Pharmacokinetics and metabolism of hydroxytyrosol, a natural antioxidant from olive oil.

3,4-Dihydroxyphenylethanol (DOPET) is the major o-diphenol detectable in extra virgin olive oil, either in free or esterified form. Despite its relevant biological effects, mainly related to its antioxidant properties, little data have been reported so far on its toxicity and metabolism. The aim of the present work is to evaluate DOPET toxicity and to investigate its molecular pharmacokinetics by using the (14)C-labeled diphenol. When orally administered to rats, the molecule does not show appreciable toxicity up to 2 g/kg b.wt. To identify and quantify its metabolites, [(14)C]DOPET has been synthesized and intravenously injected in rats. The pharmacokinetic analysis indicates a fast and extensive uptake of the molecule by the organs and tissues investigated, with a preferential renal uptake. Moreover, 90% of the administered radioactivity is excreted in urine collected up to 5 h after injection, and about 5% is detectable in feces and gastrointestinal content. The characterization of the labeled metabolites, extracted from the organs and urine, has been performed by high-pressure liquid chromatography analysis. In all the investigated tissues, DOPET is enzymatically converted in four oxidized and/or methylated derivatives. Moreover, a significant fraction of total radioactivity is associated with the sulfo-conjugated forms, which also represent the major urinary excretion products. On the basis of the reported results, an intracellular metabolic pathway of exogenously administered DOPET, implying the involvement of catechol-O-methyltransferase, alcohol dehydrogenase, aldehyde dehydrogenase, and phenolsulfotransferase, has been proposed.

Hydroxytyrosol, a natural molecule occurring in olive oil, induces cytochrome c-dependent apoptosis.

2-(3,4-Dihydroxyphenyl)ethanol (DPE), a naturally occurring phenolic antioxidant molecule found in olive oil, has been reported to exert several biological and pharmacological activities. We studied the effect of this compound on the proliferation and survival of HL60 cell line. Concentrations from 50 to 100 microM DPE, comparable to its olive oil content, caused a complete arrest of HL60 cell proliferation and the induction of apoptosis. This was demonstrated by flow cytometric analyses, poly(ADP-ribose) polymerase cleavage, and caspase 3 activation. The apoptotic effect requires the presence of two ortho-hydroxyl groups on the phenyl ring, since tyrosol, 2-(4-hydroxyphenyl)ethanol, did not induce either cell growth arrest or apoptosis. DPE-dependent apoptosis is associated with an early release of cytochrome c from mitochondria which precedes caspase 8 activation, thus ruling out the engagement of cell death receptors in the apoptotic process. 2-(3,4-Dihydroxyphenyl)ethanol induced cell death in quiescent and differentiated HL60 cells, as well as in resting and activated peripheral blood lymphocytes, while did not cause cell death in two colorectal cell lines (HT-29 and CaCo2). These results suggest that DPE down-regulates the immunological response, thus explaining the well-known antinflammatory and chemopreventive effects of olive oil at the intestinal level.

Transport mechanism and metabolism of olive oil hydroxytyrosol in Caco-2 cells.

3,4-dihydroxyphenylethanol (hydroxytyrosol; DPE) is the major phenolic antioxidant present in extra virgin olive oil, either in a free or esterified form. Despite its relevant biological effects, no data are available on its bioavailability and metabolism. The aim of the present study is to examine the molecular mechanism of DPE intestinal transport, using differentiated Caco-2 cell monolayers as the model system. The kinetic data demonstrate that [(14)C]DPE transport occurs via a passive diffusion mechanism and is bidirectional; the calculated apparent permeability coefficient indicates that the molecule is quantitatively absorbed at the intestinal level. The only labelled DPE metabolite detectable in the culture medium by HPLC (10% conversion) is 3-hydroxy-4-methoxyphenylethanol, the product of catechol-O-methyltransferase; when DPE is assayed in vitro with the purified enzyme a K(m) value of 40 microM has been calculated.

Homocysteine, a new crucial element in the pathogenesis of uremic cardiovascular complications.

Most large observational studies available today establish that moderate hyperhomocysteinemia, either genetically or nutritionally determined, is an independent risk factor for myocardial infarction, stroke, and thromboembolic disease. This is also true for chronic renal failure patients, who exhibit a high prevalence of hyperhomocysteinemia (85-100%), which reaches high plasma concentrations (20-40 microM, while control values range between 8 and 12 microM). After a renal transplant, homocysteine levels decrease, but tend to be higher than normal. The cause of hyperhomocysteinemia in renal failure is still obscure, since recent data have questioned the previous notion that a net homocysteine renal extraction and/or excretion take place in man. No matter the cause of its increase, the sulfur amino acid homocysteine is thought to induce an increment in cardiovascular risk through three basic biochemical mechanisms: (1) homocysteine oxidation, with H2O2 generation; (2) hypomethylation through S-adenosylhomocysteine accumulation, and (3) protein acylation by homocysteine thiolactone. The final result is membrane protein damage, endothelial damage, and endothelial cell growth inhibition, among other effects. Hyperhomocysteinemia, in general, is susceptible of therapeutic intervention with the vitamins involved in its metabolism. Depending on the cause, vitamin B6, vitamin B12, betaine, and/or folic acid can be effectively utilized. Chronic renal failure patients benefit from folic acid in high dosage: 1-2 mg are usually not effective ('relative folate resistance'), while 5-15 mg reduce homocysteine levels to a 'normative' range (<15 microM) in a substantial group of patients. Good results are also obtained in transplant patients, best with a combination of folic and vitamin B6. The results of the interventional trials focusing on the possible reduction in cardiovascular risk after homocysteine-lowering therapy, both in the general population and in end-stage renal disease, are expected soon, as well as the genetic and biochemical studies in suitable models, with the aim to clarify the cause-effect link suggested by the numerous observational and basic science studies.

The protective effect of the olive oil polyphenol (3,4-dihydroxyphenyl)-ethanol counteracts reactive oxygen metabolite-induced cytotoxicity in Caco-2 cells.

We investigated the injurious effects of reactive oxygen metabolites on the intestinal epithelium and the possible protective role played by two olive oil phenolic compounds, (3,4-dihydroxyphenyl)ethanol and (p-hydroxyphenyl)ethanol, using the Caco-2 human cell line. We induced oxidative stress in the apical compartment, either by the addition of 10 mmol/L H2O2 or by the action of 10 U/L xanthine oxidase in the presence of xanthine (250 micromol/L); after the incubation, we evaluated the cellular and molecular alterations. Both treatments produced significant decreases in Caco-2 viability as assessed by the neutral red assay. Furthermore, we observed a significant increase in malondialdehyde intracellular concentration and paracellular inulin transport, indicating the occurrence of lipid peroxidation and monolayer permeability changes, respectively. The H2O2-induced alterations were completely prevented by preincubating Caco-2 cells with (3,4-dihydroxyphenyl)ethanol (250 micromol/L); when the oxidative stress was induced by xanthine oxidase, complete protection was obtained at a concentration of polyphenol as small as 100 micromol/L. In contrast, (p-hydroxyphenyl)ethanol was ineffective up to a concentration of 500 micromol/L. Our data demonstrate that (3,4-dihydroxyphenyl)ethanol can act as a biological antioxidant in a cell culture experimental model and that the ortho-dihydroxy moiety of the molecule is essential for antioxidant activity. This study suggests that dietary intake of olive oil polyphenols may lower the risk of reactive oxygen metabolite-mediated diseases such as some gastrointestinal diseases and atherosclerosis.

Purification and characterization of recombinant human 5'-methylthioadenosine phosphorylase: definite identification of coding cDNA.

5'-Methylthioadenosine phosphorylase gene maps on the 9p21 chromosome, strictly linked to the important tumor suppressor gene p16INK4A. Chromosomal deletions encompassing both the phosphorylase and p16INK4A genes cause the complete absence of the enzymatic activity in a large number of tumors, thus resulting in well-defined metabolic differences between malignant and normal cells. Recently, the cloning of the phosphorylase gene has been reported on the basis of indirect evidence. In order to demonstrate definitely the identification of 5'-methylthioadenosine phosphorylase gene, we have cloned the putative enzyme coding sequence in a prokaryotic expression vector and expressed the protein in bacteria. The recombinant phosphorylase has been purified to homogeneity and its physicochemical, immunological and kinetic features have been characterized. The results obtained allowed the conclusive demonstration of 5'-methylthioadenosine phosphorylase gene cloning and the use of recombinant protein for further characterization.