Old and new targets for innovative antimalarial compounds: the different strategies of the Italian Malaria Network.

Clinical treatment-failures to affordable drugs encouraged new investigation for discovery and development of new prophylactic and therapeutic interventions against malaria. The Drug Discovery Cluster (DDcl) of the Italian Malaria Network gathers several highly integrated and complementary laboratories from different Italian Institutions to identify, synthesise, screen in vitro and in vivo new antimalarial molecules directed against the intraerythrocytic stage of P. falciparum parasites and/or with transmission blocking activity to select lead compounds for further development. Complementary research activities, both in vitro and in the clinics, aim at investigating the pathogenetic mechanisms of severe malaria anaemia and the different manifestations of the disease in malaria-HIV co-infected patients to identify new therapies and improve survival.

Urinary excretion of olive oil phenols and their metabolites in humans.

We have recently demonstrated, in humans, the bioavailability of hydroxytyrosol (3,4-dihydroxyphenylethanol; HT), one of the major antioxidant components of virgin olive oil. In particular, we reported that this compound is present in lipoproteins involved in atherosclerotic processes and is excreted in the urine mainly as glucuronide-conjugate. The aim of the present study was to elucidate, in humans, the metabolic fate of HT after ingestion of virgin olive oil. After administration of virgin olive oil, 24-hour urine collections of healthy volunteers were prepared for gas chromatography-mass spectrometry analyses in order to identify and quantify HT and its metabolites homovanillic alcohol (HVA1c) and homovanillic acid (HVA). The results indicate that this compound undergoes the action of catechol-O-methyl transferase (COMT), enzymes involved in the catecholamine catabolism, resulting in an enhanced excretion of HVA1c. We also found a significant increase of HVA, indicating an oxidation of the ethanolic residue of HT and/or of HVA1c in humans. The excretion of both metabolites significantly correlated with the dose of administered HT.

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 microg/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 (R(2) = 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.

Olive oils rich in natural catecholic phenols decrease isoprostane excretion in humans.

This study was undertaken to evaluate, in humans, the antioxidant activity of olive oil phenolics, namely hydroxytyrosol and oleuropein aglycone that share an orthodiphenolic (catecholic) structure. Human volunteers were administered olive oil samples containing increasing amounts of an olive oil phenolic extract that was characterized by gas-chromatography/mass spectrometry. The administration of phenol-rich oils was dose-dependently associated with a decreased urinary excretion of 8-iso-PGF(2alpha), a biomarker of oxidative stress. Also, a statistically significant negative correlation between homovanillyl alcohol (HValc, hydroxytyrosol's major metabolite, formed through the COMT system) and F(2)-isoprostanes excretion was found. Thus, the administration of oil samples with increasing, albeit low, concentrations of orthodiphenolic compounds, namely hydroxytyrosol and oleuropein aglycone, results in a dose-dependent reduction in the urinary excretion of 8-iso-PGF(2alpha). The statistically significant negative correlation between 8-iso-PGF(2alpha) and HValc urinary concentrations suggests that this metabolite better reflects the in vivo activities of hydroxytyrosol.

Evidence of postprandial absorption of olive oil phenols in humans.

BACKGROUND AND AIM: Olive oil phenols are potent antioxidants in vitro. If this were to be also demonstrated in vivo, it would help to explain the beneficial effects of this typical ingredient of the Mediterranean diet. This study was designed to determine the presence in lipoprotein fractions of two phenolic compounds peculiar to extra virgin olive oil, namely tyrosol and OH-tyrosol, and whether their absorption induces an antioxidant effect in vivo. METHODS AND RESULTS: Two trials were performed. In the first (Long-term), 14 healthy volunteers followed two diets, each for one month. The only difference between the diets was that the first supplied 50 g of extra virgin olive oil per day, where-as the second one supplied 50 g of refined olive oil with no simple phenols, as demonstrated by GC-MS analysis. There were no changes in LDL oxidizability and tyrosol and OH-tyrosol were not recovered in lipoproteins and plasma from fasting samples drawn at the end of each diet period. In the second study (Postprandial), eight healthy volunteers received an oral fat load consisting of 100 g of extra virgin olive oil. Blood was drawn at times 0', 30', 60', 120', 240', 360', and major plasma lipoprotein classes were separated. The concentration of tyrosol, OH-tyrosol and vitamin E was determined in lipoprotein fractions. Plasma antioxidant capacity was measured by a crocin-bleaching test and expressed as mM Trolox C equivalents. Tyrosol and OH-tyrosol were recovered in all lipoprotein fractions, except VLDL, with concentrations peaking between 60' and 120'. However, a very high variability in tyrosol and OH-tyrosol absorption was observed among subjects. Vitamin E content of LDL and HDL did not vary significantly throughout the study. Plasma antioxidant capacity increased significantly at time 120' (baseline 0.96 mM Trolox; 120' 1.19 mM Trolox; p = 0.02), and then returned almost to baseline values after 360' (1.1 mM Trolox). CONCLUSIONS: These findings suggest that phenolic compounds in olive oil are absorbed from the intestine, though not through a pathway dependent on chylomicron formation, and may exert a significant antioxidant effect in vivo, probably in the postprandial phase.

Rapid evaluation of phenolic component profile and analysis of oleuropein aglycon in olive oil by atmospheric pressure chemical ionization-mass spectrometry (APCI-MS).

Epidemiological studies have linked the Mediterranean diet with a low incidence of cardiovascular diseases. Olive oil, the major fat component of this diet, is characterized by antioxidant properties related to their content in catecholic components, particularly oleuropein aglycon. Therefore quantification of these components in edible oils may be important in determining the quality, and consequently its commercial value. The present method allows us to obtain the profile of the phenolic components of the oil from the methanolic extracts of the crude olive oil. In particular tyrosol, hydroxytyrosol, elenolic acid, deacetoxyligstroside and deacetoxyoleuropein aglycons, ligstroside and oleuropein aglycons, and 10-hydroxy-oleuropein are clearly identified by atmospheric pressure chemical ionization-mass spectrometry (APCI-MS). Moreover, oleuropein and its isomers present in the oil are quantified by APCI-MS/MS analysis of the extracts without preliminary separation from other phenolic compounds.

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.

Effect of virgin olive oil phenolic compounds on in vitro oxidation of human low density lipoproteins.

BACKGROUND AND AIM: Substantial evidence suggests that oxidative modifications of low density lipoproteins (LDL) critically contribute to the pathogenesis and progression of human atherosclerosis. Oxidized LDL (oxLDL) are present in atherosclerotic plaques and contain oxysterols that exhibit a variety of adverse biological activities. Antioxidants have also been shown to prevent LDL modification. We have therefore assessed the efficacy of virgin olive oil phenolic compounds in preventing oxidative modifications of human LDL oxidized by UV light. METHODS AND RESULTS: Cholesterol oxides formed during LDL photo-oxidation were determined by UV-HPLC in the presence of different concentrations of phenolic compounds and their pure components (tyrosol and oleuropein), and probucol, a widely used synthetic antioxidant. Electrophoretic mobility was also assayed. The results demonstrate that phenolic compounds are much more potent in preventing cholesterol oxide formation and apoproteic moiety modification than their pure components and probucol. CONCLUSIONS: The beneficial effects of a Mediterranean diet may be ascribable not only to the high unsaturated/saturated fatty acid ratio characteristic of olive oil, but also to the unique antioxidant properties of its phenolic compounds.

Antenatal rupture of a diverticular rectal duplication with neonatal perineal fistulization.

A cystic pelvic malformation was found in a fetus on antenatal sonography (US) at 26 weeks of gestational age that was no longer present 3 weeks later on control US. The male child presented at birth with a right-sided perineal mass that fistulized with meconial drainage. A radiopaque enema showed a low posterior rectal fistula filling a poorly delineated pouch. Surgery performed through a posterior sagittal approach allowed identification and closure of the fistula and pouch drainage. The diagnosis of a diverticular rectal duplication was considered, although no intestinal lining was observed macroscopically or histologically. The child's anorectal function was normal after a 20-month follow-up. Labeling of the malformation and embryological hypotheses are discussed since the case does not fulfill all the criteria of an intestinal duplication. Surgical techniques are discussed, with an emphasis on the sagittal posterior approach.

Extracts of Ginkgo biloba L. leaves and Vaccinium myrtillus L. fruits prevent photo induced oxidation of low density lipoprotein cholesterol.

Oxidation of low density lipoproteins (LDL) favours cholesterol loading in macrophages and formation of "foam cells", typical of the early atheroma lesions. LDL cholesterol oxidation generates oxysterols, extremely cytotoxic molecular species with diverse biological activities. Vegetable polyphenols are dietary components of pharmacological interest for their anti-oxidant properties. Ginkgo biloba L. (Gingkoaceae) leaves and Vaccinium myrtillus L. (Ericaceae) fruits are known for their beneficial effects in the treatment of various diseases involving free radicals and oxidative damage to biological lipids. In this study we investigated the effect of Ginkgo biloba L. and Vaccinium myrtillus L. extracts on the formation of cholesterol oxides during the photo induced oxidation of human LDL. The results demonstrate a concentration dependent inhibition of oxysterol formation in the presence of both extracts. Protection against oxidation was confirmed by the partial restoration of the normal electrophoretic mobility of LDL, which has been influenced by the UV irradiation. These effects extend knowledge of the therapeutic action of Ginkgo biloba L. and Vaccinium myrtillus L. as agents in anti-atherosclerotic regimens.

Time course and concentration dependence of the incorporation of deuterated/tritiated arachidonic acid and derived fatty acids in THP-1 cell lipids.

We have supplemented THP-1 cells, a human monocytic leukemia cell line, with arachidonic acid (AA), containing [3H8] AA, 1-25 microM, for up to 24 hours, and explored the time and concentration dependent patterns of incorporation in cell lipid classes and subclasses. Twenty-five microM AA consisted of deuterated AA ([2H8] AA), containing also [3H8] AA. Phospholipids (PL) were separated by HPLC with UV and radiodetection, and the fatty acids (FA) methyl esters were analyzed by GC. [2H8] AA pentafluorobenzyl-esters from individual lipid classes were obtained and analyzed by GC-MS. Incorporation of AA in cell lipids increased linearly with increasing concentrations, whereas 22:4 and 22:5 accumulated only at 25 microM AA. Up to 10 microM AA, more than 95% of the FA was incorporated in PL, whereas at 25 microM AA a significant proportion of the exogenous FA was incorporated in triglycerides (TG) and in diacyl phosphatidylcholine (PC). The time-course of AA incorporation showed that the peak was at 3 hours, with minimal incorporation in TG, in the presence of 5 microM, whereas the peak occurred at 6 hours, with about 50 percent incorporation in TG, with 25 microM. The data indicate that the range of AA concentrations and the time course of the incorporation of this FA in cell structural lipids are critical.

n-6 and n-3 fatty acid accumulation in thp-1 cell phospholipids.

The human monocytic leukemia cell line THP-1 is depleted of the long-chain n-6, AA, when compared to human monocytes. This reflects the low availability of this FA in the growth medium generally used for cultured cells. The effects of AA, as well as EPA, supplementation of THP-1 cells on the incorporation of these FA in cell PL, especially in PC and PE, was investigated. In addition the incorporation of labeled AA in PL from THP-1 cells was compared to that in human monocytes. Measurements were done through HPLC separation of PL, detected by UV absorption and radioactivity, FA analysis by GC and characterization of PC subclasses by FAB-MS. Marked differences were observed in the incorporation of the two FA in cell PL, particularly two PC subclasses, and in the accumulation in individual PL after supplementation of THP-1 cells. Accumulation of AA and EPA in THP-1 cells appeared to be mutually independent. The incorporation of AA was also quite different in THP-1 from that in monocytes. Thus, characterization of the FA content in lipids of cultured cells is an essential requirement for optimal utilization of these cells.

Radioisotopic study of the adrenal glands using 131I-19-iodocholesterol.

Scintigraphy of the adrenal gland with 131I-19-iodocholesterol has recently been added to radiological techniques in adrenal imaging and has been used successfully to demonstrate anatomical and functional disorders of the adrenals in a variety of clinical situations. A review of the authors' experience stresses the diagnostic value of this method. Radiological findings and results of scintillation imaging are complementary: their comparison improves and clarifes indications for scintigraphy. Hyperadrenal cortical diseases always gave satisfactory scintigrams, the most interesting results being obtained in adrenal cortical hyperplasia and unilateral hyperfunctioning adenomas. In these cases the evaluation of the response to stimulation or suppression tests was very useful. On the other hand scintigraphy was less valuable in demonstrating malignant and non malignant tumours.