G. Galli

Olive oil phenolics are dose‐dependently absorbed in humans

Olive oil phenolic constituents have been shown, in vitro, to be endowed with potent biological activities including, but not limited to, an antioxidant action. To date, there is no information on the absorption and disposition of such compounds in humans. We report that olive oil phenolics, namely tyrosol and hydroxytyrosol, are dose‐dependently absorbed in humans after ingestion and that they are excreted in the urine as glucuronide conjugates. Furthermore, an increase in the dose of phenolics administered increased the proportion of conjugation with glucuronide.

Coordinated Control of Cholesterol Catabolism to Bile Acids and of Gluconeogenesis via a Novel Mechanism of Transcription Regulation Linked to the Fasted-to-fed Cycle

Bile acid metabolism plays an essential role in cholesterol homeostasis and is critical for the initiation of atherosclerotic disease. However, despite the recent advances, the molecular mechanisms whereby bile acids regulate gene transcription and cholesterol homeostasis in mammals still need further investigations. Here, we show that bile acids suppress transcription of the gene (CYP7A1) encoding cholesterol 7α-hydroxylase, the rate-limiting enzyme in bile acid biosynthesis, also through an unusual mechanism not involving the bile acid nuclear receptor, farnesoid X receptor. By performing cell-based reporter assays, protein/protein interaction, and chromatin immunoprecipitation assays, we demonstrate that bile acids impair the recruitment of peroxisome proliferator-activated receptor-γ coactivator-1α and cAMP response element-binding protein-binding protein by hepatocyte nuclear factor-4α, a master regulator of CYP7A1. We also show for the first time that bile acids inhibit transcription of the gene (PEPCK) encoding phosphoenolpyruvate carboxykinase, the rate-limiting enzyme in gluconeogenesis, through the same farnesoid X receptor-independent mechanism. Chromatin immunoprecipitation assay revealed that bile acid-induced dissociation of coactivators from hepatocyte nuclear factor-4α decreased the recruitment of RNA polymerase II to the core promoter and downstream in the 3′-untranslated regions of these two genes, reflecting the reduction of gene transcription. Finally, we found that Cyp7a1 expression was stimulated in fasted mice in parallel to Pepck, whereas the same genes were repressed by bile acids. Collectively, these results reveal a novel regulatory mechanism that controls gene transcription in response to extracellular stimuli and argue that the transcription regulation by bile acids of genes central to cholesterol and glucose metabolism should be viewed dynamically in the context of the fasted-to-fed cycle.

Hydroxytyrosol, as a component of olive mill waste water, is dose- dependently absorbed and increases the antioxidant capacity of rat plasma

Hydroxytyrosol is the most potent phenolic antioxidant of olive oil and olive mill waste water (OMWW) and its biological activities have stimulated research on its potential role in cardiovascular protection. However, evidence of the absorption of OMWW phenolics and on their possible in vivo activity has, until now, never been provided. Three groups male Sprague-Dawley rats were administered 1, 5, or 10 mg/Kg of the OMWW extract, respectively, providing 41.4, 207, and 414 μg/Kg of hydroxytyrosol, respectively. Urine was collected for 24 h and the urinary levels of hydroxytyrosol were quantified by mass spectrometry. Hydroxytyrosol was dose-dependently (R2=0.95) absorbed and excreted in the urines mostly as a glucuronide conjugate. Further, the administration of an hydroxytyrosol-rich OMWW extract (10 mg/kg) to the rats was also associated with an increase of their plasma antioxidant capacity. Future experiments will eventually further clarify its metabolic fate and its in vivo actions.

Correlation between release of free arachidonic acid and prostaglandin formation in brain cortex and cerebellum.

Levels of free arachidonic acid and of prostaglandin F2alpha and E2 have been measured in both brain cortex and cerebellum of rats killed by focussed microwave irradiation, and after decapitation followed by ischemia. The same parameters were studied during incubation assays. It was found that: a) after ischemia levels of both free arachidonic acid and of prostaglandins in cerebellum are lower than in brain cortex, b) formation of prostaglandins from endogenous precursor in incubated cortex is higher than in cerebellum, c) release of free arachidonic acid occurs mainly during the time interval between the sacrifice of the animals and the beginning of the incubation, whereas prostaglandins are formed mainly during the incubation assay. The correlation between release of free arachidonic acid and prostaglandin formation is discussed.

Mass spectrometric investigations of some unsaturated sterols biosynthetically related to cholesterol

Abstract The mass spectra of the acetate derivatives of several C-29, C-28 and C-27 unsaturated sterols which are pr o bable precursor in the biosynthetic pathway of cholesterol are investigated. The identification of characteristic peaks indicating the position of the double bonds in the side chain or in the nucleus is described.

Biological activities and metabolic fate of olive oil phenols

We have been studying the biological activities of olive oil phenols in vitro and in vivo including the bioavailability of phenols and their effects in animals and humans. In vitro, ortho-diphenolic (catecholic) compounds such as oleuropein and hydroxytyrosol exert potent antioxidant activities, such as inhibition of low density lipoproteins oxidation and free radical scavenging. They modify pathophysiological processes at cellular level favorably, e.g. by inhibiting not only the production of superoxide anions and the respiratory burst of neutrophils, but also platelet aggregation and the production of thromboxane and leukotriene B 4 by neutrophils. Finally, oleuropein stimulates the release of nitric oxide by macrophages. Most important, recent data demonstrate that oleuropein and hydroxytyrosol, active at concentrations in the low μM range, are dose-dependently absorbed by humans and rats and, at very low doses, maintain their antioxidant activities in vivo. This is indicated by the increase in plasma antioxidant capacity and the reduced excretion of isoprostanes in humans and in sidestream smoke-exposed rats. To this end, our studies demonstrated that high quality, phenol-rich olive oil provides bioactive compounds that exert salubrious effects in humans and thus may contribute to the beneficial properties of the Mediterranean diet.

PGF2α, thromboxane B2 and hete levels in gerbil brain cortex after ligation of common carotid arteries and decapitation

The effects of ligation of both common carotid arteries in the gerbil on the levels of PGF2 alpha, TXB2, HETE and of energy metabolites in brain cortex, have been investigated. Also, in the same experimental conditions the changes of cyclic AMP in brain cortex, cerebellum, striatum and hippocampus have been monitored. ATP, glycogen, glucose and phosphocreatine decrease whereas, lactate and cyclic AMP are enhanced in the ischemic brain, as previously reported. In contrast, levels of arachidonic acid metabolites are not modified. During ischemia following decapitation, instead, PGF2 alpha, and TXB2, show considerable increase.

Platelet formation of 12-hydroxyeicosatetraenoic acid and thromboxane B2 is increased in type IIA hypercholesterolemic subjects

The formation of the major metabolic products of endogenous arachidonic acid (AA) via cyclooxygenase and lipoxygenase pathways in platelets from normal and type IIA hypercholesterolemic subjects was evaluated. 12-Hydroxyeicosatetraenoic acid (12-HETE) and thromboxane B2(TXB2) were determined by selected ion monitoring (SIM) after extraction and purification of collagen stimulated platelet-rich plasma (PRP). The levels of both arachidonic acid metabolites in the non-stimulated PRP of control and type IIA subjects were below the detection limit of the method, rising significantly after collagen stimulation. Both 12-HETE and TXB2 levels in collagen-stimulated PRP samples from the patients were significantly higher than levels in controls (P less than 0.001). In view of the key role of 12-HETE in mediating smooth muscle cell migration and proliferation and in stimulating macrophage activity, these data may provide information for the understanding of the elevated incidence of thrombosis and atheromatous lesion in patients with type IIA hypercholesterolemia.

Evidence for the presence of 7-hydroperoxycholest-5-en-3β-ol in oxidized human LDL

Low density lipoprotein (LDL) cholesterol is known to be oxidized both in vitro and in vivo giving rise to oxygenated sterols. Conflicting results, however, have been reported concerning both the nature and the relative concentrations of these compounds in oxidized human LDL. We examined the extracts obtained from Cu(2+)-oxidized LDL. Thin layer chromatography analysis showed that the sterol mixture became more complex with reaction time. Analysis of the components by thin layer chromatography and mass spectrometry allowed to establish that 7 alpha- and 7 beta-hydroperoxycholest-5-en-3 beta-ol (7 alpha OOH and beta OOH) are largely prevalent among the oxysterols at early times of oxidation. These hydroperoxy derivatives have not been previously identified in oxidized LDL. The concentration of 7-hydroperoxycholest-5-en-3 beta-ol decreased with oxidation time with a concomitant increase of cholest-5-en-3 beta, 7 alpha-diol (7 alpha OH), cholest-5-en-3 beta, 7 beta-diol (7 beta OH), cholesta-3,5-dien-7-one (CD) and cholest-5-en-3 beta-ol-7-one (7CO). After 24 h of oxidation a minor component of the LDL sterols was cholestan-3 beta-ol-5,6-oxide (EP).

A rapid gas chromatographic-mass spectrometric method for prostaglandin analysis at picomole levels

Abstract A rapid method for the qualitative and quantitative determinations of PGE 1 , E 2 , F 1α , and F 2α is described. Tris-TMS-PGF and mono-TMS-PGB methyl esters are obtained by the reaction of the corresponding PGF and PGE methyl esters with N -trimethylsilylimidazole (TSIM) and piperidine. The reaction is instantaneous, and a single derivative with excellent gas chromatographic properties is obtained for each prostaglandin tested. The presence of very prominent peaks in the mass spectra of the derivatives allows the determination of prostaglandins at picomole levels using multiple ion detection (MID).