production of malondialdehyde in response to collagen and thrombin. Inhibition of thrombin-induced activity was greater in the presence of plasma. However, heparin enhanced aggregation and release evoked by ADP and did not consistently inhibit MDA synthesis produced by arachidonate. The results indicate that in addition to the effects of heparin on platelet function mediated by anti-thrombin activity and the previously described augmentation of responses to ADP, heparin has weak inhibitory activity against platelet-collagen interactions. Binding of heparin to the platelet membrane (and to surfaces to which platelets adhere) could account for these findings by causing non-specific interference with agonist-receptor interactions.

Vincristine inhibits the synthesis of malondialdehyde by human platelets in vitro.

When platelet-rich plasma was preincubated with vincristine, a depression in malondialdehyde production was seen when compared with saline controls. Colchicine caused an increase in malondialdehyde production. If prostaglandins are important in tumor-cell metabolism, then vincristine might exert some of its cytotoxic effect by depressing prostaglandin production.

Platelet aggregation, malondialdehyde generation and production time in children with sickle cell anaemia.

In view of prior reports of platelet activation in the sickling disorders, platelet aggregation, malondialdehyde (MDA) production following stimulation with N-ethylmaleimide, and/or production time (survival) measurements were examined in 44 children with homozygous sickle cell disease. Aggregation in response to epinephrine, collagen, and adenosine diphosphate was similar to or only slightly less than in normal black controls, rendering highly unlikely the circulation of a sizable population of refractory or "exhausted" platelets. The platelets from the normal blacks aggregated less in response to epinephrine than those from white control subjects. MDA generation in sickle cell platelets was not increased, and platelet production time was not shortened in 6 patients studied during crisis. These observations do not support the occurrence of a marked degree of platelet activation and consumption in sickle cell anaemia.

Platelet malondialdehyde production and aggregation responses induced by arachidonate, prostaglandin-G2, collagen, and epinephrine in 12 patients with storage pool deficiency.

We assessed the integrity of the prostaglandin synthetic pathway by measuring malondialdehyde (MDA) production and studied platelet aggregation responses to arachidonic acid and PGG2 in 12 patients with storage pool deficiency (SPD). Eight patients were deficient only in dense granules (delta-SPD) and four were deficient in both dense and alpha-granules (alpha delta-SPD). Production of MDA in response to arachidonic acid (AA), epinephrine, and collagen suggested that the transformation of AA to prostaglandin metabolites was normal in delta-SPD but abnormal in alpha delta-SPD and that the liberation of AA from phospholipids were abnormal in the majority of patients with SPD. Since the content of secretable adenosine diphosphate (ADP) is diminished in SPD platelets, the aggregation responses of these platelets to AA and PGG2 were studied to help answer the question whether these agents aggregate platelets directly or through release of endogenous ADP. Among patients with delta-SPD, aggregation by both AA and PGG2 was decreased in four albinos whose platelets were markedly deficient in ADP. In contrast, normal, or less strikingly abnormal, responses were observed in patients whose platelets either contained higher levels of platelet ADP or showed increased sensitivity to ADP. The more marked impaired responses to AA and PGG2 in patients with alpha delta-SPD suggest that substances derived from alpha-granules may also play a role in platelet aggregation by these agents. The aggregation responses in these patients with various types of SPD is consistent with a theory that granule-derived ADP mediates platelet aggregation by AA and PGG2.