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.

Diagnosis and follow-up of cystinuria: use of proton magnetic resonance spectroscopy.

Proton Nuclear Magnetic Resonance (NMR) Spectroscopy of urine (as well as of other biological fluids) is a very powerful technique enabling multi-component analysis useful in both diagnosis and follow-up of a wide range of inherited metabolic diseases. Among these pathologies, cystinuria is characterised by accumulation in urine of four dibasic amino acids, namely lysine, arginine, ornithine and cystine; the last one, being only slightly water soluble, generates urolithiasis. The mentioned aminoacids can be detected in the urine NMR spectrum of cystinuric patients, the most abundant being the lysine (5 mM and over are often detected), whose typical signals become very high; arginine and ornithine are also usually detectable, although pathologic concentrations are lower (usually below 2mM). The proposed NMR technique is also suitable in monitoring the therapy with alpha-mercaptopropionylglycine (MPG), providing quantitation of several metabolites of interest in the follow-up of the pathology, like cystine, creatinine and citrate.

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.

Altered intestinal permeability to mannitol in diabetes mellitus type I.

BACKGROUND: Intestinal permeability has seldom been investigated in diabetes mellitus, even though patients frequently report gastrointestinal symptoms, and it has recently been shown that the prevalence of celiac disease associated with diabetes mellitus is higher than expected. METHODS: Intestinal permeability to cellobiose and mannitol was investigated in 31 patients affected by type I uncomplicated diabetes mellitus. Values were compared with those obtained in 32 normal subjects. RESULTS: The percentage of mannitol recovery was far higher than normal in two thirds of the investigated patients and correlated with the length of disease, even though the probes' ratio (cellobiose/mannitol) was in the normal range. CONCLUSIONS: A not previously reported increase of intestinal permeability to mannitol, clear-cut and not associated with that of the larger probe, is found in type I uncomplicated diabetes mellitus. These results may describe a primary feature of type I diabetes mellitus and the initial steps of evolution to celiac disease.

Biocompatibility studies on glass ionomer cements by primary cultures of human osteoblasts.

Glass ionomer cements (GICs) are materials largely employed in the dental field that have been considered recently as cements in orthopaedic surgery for their proven osteogenic features. The aim of this study was to compare the response of cultured human osteoblastic cells to a number of commercial glass ionomer cements in order to provide indications useful for the further development of formulations that have potential for use as cements or implants in repair and replacement of bone tissue. The GICs tested were: Ketac-Fil Aplicap, lonocem lonocap 1,0, GC Fuji II, GC Fuji II LC and Vitremer 3M. Several features such as plating efficiency, adhesion and morphology of the cells were studied, as well as the only specific biochemical parameter of osteoblastic phenotype, namely osteocalcin production. In addition, the colonisation of materials by osteoblastic cells was verified by means of scanning electron microscopy. Altogether, the results obtained indicate that four of the five glass ionomer cements tested are biocompatible, showing vital cells adhering to the materials, proliferating and expressing the biochemical markers of osteoblastic phenotype, whereas Vitremer 3M, although currently employed in the dental field, exhibits a great cytotoxicity toward the cells. The adverse reaction of this GIC can be attributed to the leaching of at least two components of the polyacidic phase evidenced by protonic magnetic resonance analysis (PMR), namely 2-hydroxyethylmethacrylate (HEMA), and an unidentified acidic species. The addition of pure HEMA at the same concentrations found by means of PMR to cultures of osteoblastic cells resulted in a complete cell death. Our results also show that in vitro methods employing primary cultures of human cells specific to the implant sites of prostheses are appropriate and suitable tools for evaluating biocompatibility of materials. Furthermore, this kind of approach can provide indications useful in the design of novel materials as well as in improving the characteristics of the formulations already available.

Diagnosis and follow-up of inborn errors of amino acid metabolism: Use of proton magnetic resonance of biological fluids.

Proton magnetic resonance spectra of biological fluids such as urine, plasma and cerebro-spinal fluid can be used for multi-component analysis of highly concentrated species, thus providing information about the general metabolism of the patient. Hydrogen containing analytes in concentration higher than 10µM are indeed often detectable in biological fluid in 15 minutes by means of an unexpensive 200 MHz spectrometer essentially without sample manipulation. Amino acids, keton bodies, organic acids and other metabolites can be easily estimated by this approach; consequently this technique represents a powerful tool particularly in the diagnosis of inborn errors of amino acid metabolism, when improving the prognosis often depends on a very early diagnosis and on an effective method for monitoring the effects of therapy.In the present paper, several cases of inherited diseases related to amino acid impaired metabolism will be presented to illustrate the importance in the diagnosis. Phenylketonuria, tyrosinemia, cystinuria, ornithinemia, argininosuccinic aciduria, maple syrup urine disease (MSUD), alkaptonuria, lysinuria and other genetic pathologies were in fact unambiguously and rapidly diagnosed by means of the identification in the biological fluids of the relevant accumulating amino acids and/or of their metabolites. The proposed technique is suitable to become, in the future, a useful routine tool for a wide neonatal screening.

Diet treatment of branched chain ketoaciduria studied by proton magnetic resonance spectroscopy.

A novel nuclear magnetic resonance method is proposed for the diagnosis and follow-up of patients affected by branched chain ketoaciduria. The method allows quantitation of the branched chain amino acids (BCAA's) such as leucine, isoleucine and valine and of related keto- and hydroxy acids by means of a single spectrum. The method implies short time of analysis, as opposed to the very long time required by the techniques currently in use (amino acid analyzer combined with gaschromatography/mass spectrometry of keto- and hydroxyacids), it is easy and suitable for adjustements of the dietary treatment even on a daily basis. The case of a 15 days old newborn child, presenting muscular hypertonicity was unambiguously diagnosed in few minutes by means of one single NMR spectrum of urine. More interestingly, NMR spectra of serum in the following days were suitable for quantitating amino-, and keto acids as well as other metabolites of relevance in the follow up of the dietary treatment of the disease. After a diet lacking of BCAA's, to eliminate keto acids, a low BCAA diet was introduced, that succeeded in keeping the serum levels of the three amino acids within the normal range, while dropping the related keto acids.

Enzymatic basis for the calcium-induced decrease of membrane protein methyl esterification in intact erythrocytes. Evidence for an impairment of S-adenosylmethionine synthesis.

The effect of Ca2+ loading, induced by the ionophore A23187, on methyl esterification of membrane proteins (i.e. bands 2.1, 3, 4.1 and 4.5) has been investigated in intact human erythrocytes. When the cells were incubated with L-[methyl-3H]methionine, 40 microM CaCl2 and 10 microM A23187 induce a 50% inhibition of membrane protein methyl esterification. This effect is selectively due to the increased intracellular Ca2+ concentration, as it is antagonized by 10 mM EGTA, and other divalent cations such as Mn2+ do not exert any inhibition. In order to clarify the mechanism(s) of the reported inhibition, the various events involved in the methyl esterification process in vivo were analyzed. L-Methionine uptake as well as protein methylase II activity are not directly affected by altered intracellular Ca2+ concentrations. Conversely in the Ca2+-loaded erythrocytes the conversion of [3H]methionine into [3H]AdoMet, catalyzed by AdoMet synthetase, decreases up to 25%. When the undialyzed erythrocyte cytosolic fraction is assayed in vitro for AdoMet synthetase the activity of the enzyme from the CaCl2/A23187-treated erythrocytes is significantly lower than the control, up to 5 mM ATP. This result suggests that in the Ca2+-loaded erythrocytes the ATP intracellular concentration is significantly lowered. The direct evaluation of ATP intracellular concentration, by HPLC, confirms a significant drop of ATP level, as a consequence of the Ca2+ loading. The removal of Ca2+ from the cells quantitatively restores both the AdoMet synthesis and the methyl esterification levels. The possible role of altered ATP intracellular concentrations as a regulatory factor in the AdoMet-dependent reactions as well as in post-translational protein methylation related to the ageing process is also discussed.

Transport and metabolism of double-labelled CDPcholine in mammalian tissues.

Double-labelled [methyl-14C,5-3H]CDPcholine has been synthesized and subjected to a pharmacokinetic analysis in several biological systems. In transport experiments with intact human erythrocytes no incorporation of radioactivity is observable. On the other hand the results obtained with perfused rat liver suggest a rapid cleavage of the pyrophosphate bridge of the molecule, followed by a rapid uptake of the hydrolytic products. The plasma half-lives of intravenously injected CDPcholine and of its metabolites have been evaluated within 60 sec range. Renal and fecal excretion of the injected radioactivity is negligible: only 2.5% of administered 14C- and 6.5% of the 3H- is excreted up to 48 hr after administration. Liver and kidney are the major CDPcholine metabolizing organs, characterized by a fast and extensive uptake of choline metabolites, followed by a slow release; conversely the rate of uptake of both 3H and 14C-labelled moieties by rat brain is significantly slower, reaching a steady-state level after 10 hr. The characterization of the labelled compounds detectable in the investigated organs provides some insights on the metabolism of the drug: the 3H-cytidine moiety in all the examined organs appears to be incorporated into the nucleic acid fraction via the cytidine nucleotide pool; the [14C]choline moiety of the molecule is in part converted, at the mitochondrial level, into betaine which accounts for about 60% of the total 14C-radioactivity associated with liver and kidney 30 min after administration; [14C]betaine in turn acts as methyl donor to homocysteine yielding [14C]methionine subsequently incorporated into proteins; the time dependent increase in labelled phospholipids is indicative of a recycling of the choline methyl-groups in this lipid fraction via CDPcholine and/or S-adenosylmethionine; the rather extensive amount of labelled methionine detectable in brain probably arises from its uptake from the blood stream, since the enzyme catalyzing the conversion of betaine into methionine is lacking in brain.

Studies on enzyme-substrate interactions of cholinephosphotransferase from rat liver.

In order to elucidate the reaction mechanism and the substrate-binding sites, CDPcholine:1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2), prepared from rat liver microsomal fraction, has been subjected to kinetic analysis and substrate specificity studies. Kinetic evidence supports the hypothesis of a Bi-Bi sequential mechanism, involving a direct nucleophilic attack of diacylglycerol on CDPcholine during the reaction. To investigate the substrate requirements for recognition and catalysis, several CDPcholine analogs, modified in the nitrogen base or in the sugar or in the pyrophosphate bridge, have been synthesized, characterized and assayed as substrates and/or inhibitors of the reaction. The amino group on the pyrimidine ring, the 2'-alcoholic function of the ribose moiety as well as the pyrophosphate bridge have been identified as critical sites for enzyme-substrates interactions.

Mechanism of propylamine-transfer reactions. Kinetic and inhibition studies on spermidine synthase from Escherichia coli.

Spermidine synthase (EC 2.5.1.16) purified from Escherichia coli has been subjected to a kinetic analysis including initial velocity and substrate analogs inhibition studies. Evidence is reported for a ping-pong mechanism, indicating that a propylaminated form of the enzyme is an obligatory intermediate in the reaction mechanism. S-Adenosyl(5')-3-methylthiopropylamine exerts a competitive substrate inhibition by combining with the improper stable enzyme form, while putrescine does not show any inhibitory effect. In order to investigate the substrate binding sites, new sulfonium-deaminated analogs of S-adenosyl(5')-3-methylthiopropylamine have been synthesized and assayed as substrates and as inhibitors of the reaction. The replacement of the amino group of adenine, or propylamine moiety of the sulfonium compound by the hydroxyl group, or both, resulted in a complete loss of activity as substrate. On the other hand, the deaminated analogs exert a competitive inhibition with respect to putrescine. On the basis of these results and in analogy with methyltransfer reactions, three recognition sites for S-adenosyl(5')-3-methylthiopropylamine on propylamine transfer enzymes are proposed.

[New 5'-sulfurated nucleosides as inhibitors of the biosynthesis of polyamines]. / Nuovi nucleosidi 5'-solforati quali inibitori della biosintesi delle poliammine.

The inhibitory effect of the deaminated analogs of S-adenosyl(5')-3-methylthiopropylamine on spermidine synthase (EC 2.5.1.16.) from E. coli, AdoMet decarboxylase (EC 4.1.1.50.) from human placenta and E. coli and AdoMet lyase (EC 3.3.1.-) from rat liver have been reported. 5'-isobutylthioadenosine, a new powerful antiproliferative drug has been assayed as substrate of MTA phosphorylase (EC 2.4.2.1). The apparent Km is 1,8x10(-5) M. The inhibitory effect of 5'-isobutylthiadenosine on AdoMet lyase and spermidine synthase was also demonstrated.