M. Blasevich

Inhibition of platelet aggregation and eicosanoid production by phenolic components of olive oil

This study was designed to investigate the in vitro effects of phenolic compounds extracted from olive oil and from olive derived fractions. More specifically, we investigated the effects on platelets of 2-(3,4-di-hydroxyphenyl)-ethanol (DHPE), a phenol component of extra-virgin olive oil with potent antioxidant properties. The following variables were studied: aggregation of platelet rich plasma (PRP) induced by ADP or collagen, and thromboxane B2 production by collagen or thrombin-stimulated PRP. In addition, thromboxane B2 and 12-hydroxyeicosatetraenoic acid (12-HETE) produced during blood clotting were measured in serum. Preincubation of PRP with DHPE for at least 10 min resulted in maximal inhibition of the various measured variables. The IC50s (concentration resulting in 50% inhibition) of DHPE for ADP or collagen-induced PRP aggregations were 23 and 67 microM, respectively. At 400 microM DHPE, a concentration which completely inhibited collagen-induced PRP aggregation, TxB2 production by collagen- or thrombin-stimulated PRP was inhibited by over 80 percent. At the same DHPE concentration, the accumulation of TxB2 and 12-HETE in serum was reduced by over 90 and 50 percent, respectively. We also tested the effects of PRP aggregation of oleuropein, another typical olive oil phenol, and of selected flavnoids (luteolin, apigenin, quercetin) and found them to be much less active. On the other hand a partially characterized phenol-enriched extract obtained from aqueous waste from olive oil showed rather potent activities. Our results are the first evidence that components of the phenolic fraction of olive oil can inhibit platelet function and eicosanoid formation in vitro, and that other, partially characterized, olive derivatives share these biological activities.

Conjugated linoleic acids (CLA) as precursors of a distinct family of PUFA

One of the possibilities for distinct actions of c9,t11- and the t10,c12-conjugated linoleic acid (CLA) isomers may be at the level of metabolism since the conjugated diene structure gives to CLA isomers and their metabolites a distinct pattern of incorporation into the lipid fraction and metabolism. In fact, CLA appears to undergo similar transformations as linoleic acid but with subtle isomer differences, which may account for their activity in lowering linoleic acid metabolites in those tissues rich in neutral lipids where CLA is preferentially incorporated. Furthermore, c9,t11 and t10,c12 isomers are metabolized at a different rate in the peroxisomes, where the shortened metabolite from t10,c12 is formed at a much higher proportion than the metabolite from c9,t11. This may account for the lower accumulation of t10,c12 isomer into cell lipids. CLA isomers may therefore be viewed as a “new” family of polyunsaturated fatty acids (PUFA) producing a distinct range of metabolites using the same enzymatic system as the other (i.e., n−3, n−6 and n−9) PUFA families. It is likely that perturbation of PUFA metabolism by CLA will have an impact on eicosanoid formation and metabolism, closely linked to the biological activities attributed to CLA.

INHIBITION OF LEUKOCYTE LEUKOTRIENE B4 PRODUCTION BY AN OLIVE OIL-DERIVED PHENOL IDENTIFIED BY MASS-SPECTROMETRY

We have evaluated the effects of hydroxytyrosol (HT), a potent antioxidant present in olive oil, on the formation of arachidonic acid 5-lipoxygenase metabolites by leukocytes in vitro. HT, a simple phenolic compound, extracted from first-pressure oil, was isolated by HPLC and characterized by gas chromatography/mass spectrometry. HT inhibited in a dose-related manner the production of leukotriene B4 (LTB4) by calcium ionophore-stimulated leukocytes. As expected, similar inhibition was observed for omega-oxidized metabolites of LTB4, namely 20-hydroxy and 20-carboxy-LTB4. The results disclose a new biological activity of olive oil-derived phenols on leukocyte eicosanoid production.

Differential effects of probenecid on the levels of endogenous PGF2α and TXB2 in brain cortex

Abstract Probenecid in single or repeated doses does not modify levels of PGF 2α and TXB 2 in rat brain cortex. After administration of subconvulsant dose of pentamethylene tetrazone (PMT) PGF 2α increases sharply and rapidly declines subsequently, whereas the elevation of TXB 2 is smaller but of longer duration. After probenecid pretreatment PGF 2α levels do not decline up to 30 minutes after the initial peak and are still elevated after 60 minutes. Levels of TXB 2 tend to be reduced after pretreatment. Differences in transport process or in biosynthetic compartments for these arachidonic acid (AA) metabolites may account for the observed data.

Inhibition by n-3 Fatty Acids of Arachidonic Acid Metabolism in a Primary Culture of Astroglial Cells

Docosahexaenoic acid (22∶6 n-3) was present in low concentrations in a primary culture of rat brain astroglial cells, when compared to brain cortex. We have thus supplemented these cells with this fatty acid and investigated the effects of its incorporation in cell phospholipids on the conversion of arachidonic acid, 20∶4 n-6, through the cyclo and lipoxygenase pathways, after cell stimulation. Docosahexaenoic acid-enriched cells produced less thromboxane B2 and 6-keto-Prostaglandin F1α and markedly less 12-hydroxyeicosatetraenoic acid than unsupplemented cells, after stimulation with the Ca2+-ionophore A23187. The production of 15-hydroxyeicosatetraenoic acid from arachidonic acid was slightly increased in docosahexaenoic acid-supplemented cells. We have also supplemented these cells with eicosapentaenoic acid (20∶5 n-3) and, in addition to accumulation of this fatty acid in cell phospholipids, we found elevation of 22∶5 n-3 and some increment of 22∶6, confirming that glial cells are able to convert eicosapentaenoic acid to the long chain, more unsaturated derivatives. In conclusion, n-3 fatty acids, when supplemented to glial cells, appear to modulate the arachidonic acid cascade and to be converted through the elongation and desaturation pathways.

Arachidonic acid cycloxygenase and lipoxygenase pathways are differently activated by platelet activating factor and the calcium-ionophore A23187 in a primary culture of astroglial cells

The aim of our study has been to investigate the metabolism of endogenous arachidonic acid or that of radiolabeled arachidonate in astroglial cells, stimulated with platelet activating factor (PAF) and with the calcium-ionphore A23187. Primary cultures of astroglial cells were obtained from brain cortex of one-day-old rats and were characterized by immunofluorescent staining vs glial fibrillary acidic protein. In labeled cells, diacylglycerol was formed after stimulation with platelet activating factor, whereas mainly the release of labeled arachidonic acid from phospholipids was observed after stimulation with calcium-ionophore. Both PAF and the calcium-ionophore A23187 actively stimulated the formation of the cycloxygenase products PGD2, TXB2 and 6-keto-PGF1 alpha, measured by radio- or enzyme-immunoassay. Differences were observed, instead, in the formation of the lipoxygenase metabolites, the hydroxyeicosateraenoic acids, which were measured by high pressure liquid chromatography (HPLC) with on line radiodetection for the labeled products, and Leukotriene C4, measured by radioimmunoassay. The formation of hydroxyacids by stimulated cells was confirmed by gas chromatography-mass spectrometry (GC-MS). In labeled cells, both agonists induced the formation of 12- and 15-hydroxyeicosatetraenoic acids, whereas stimulation of unlabeled cells with calcium ionophore resulted in formation of 12-hydroxyeicosatetraenoic acid and Leukotriene C4. Our results suggest that in astroglial cells, PAF, a compound which is produced in several tissues including brain, mobilizes a selected arachidonic acid pool, possibly associated with diacylglycerol production, from phospholipids, thus activating the conversion of the released fatty acid via the cyclo and the 12-lipoxygenase pathways.

Changes in arachidonic acid levels and formation and in lipid synthesis in the human neuroblastoma SK-N-BE during retinoic acid-induced differentiation.

Abstract: We observed that retinoic acid, which differentiates the human neuroblastoma SK‐N‐BE into mature neurons, induced an elevation in levels of polyunsaturated fatty acids, especially arachidonic acid (20:4 n‐6). This effect was not induced by phorbol myristate acetate, another differentiating agent. We then explored the effects of retinoic acid on the formation of arachidonic acid and of docosahexaenoic acid from precursors and on the de novo lipid synthesis from acetate at various stages of differentiation, which was assessed by morphological (cell number and neurite outgrowth) and biochemical (protein content and thymidine incorporation) criteria. At 3 days of incubation with retinoic acid, in the n‐6 series, total conversion of linoleic acid, especially to 20:3 n‐6, was elevated, in association with preferential incorporation of acetate into phospholipids; in contrast, at 8 days, synthesis of 20‐carbon polyunsaturated fatty acids declined, in association with enhanced incorporation in triglycerides. In the n‐3 series, eicosapentaenoic acid was converted to docosahexaenoic acid in SK‐N‐BE, but the conversion was not affected by retinoic acid. During the early stage of neuronal differentiation, therefore, enhanced production of 20‐carbon polyunsaturated fatty acids from their precursors occurred, and newly formed fatty acids were preferentially incorporated in phospholipids, possibly in association with membrane deposition. When differentiation was completed, arachidonic acid formation and incorporation of acetate in phospholipids and cholesterol declined with enhanced labeling of storage lipids.

The β-oxidation of arachidonic acid and the synthesis of docosahexaenoic acid are selectively and consistently altered in skin fibroblasts from three Zellweger patients versus X-adrenoleukodystrophy, Alzheimer and control subjects

Abstract The β -oxidation of [ 3 H] arachidonic acid (AA; 20:4 n-6) and the conversion of [1– 14 C]eicosapentaenoic acid (EPA, 20:5 n-3) to docosahexaenoic acid (DHA, 22:6 n-3) have been studied in skin fibroblasts from patients with inherited peroxisomal diseases, such as Zellweger (ZW) and X-linked adrenoleukodystrophy (X-ALD), from patients with Alzheimers disease (AD), a non-inherited neuropathology, and from controls. EPA is not converted to DHA, while there is enhanced formation of the intermediate product 22:5 n-3 in ZW, when compared to X-ALD, AD and controls. We also confirmed that AA is not β -oxidized to 4,7,10-hexadecatrienoic acid (16:3), a metabolite produced by peroxisomes, while being more effectively converted to the elongation product 22:4, in ZW, in comparison to X-ALD, AD and controls. The data demonstrate a defect in DHA synthesis and in AA β -oxidation, and the occurrence of associated adaptative modifications in the metabolism of these long chain PUFA, in three Italian ZW patients.

Effects of hypoxia and recovery on brain eicosanoids and carbohydrate metabolites in rat brain cortex.

The effects of hypoxia (respiration of 5% O2 for 30 min) and recovery (respiration of air up to 30 min) on brain levels of carbohydrate metabolites, of free arachidonate (FAA) and of several eicosanoids (E) were studied in the rat. Animals were sacrificed before or after 30 min of hypoxia, and during recovery, by microwave radiation (MW). At the end of the hypoxic period, arterial pO2 was reduced to 28 mm Hg and glycemia was elevated. Brain lactate and glucose levels were also elevated, whereas glycogen was unchanged. Levels of free FAA and of E were practically unchanged. During recovery, arterial pO2 values were raised above prehypoxic levels at 5 min and returned to normal within 30 min. The elevated serum glucose declined at 5 min and values returned to normal at 30. Brain glucose was still elevated at 5 min and returned to normal, together with lactate, at 30 min. Brain FAA did not change during recovery, but levels of prostaglandin F2 alpha (PGF2 alpha), prostaglandin E2 (PGF2) and thromboxane B2 (TxB2) were raised, at 5 min, and those of 6-keto-PGF1 alpha were reduced with respect to pre-hypoxia. At the end of recovery, all E were lower than during hypoxia. The results indicate that changes of brain E occur only during recovery from hypoxia. At 5 min of recovery, at high arterial pO2 values, concentrations of all E were elevated, with the exception of 6-keto-PGF1 alpha which was reduced. At 30 min a marked reduction of E levels was observed, possibly resulting from increased clearance following elevation of cerebral blood flow.

Eicosanoid and inositol phosphate response to platelet-activating factor (PAF) and to a PAF antagonist in rat astroglial cells

The effects of different concentrations of exogenous platelet-activating factor (PAF) on the formation of arachidonic acid-cyclooxygenase metabolites and on the production of inositol phosphates have been investigated in a primary culture of rat astroglial cells. The cells were used at confluence and the purity was checked by immunostaining of the culture with specific antibodies against glial fibrillary acidic protein. Incubation of the cells with PAF (range 10(-9) to 10(-6) M) resulted in maximal accumulation of total inositol phosphate (620 +/- 60% increment over basal values, P < 0.001) at the concentration of 10(-8) M, after 1 min of stimulation. Smaller inositol phosphate accumulation occurred at higher concentrations of the agonist and at longer stimulation time. After 1 min of stimulation with PAF, the accumulation of the cyclooxygenase metabolites, thromboxane B2 (630 +/- 58 vs 20 +/- 2 pg/mg protein in non-stimulated samples) and 6-keto-prostaglandin F1 alpha (132 +/- 15 vs 55 +/- 7 pg/mg protein in non-stimulated samples) was also maximal at 10(-8) M concentration of the agonist. When the cultures were stimulated with PAF or Ca(2+)-ionophore after preincubation with equimolar concentration of the PAF inhibitor BN 52021, a significant inhibition in the synthesis of both inositol phosphates and cyclooxygenase metabolites occurred only in the PAF-stimulated cells.