Evelyne Véricel

The influence of low intake of n-3 fatty acids on platelets in elderly people

A total of ten healthy elderly subjects ingested one capsule of 600 mg (corresponding to 150 mg docosahexaenoic acid and 30 mg eicosapentaenoic acid) RO-PUFA triglycerides per day and ten others ingested one capsule of 600 mg sunflower oil as a placebo for 42 days. In the n-3 polyunsaturated fatty acids (PUFA) group, a significant decrease of systolic blood pressure was observed, as well as a trend towards a decrease in both platelet activation and basal formation of thromboxane B(2). Also, a slight but significant increase of docosahexaenoic acid was observed in the phosphatidylethanolamine fraction as well as a significant increase of vitamin E level after the n-3 PUFA intake. Moreover, the basal production of malondialdehyde significantly decreased. No modification was observed for all these parameters in the placebo group. We conclude that a small intake of n-3 PUFA decreased the oxidative stress in platelets of elderly people and could be beneficial in subjects with atherothrombotic tendencies by lowering the cell peroxide tone.

Age-related changes in arachidonic acid peroxidation and glutathione-peroxidase activity in human platelets

Lipid peroxidation, vitamin E level and glutathione-peroxidase activity were determined in platelets from elderly (greater than 68 years) and young (21-43 years) people. To further assess the platelet lipid peroxidation, the metabolism of endogenous arachidonic acid in unstimulated platelets as well as that of the exogenous one were measured in the two groups. The oxygenated metabolites of arachidonic acid were enhanced in the elder population under both conditions tested. In addition, the platelet malondialdehyde content, a marker of the overall lipid peroxidation, was also found significantly increased in platelets from aged subjects. On the other hand, the platelet vitamin E level and the glutathione-peroxidase activity were significantly depressed in the elder group compared to the young one. These results suggest that the increased platelet activation observed with age could be linked to the accumulation of lipoxygenase-dependent peroxides associated with the decreased antioxidative defence of the cells, especially glutathione-peroxidase activity.

Poxytrins, a class of oxygenated products from polyunsaturated fatty acids, potently inhibit blood platelet aggregation

Docosahexaenoic acid (DHA), an important component of marine lipids, exhibits antiinflammatory activity related to some of its oxygenated metabolites, such as neuroprotectin/protectin D1 [NPD1/PD1;10(R),17(S)‐dihydroxy‐docosa‐4Z,7Z, 11E,13E,15Z,19Zhexaenoic acid] produced through the 15‐lipoxygenase pathway. However, other metabolites from DHA can be produced through this pathway, and other polyunsaturated fatty acids (PUFAs) of nutritional value may be oxygenated as well. Their biological activities remain unknown. Isomers of protectin D1 were synthesized using soybean lipoxygenase and tested for their ability to inhibit human blood platelet aggregation. A geometric isomer called PDX, previously described with the 11E,13Z,15E geometry, instead of 11E,13E,15Z in PD1, inhibited platelet aggregation at submicromolar concentrations when induced by either collagen, arachi‐donic acid, or thromboxane. The inhibition occurred at the level of both the cyclooxygenase activity and throm‐boxane receptor site. Interestingly, all the metabolites tested exhibiting the E,Z,E‐conjugated triene were active, whereas E,E,Z trienes (as in PD1) or all‐írαn* (E,E,E) trienes were inactive. We conclude that PDX and other oxygenated products from PUFAs of nutritional interest, having the E,Z,E‐conjugated triene motif and collectively named poxytrins (PUFA oxygenated trienes), might have antithrombotic potential.—Chen, P., Vãricel, E., Lagarde, M., Guichardant, M. Poxytrins, a class of oxygenated products from polyunsaturated fatty acids, potently inhibit blood platelet aggregation. FASEB J. 25, 382–388 (2011). www.fasebj.org

Influence of very low dietary intake of marine oil on some functional aspects of immune cells in healthy elderly people

Ageing is a multifactorial process involving decreased antioxidant defences and immune functions. n-3 Polyunsaturated fatty acids have been associated with human health benefits, especially against inflammatory and autoimmune diseases. However, their immunomodulatory effects were usually observed with high dosages (>2 g/d) known to increase lipid peroxidation. In contrast, very low doses, that may prevent lipid peroxidation, might affect the immune system differently. To study the latter hypothesis further, we investigated whether the supplementation of healthy elderly people with very low doses of marine oil (MO), a docosahexaenoate (DHA)- and eicosapentaenoate (EPA)-rich triacylglycerol, was able to affect lymphocyte proliferation and biochemical markers known to be altered with age. In a randomized, double-blind design, twenty healthy elderly subjects were assigned to a placebo group (600 mg sunflower oil/d) or to a group consuming 600 mg MO/d providing 150 mg DHA + 30 mg (EPA) for 6 weeks. At day 42, the proliferative responses of lymphocytes to several mitogens were significantly (P<0.01) decreased in the MO group compared with control values. This was accompanied by a slight lowering of their cytosolic cyclic nucleotide phosphodiesterase (PDE) activity, a marked and significant (P<0.05) increase of their particulate PDE activity (+56-57 %) and a slight but significant (P<0.05) increase in cyclic nucleotide intracellular levels. At the same time, the glutathione peroxidase activity was markedly and significantly (P<0.01) depressed in the MO group. None of these modifications could be seen in the placebo group. Collectively, these results demonstrate that even very low doses of n-3 fatty acids are sufficient to affect the immune responses of elderly subjects.

Lower levels of lipid peroxidation in human platelets incubated with eicosapentaenoic acid

The modulatory effects of eicosapentaenoic acid (EPA) on platelet arachidonic acid (AA) metabolism were applied to an in vitro model of oxidant stress. Unstimulated normal human blood platelets were first treated with a thiol-oxidizing agent, azodicarboxylic acid bis(dimethylamide) (diamide) (1 microM), and then incubated with a low concentration of EPA (100 nM). Diamide treatment led to a lower alpha-tocopherol content compared to control. Formation of MDA, a marker of the overall lipid peroxidation, as well as formation of 12-hydroxyeicosatetraenoic acid (12-HETE), the 12-lipoxygenase end-product of AA, were both higher in diamide-treated platelets. Subsequent incubation of diamide-treated platelets with EPA counteracted the effects of oxidant stress induced pharmacologically by diamide. Interestingly, EPA prevented the alpha-tocopherol level from falling and the overall lipid peroxidation from increasing as it did during diamide treatment. In particular, incubation of diamide-treated platelets with EPA led to significantly lower amounts of 12-HETE. Conversely, preincubation of platelets with 100 nM EPA protected cells from oxidizing effects induced by diamide treatment, either on the level of lipid peroxides or on the antioxidant status. These results indicate that, in this particular model, EPA permitted platelets to have control levels of tocopherol, MDA and 12-HETE despite diamide treatment. Low concentrations of EPA might have prevented the increase of lipid hydroperoxides and especially the transient accumulation of 12-hydroperoxyeicosatetraenoic acid (12-HPETE).

Decrease in platelet reduced glutathione increases lipoxygenase activity and decreases vitamin E

Unstimulated normal human blood platelets were treated with azodicarboxylic acidbis(dimethylamide) (diamide), a thiol-oxidizing agent. Oxygenated arachidonic acid (AA) metabolites, malondialdehyde (MDA), and tocopherols were then quantified by high-performance liquid chromatography (HPLC). Diamide treatment partially decreased the amount of reduced glutathione (GSH) content and induced a subsequent decrease in peroxidase activity. However, formation of 12-hydroxy-eicosatetraenoic acid (12-HETE), the end-product of lipoxygenation of AA, increased. Formation of MDA, a marker of overall lipid peroxidation, was also enhanced. Furthermore, platelet α-tocopherol, but not γ-tocopherol, significantly decreased. These results indicate that enhanced “basal” lipoxygenase activity, as a marker of specific AA oxygenation, may be linked to decreased platelet antioxidant status.

Effects of small concentrations of eicosapentaenoic acid on platelets

Abstract. Increased platelet functions have been associated with enhanced arachidonic acid (AA) metabolism in diabetics and elderly people. We investigated these parameters in both groups after feeding low doses of pure eicosapentaenoic acid (EPA). After such an intake, platelet aggregation induced by various agonists was decreased whereas the oxygenated metabolism of endogenous AA was not substantially altered, and the fatty acid composition of plasma and platelet lipid pools did not change. In the most recent study, the measurement of vitamin E revealed that although its level in plasma was not modified, it was increased in platelets after EPA intake. It is concluded that small amounts of EPA intake do not reduce platelet aggregation in competing with AA metabolism but rather in depressing the peroxide tone of the cell.

Inhibitory effects of in vivo oxidized high-density lipoproteins on platelet aggregation: evidence from patients with abetalipoproteinemia

There is evidence that high‐density lipoproteins (HDLs) may regulate platelet function, but disparate results exist regarding the effects of oxidized HDLs on platelets. The objective of our study was to determine the role of in vivo oxidized HDLs on platelet aggregation. Platelet aggregation and redox status were investigated in 5 patients with abetalipoproteinemia (ABLP) or homozygous hypobetalipoproteinemia, two rare metabolic diseases characterized by the absence of apolipoprotein B‐containing lipoproteins, compared to 5 control subjects. Platelets isolated from plasma of patients with ABLP aggregated 4 to 10 times more than control platelets, depending on the agonist. By contrast, no differences in the extent of platelet aggregation were observed between ABLP platelet‐rich plasma (PRP) and control PRP, suggesting the presence of a protective factor in ABLP plasma. ABLP HDLs inhibited agonist‐induced platelet aggregation by binding to SR‐BI, while control HDLs had no effect. On the other hand, lipoprotein‐deficient plasma from patients with ABLP did not inhibit platelet aggregation. Severe oxidative stress was evidenced in patients with ABLP. Compared to control HDLs, ABLP HDLs showed a 40% decrease of α‐tocopherol and an 11‐fold increased malondialdehyde concentration. These results demonstrate that in vivo oxidized HDLs do not lose their antiaggregatory properties despite oxidation.—Calzada, C., Véricel, E., Colas, R., Guillot, N., El Khoury, G., Drai, J., Sassolas, A., Peretti, N., Ponsin, G., Lagarde, M., Moulin, P. Inhibitory effects of in vivo oxidized high‐density lipoproteins on platelet aggregation: evidence from patients with abetalipoproteinemia. FASEB J. 27, 2855–2861 (2013). www.fasebj.org

Variations in the fatty acid composition of lipid classes from lipoproteins in elderly women.

Fatty acid composition of lipid classes and NMR spectra of lipoproteins were compared in 6 young (24-35-year-old) and 6 elderly (79-90-year-old) women. Cholesteryl ester, triglyceride and protein content of LDL in elderly women were significantly higher (+52-57% and +20% for lipids and proteins, respectively) than those observed in young women. HDL lipid levels were similar in the two groups. The proportion of linoleic acid (mainly in cholesteryl esters and phospholipids) of each lipoprotein species was always lower in octogenarians when compared with young females (lowering of 13-28% and 27-46% for cholesteryl esters and phospholipids, respectively). Conversely, the proportions of mono-unsaturated fatty acids (mainly oleic acid) increased in all lipid classes, although this was only significant in cholesteryl esters from each lipoprotein species. NMR spectra of lipoproteins showed a restricted mobility of acyl chain terminal CH3 groups in old women which was significant only in VLDL and HDL3. This suggests that the decrease of linoleic acid could affect the lipid mobility in lipoproteins of elderly women.

Glycoxidized HDL, HDL Enriched With Oxidized Phospholipids and HDL From Diabetic Patients Inhibit Platelet Function

CONTEXTnHigh-density lipoproteins (HDL) possess atheroprotective properties including anti-thrombotic and antioxidant effects. Very few studies relate to the functional effects of oxidized HDL on platelets in type 2 diabetes (T2D).nnnOBJECTIVEnThe objective of our study was to investigate the effects of in vitro glycoxidized HDL and HDL from patients with T2D on platelet aggregation and arachidonic acid signaling cascade. At the same time, the contents of hydroxylated fatty acids were assessed in HDL.nnnRESULTSnCompared with control HDL, in vitro glycoxidized HDL had decreased proportions of linoleic (LA) and arachidonic (AA) acids in phospholipids and cholesteryl esters, and increased concentrations of hydroxy-octadecadienoic acids (9-HODE and 13-HODE) and 15-hydroxy-eicosatetraenoic acid (15-HETE), derived from LA and AA respectively, especially hydroxy derivatives esterified in phospholipids. Glycoxidized HDL dose-dependently decreased collagen-induced platelet aggregation by binding to scavenger receptor BI (SR-BI). Glycoxidized HDL prevented collagen-induced increased phosphorylation of platelet p38 MAPK and cytosolic phospholipase A2, as well as intracellular calcium mobilization. HDL enriched with oxidized phosphatidylcholine (PC), namely PC(16:0/13-HODE) dose-dependently inhibited platelet aggregation. Increased concentrations of 9-HODE, 13-HODE, and 15-HETE in phospholipids (2.1-, 2.1-, and 2.4-fold increase, respectively) were found in HDL from patients with T2D, and these HDL also inhibited platelet aggregation via SR-BI.nnnCONCLUSIONSnOur results suggest that in vitro glycoxidized HDL as well as HDL from patients with T2D inhibit platelet aggregation, and suggest that oxidized LA-containing phospholipids may contribute to the anti-aggregatory effects of glycoxidized HDL and HDL from patients with T2D.