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M Lagarde

Publications and source records attributed to M Lagarde.

At least 199 records · Page 11Linked to original sources
PubMed1988

Occurrence of the 15-hydroxy derivative of dihomogammalinolenic acid in human platelets and its biological effect.

Various polyunsaturated fatty acids are oxygenated by platelet lipoxygenase at the n - 9 position. The present paper reports that platelets may also oxygenate dihomogammalinolenic acid (20:3(n - 6)) at the n - 6 position, leading to the formation of substantial amounts of 15-OH-8,11,13-20:3 characterized by its ultraviolet spectrum, HPLC and GC-MS analysis. Its formation was inhibited by aspirin and eicosatetraynoic acid, but not by heneicosatetraynoic acid, a specific inhibitor of platelet lipoxygenase. The time-course of its synthesis was very close to that of 12-OH-8,10-17:2 (HHD), the non-cyclic cyclooxygenase side-product, but different from that of 12-OH-8,10,14-20:3, the platelet lipoxygenase end-product of 20:3 (n - 6). Overall, these results indicate that 15-OH-20:3 could be a cyclooxygenase metabolite generated in an aborted process. Like other monohydroxy derivatives of polyenoic fatty acids, 15-OH-20:3 was able to modulate thromboxane-induced platelet aggregation. The derivative exhibited a biphasic effect on the aggregation. It potentiated at concentrations below 2.10(-7) M and inhibited at higher doses. It is concluded that the potentiating activity might explain at least part of the transient enhancement of the platelet activation observed in adding exogenous 20:3(n - 6).

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1988

Different metabolic behavior of long-chain n-3 polyunsaturated fatty acids in human platelets.

Whereas numerous studies deal with the effects and metabolism of eicosapentaenoic acid (20:5(n - 3)) in platelets, very few concern docosahexaenoic acid (22:6(n - 3)), although both acids are consumed in equal amounts from most fish fat. The present paper reports the modulation of 22:6(n - 3) oxygenation as well as that of endogenous arachidonic acid (20:4(n - 6)) in 22:6(n - 3)-rich platelets. Like the oxygenation of 20:5(n - 3), the lipoxygenation of 22:6(n - 3) occurred at a low level when incubated alone, but was markedly increased in the presence of 20:4(n - 6), suggesting a similar peroxide tone dependency. 20:5(n - 3) could not replace 20:4(n - 6) in the increasing 22:6(n - 3) lipoxygenation, whereas 22:6(n - 3) shared the potentiating effect of 20:4(n - 6) on both the cyclooxygenation and the lipoxygenation of 20:5(n - 3). On the other hand, 20:5(n - 3), 22:6(n - 3) or 20:5(n - 3) + 22:6(n - 3) enrichment of platelet phospholipids inhibited the formation of cyclooxygenase but not lipoxygenase products from endogenous 20:4(n - 6) in thrombin-stimulated platelets. In doing so, 22:6(n - 3) appeared even more potent than 20:5(n - 3), although it was not liberated after acylation in phospholipids, the opposite of what was observed with 20:5(n - 3). Therefore, it seems that, in contrast to 20:5(n - 3), which may compete with endogenous 20:4(n - 6) at the cyclooxygenase level, 22:6(n - 3) would affect the latter enzyme activity in a different way. We conclude that 20:5(n - 3) and 22:6(n - 3) behave differently and might act synergistically on the inhibition of platelet functions after fish fat intake.

Arachidonic Acids↗
PubMed1988

Inhibition by lipoxygenase products of TXA2-like responses of platelets and vascular smooth muscle. 14-Hydroxy from 22:6n-3 is more potent than 12-HETE.

Lipoxygenase products, which are formed in great amounts in platelets during their activation, have been prepared from arachidonic acid (20:4n-6), the main polyunsaturated fatty acid (PUFA) esterified in platelet phospholipids, and from two major PUFAs of fish fat, eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acids. These compounds have been synthesized using platelet suspension as enzymic source, purified by high performance liquid chromatography, and their structure were checked by gas chromatography-mass spectrometry. Their effects were investigated in vitro upon human platelet aggregation induced by 11,9-epoxy-methano-analogue of PGH2 (U-46619) and upon thromboxane A2-induced vasoconstriction of rabbit aorta. All hydroxylated fatty acids inhibited U-46619-induced aggregation in a concentration-dependent fashion. Compounds issued from 22:6n-3 were the most potent inhibitors and their IC50 differed significantly from that of 12-hydroxy-eicosatetraenoic acid (12-HETE). Among them, 14-hydroxy-docosahexaenoic acid (14-OH-22:6) was the most effective anti-aggregating molecule (IC50:0.45 microM). 10 microM 12-HETE and 14-OH-22:6 inhibited 60% and 75% of smooth muscle contraction induced by TXA2-like material, respectively. At 1 microM, solely 14-OH-22:6 had an inhibitory effect on adrenaline-, angiotensine- or histamine-induced contraction. Since thromboxane receptors in platelets and vascular smooth muscle cells present strong similarities, it is concluded that hydroxylated fatty acids can antagonize prostanoid action probably by interfering with their receptor sites.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1988

Thermospray-mass spectrometric analysis of underivatized monohydroxy fatty acids: application to stimulated platelets.

Monohydroxylated fatty acids prepared from polyunsaturated fatty acids of nutritional value were analysed by thermospray-mass spectrometry without prior chemical derivatization. Positive and negative ionization modes were compared. The highest sensitivity was observed with the negative ionization mode with detection limits of 10 pmol based on the 12-hydroxy derivative of eicosatrienoic acid (12-OH-8,10,14-20:3). This is comparable to that obtained by high-performance liquid chromatography with UV detection at 234 nm. Selected ion monitoring based on the fragment [M-H]- allowed a variety of standard monohydroxy fatty acids to be detected. This approach makes possible the analysis of various derivatives generated by thrombin-stimulated platelets (10(9) cells) pre-enriched with minor polyunsaturated fatty acids, even when these derivatives co-elute from the column (e.g., 12-HETE and 14-OH-22:6).

Blood Platelets↗
PubMed1988

Platelets and aging. II--Plasma lipoproteins and fatty acid profiles.

Lipid composition of both plasma and platelets were investigated in sixteen old (78-94 years) and eight young (25-35 years) subjects. No age-related change was noted in plasma total and HDL cholesterol whereas a slightly increase of triglycerides was observed in the elderly population. Level of apo AI tended to decrease while apo AII decreased significantly in the elderly. These results led to a higher apo AI/apo AII ratio in elder subjects. However, no difference was detected in the level of apo B. In contrast, several modifications appeared in fatty acid composition of plasma lipids. Primarily, monounsaturated fatty acids content was increased while level of linoleic acid (and arachidonic acid in phospholipids) decreased in each class of plasma lipids of elderly subjects. In platelets from the elderly, we found an enhancement of monounsaturated fatty acids in phosphatidyl-inositol (PI), -ethanolamine (PE) and -choline (PC). The decrease of linoleic acid was detected in PC while the reduction of AA was noted in PE. These results indicate that modifications of fatty acid composition in both plasma and platelet lipids appeared with aging. They might be linked to the enhanced platelet activation in vivo observed in elderly people.

Adult↗
PubMed1988

Hydroperoxides produced by n-6 lipoxygenation of arachidonic and linoleic acids potentiate synthesis of prostacyclin related compounds.

In a previous paper we reported that arachidonic acid (20:4(n-6] strongly enhances the endothelial cell synthesis of prostaglandin I3 (PGI3) from eicosapentaenoic acid (20:5(n-3], in stimulating the cyclooxygenase rather than the prostacyclin synthase (Bordet et al. (1986) Biochem. Biophys. Res. Commun. 135, 403-410). In the present study, endothelial cell monolayers were co-incubated with exogenous 20:5(n-3) or docosatetraenoic acid (22:4(n-6], and n-6 lipoxygenase products of 20:4(n-6) or linoleic acid (18:2(n-6], namely 15-HPETE and 13-HPOD, respectively. Prostaglandins or dihomoprostaglandins were then measured by gas chromatography-mass spectrometry. Both hydroperoxides, up to 20 microM, stimulated the cyclooxygenation of 20:5(n-3) and 22:4(n-6), in particular the formation of PGI3 and dihomo-PGI2, respectively. Higher concentrations inhibited prostacyclin synthetase. In contrast, the reduced products of hydroperoxides, 15-HETE and 13-HOD, failed to stimulate these cyclooxygenations, 13-HPOD appeared more potent than 15-HPETE and the cyclooxygenation of 22:4(n-6) seemed to require higher amounts of hydroperoxides to be efficiently metabolized than 20:5(n-3). These data suggest that prostacyclin potential of endothelium might be enhanced by raising the peroxide tone.

Arachidonic Acid↗
PubMed1988

Platelets and aging. I--Aggregation, arachidonate metabolism and antioxidant status.

Platelet functions were investigated in sixteen old (78-94 years) and eight young (25-35 years) subjects. Whole blood platelet aggregation induced by collagen was higher in the elderly. Similarly, aggregation of platelet rich plasma and plasma-free platelets induced by various agents was increased but the collagen-induced release of ATP was reduced. In agreement with the enhanced platelet aggregability, the increase of thromboxane formation (under thrombin stimulation) was also noted in platelets from elderly people. To further assess platelet and vascular function in vivo, we measured the excretion of urinary TXB2, 2,3-dinor TXB2, 6-keto-PGF1 alpha and 2,3-dinor-6-keto-PGF1 alpha. The four metabolites were all increased in the elder population. In addition, a significant reduction of platelet vitamin E was observed in the elderly people, although the plasma content was normal. These results indicate numerous modifications of platelet behaviour with aging. They include the increased platelet susceptibility to aggregation, and the depletion of ATP granule content, which could reflect an activation in vivo in agreement with the enhanced urinary excretion of thromboxane and prostacyclin metabolites. We hypothesize that platelet hyperactivity associated with the enhanced oxygenated metabolism of arachidonic acid could be linked to vitamin E depletion. These changes may reveal a prethrombotic state in the elderly population.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1988

Liquid chromatography and gas chromatography/mass spectrometry of lipoxygenase and cyclooxygenase products from platelets and endothelial cells.

Liquid chromatography coupled with mass spectrometry using either positive or negative ionization was used for measuring various lipoxygenase products of polyunsaturated fatty acids. The negative ionization appeared as the most sensitive mode and allowed to detect pmol amounts of products from biological extracts. Gas chromatography/mass spectrometry with the negative chemical ionization mode was also used for measuring prostacyclin synthetase products, namely the stable metabolites of PGI2, PGI3 and dihomo PGI2. In this way, fmol amounts of metabolites could be measured in biological extracts.

Blood Platelets↗
PubMed1988

Vitamin E fails to alter the aggregation and the oxygenated metabolism of arachidonic acid in normal human platelets.

Using low doses of vitamin E, either in vitro or in vivo, we have succeeded in almost doubling plasma and platelet alpha-tocopherol in healthy humans. Despite such an enrichment, platelet aggregation induced by collagen and thromboxane A2 minetic U46619 was not much affected, although that induced by exogenous arachidonic acid was significantly decreased. Similarly, the oxygenation of exogenous arachidonic acid was not modified. When incubated with thrombin some variations in the formation of endogenous cyclooxygenase and lipoxygenase products could be observed, although rarely significantly. The tendency was a decrease after in vivo enrichment and an increase when enrichment occurred in vivo. Serum oxygenated metabolites of arachidonic acid as well as urinary metabolites of thromboxane and prostacyclin were also not affected after vitamin E supplementation. Since the lipoxygenation of eicosapentaenoic acid was very strongly peroxide-dependent, the effect of alpha-tocopherol enrichment was tested and the 12-hydroperoxide derivative of arachidonic acid was used as a physiological peroxide. No modification could be observed, confirming that vitamin E does not alter the specific peroxidation of polyunsaturated fatty acids in normal platelets. We conclude that vitamin E supplementation neither affects arachidonic acid-dependent aggregation nor the oxygenated metabolism of arachidonic acid in normal human platelets.

Adult↗
PubMed1988

Liberation and oxygenation of polyenoic acids in stimulated platelets.

Radiolabeled polyenoic acids were incorporated into human platelet lipids using albumin as vector. Platelets were then triggered with 0.1 or 1 U/ml thrombin, and 0.5 or 2 x 10(-6) M calcium ionophore A23187. Lipid extracts were analyzed for neutral lipids, free fatty acids, monohydroxylated acids, prostanoids and glycocerophospholipid subclasses. During platelet activation induced by thrombin or by ionophore, arachidonic and eicosapentaenoic acids were liberated from phospholipids in large amounts and were subsequently oxygenated via platelet oxygenases. Substantial amounts of lipoxygenase products and thromboxanes were produced from these acids. Liberation and oxygenation of linoleic, alpha-linolenic, and docosahexaenoic acids were much less pronounced. Polyenoic acid liberation from phospholipid subclasses also behaved quite differently. Apart from alpha-linolenic and adrenic acids, which were poorly liberated, all the others were freed from phosphatidylinositol. In addition, arachidonic, eicosapentaenoic, and 5, 8, 11-eicosatrienoic acids were liberated from phosphatidylcholine at high concentrations of agonists and partially reincorporated into phosphatidylethanolamine. Finally, linoleic acid was deacylated from phosphatidylinositol and phosphatidylserine and almost entirely reacylated into phosphatidylcholine, whereas docosahexaenoic acid was deacylated from phosphatidylcholine and phosphatidylinositol reacylated into phosphatidylethanolamine, respectively. It is concluded that these polyenoic acids, all for which modulate platelet functions, exhibit very different metabolisms. They may act via their oxygenated derivatives and/or at the membrane phospholipid level.

Biotransformation↗
PubMed1988

Fatty acid composition of HL-60 cells is modified upon proliferation arrest and differentiation.

The human leukemic cell line HL-60 undergoes differentiation to granulocytic-like cells in response to dimethyl sulfoxide (DMSO) or retinoic acid (RA). This differentiation is accompanied by an arrest in cell proliferation. Studies have implicated alterations in the phospholipid fatty acid (FA) composition as a result of HL-60 differentiation. However, changes in FA's are also known to occur during the arrest of cellular proliferation. Using a highly efficient capillary gas-liquid chromatography technique, the phospholipid FA composition of HL-60 and of DMSO-resistant and RA-resistant HL-60 subclones was determined in proliferating cells, in density-arrested cells, and in terminally differentiated cells. The same specific modifications in some of the FAs of the three cell lines were observed when proliferation was inhibited by cell density; 16:0 and 18:2n-6 were decreased and 22:6n-3 increased. Moreover, 16 and 18 dimetylacetals were both increased when proliferation was decreased, indicating modifications in plasmalogen contents. Granulocytic differentiation of HL-60 cells and of its subclones with DMSO and/or RA provoked modifications in phospholipid FAs different from that found in density-arrested, undifferentiated cells such as decreases in monoenoic FAs of 16 and 18 carbons as well as an increase in arachidonic acid, the major polyunsaturated FA. The biological significance of these changes upon arrest of proliferation and differentiation are discussed. These results indicate that, when arrest of proliferation accompanies differentiation, these two phenomena can be responsible for different changes and, whenever possible, they have to be considered separately in order to know which modifications are effectively due to differentiation itself.

Cell Differentiation↗
PubMed1987

Acute myocardial infarction: measurement of arachidonate end-products in whole blood as an index of platelet cyclo-oxygenase activity in vivo.

The endogenous arachidonic acid metabolism was investigated ex vivo, in separated serum from clotted whole blood, soon after the onset of acute myocardial infarction (3.3 +/- 0.7 hr). A group of eight consecutive male patients was selected, since no evidence was obtained of any associated disease known to increase platelet activity or any recent exposure to cyclo-oxygenase inhibitors. This group of patients compared to an age and sex matched control group showed a large decrease in the platelet cyclo-oxygenase end-products in whole blood: thromboxane B2 (TXB2), 12-hydroxy-5-cis, 8-cis, 10-trans-heptadecatrienoic acid (HHT) and 6-keto-PGF1 alpha (p less than .01). In addition, platelet lipoxygenase produced an increased amount of 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE) as measured by its reduced metabolite 12-HETE (p less than .05). Furthermore, the TXB2 plasma concentration was significantly elevated in patients (p less than .01), confirming the enhanced platelet reactivity during the early stages of acute myocardial infarction. These results point out that a decreased level of cyclo-oxygenase end-products and an increased level of lipoxygenase end-product in serum is consistent with a previous in vivo cyclo-oxygenase hyperactivity.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1986

In vitro incorporation and metabolism of icosapentaenoic and docosahexaenoic acids in human platelets--effect on aggregation.

Washed human platelets were pre-loaded with icosapentaenoic acid (EPA), docosahexaenoic acid (DHA) or EPA + DHA and tested for their aggregation response in comparison with control platelets. In fatty acid-rich platelets, an inhibition of the aggregation could be observed when induced by thrombin, collagen or U-46619. The strongest inhibition was observed with DHA-rich platelets and it was reduced when DHA was incorporated in the presence of EPA. Study of fatty acid distribution in cell lipids after loading showed that around 90% of EPA or DHA taken up was acylated into phospholipids and a very small amount (less than 2%) remained in their free and hydroxylated forms. DHA was more efficiently acylated into phosphatidylethanolamine (PE) than into phosphatidylinositol (PI) in contrast to what observed with EPA, and both acids were preferentially incorporated into phosphatidylcholine (PC). EPA inhibited total incorporation of DHA and increased its relative acylation into PE at the expense of PC. In contrast, DHA did not affect the acylation of EPA. Upon stimulation with thrombin, EPA was liberated from phospholipids and oxygenated (as judged by the formation of its monohydroxy derivative) whereas DHA was much less metabolized, although consistently transferred into PE. It is concluded that EPA and DHA might affect platelet aggregation via different mechanisms when pre-loaded in phospholipids. Whereas EPA is known to alter thromboxane A2 metabolism from endogenous arachidonic acid, by competing with it, DHA might act directly at the membrane level for inhibiting aggregation.

Biological Transport↗