Antioxidant and Biological Activities of Hydroxytyrosol and Homovanillic Alcohol Obtained from Olive Mill Wastewaters of Extra-Virgin Olive Oil Production.

Some constituents of the Mediterranean diet, such as extra-virgin olive oil (EVOO) contain substances such as hydroxytyrosol (HT) and its metabolite homovanillic alcohol (HA). HT has aroused much interest due to its antioxidant activity as a radical scavenger, whereas only a few studies have been made on the HA molecule. Both chemical synthesis and extraction techniques have been developed to obtain these molecules, with each method having its advantages and drawbacks. In this study, we report the use of tyrosol from olive mill wastewaters as a starting molecule to synthesize HT and HA, using a sustainable procedure characterized by high efficiency and low cost. The effects of HT and HA were evaluated on two cell lines, THP-1 human leukemic monocytes and L-6 myoblasts from rat skeletal muscle, after treating the cells with a radical generator. Both HT and HA efficiently inhibited ROS production. In particular, HT inhibited the proliferation of the THP-1 leukemic monocytes, while HA protected L-6 myoblasts from cytotoxicity.

Polypyrrole hollow fiber for solid phase extraction.

We have developed a solid-phase extraction method based on conductive polypyrrole (PPy) hollow fibers which were fabricated by electrospinning and in situ polymerization. The electrospun poly (e-caprolactone) (PCL) fibers were employed as templates for the in situ surface polymerization of PPy under mechanical stirring or ultrasonication to obtain burr-shaped or smooth fiber shells, respectively. Hollow PPy fibers, achieved by removing the PCL templates, were the ideal sorbents for solid phase extraction of polar compounds due to their inherent multi-functionalities. By using the hollow PPy fibers, two important neuroendocrine markers of behavioural disorders, 5-hydroxyindole-3-acetic acid and homovanillic acid, were successfully extracted. Under the optimized conditions, the absolute recoveries of the above two neuroendocrine markers were 90.7% and 92.4%, respectively, in human plasma. Due to its simplicity, selectivity and sensitivity, the method may be applied to quantitatively analyse the concentrations of polar species in complex matrix samples.

Electrophoretic analysis of biomarkers using capillary modification with gold nanoparticles embedded in a polycation and boron doped diamond electrode.

Field-amplified sample stacking using a fused silica capillary coated with gold nanoparticles (AuNPs) embedded in poly(diallyl dimethylammonium) chloride (PDDA) has been investigated for the electrophoretic separation of indoxyl sulfate, homovanillic acid (HVA), and vanillylmandelic acid (VMA). AuNPs (27 nm) exhibit ionic and hydrophobic interactions, as well as hydrogen bonding with the PDDA network to form a stable layer on the internal wall of the capillary. This approach reverses electro-osmotic flow allowing for fast migration of the analytes while retarding other endogenous compounds including ascorbic acid, uric acid, catecholamines, and indoleamines. Notably, the two closely related biomarkers of clinical significance, HVA and VMA, displayed differential interaction with PDDA-AuNPs which enabled the separation of this pair. The detection limit of the three analytes obtained by using a boron doped diamond electrode was approximately 75 nM, which was significantly below their normal physiological levels in biological fluids. This combined separation and detection scheme was applied to the direct analysis of these analytes and other interfering chemicals including uric and ascorbic acids in urine samples without off-line sample treatment or preconcentration.

High-performance liquid chromatography of monoamines on phenyl-bound sorbents using organic free mobile phases.

Tryptophan and its metabolites, 5-hydroxytryptophan, 5-hydroxytryptamine, 5-hydroxyindolacetic acid, as well as dopamine, homovanilic acid and 2,3-dihydroxyphenylacetic acid, were separated on phenyl bound silica gel using isocratic elution with phosphate buffer. The method was successfully transferred to several other phenyl HPLC columns from different manufacturers simply by adjusting the pH of the buffer. The method has been validated by the determination of the level of monoamines in rat hypothalamus.

HPLC-ED determination of catecholamines and their metabolites in urine.

High performance liquid chromatography (HPLC) with electrochemical detection (ED) was applied for the monitoring of catecholamines (epinephrine - E, norepinephrine - NE, dopamine - DA) and their main metabolites (homovanillic acid - HVA, vanillylmandelic acid - VMA) in urine samples of patients with the pheochromocytoma diagnosis. Both amperometric and colorimetric detectors were tested for the clinical applications and the results were compared. The optimization of separation conditions was worked out, especially the effects of mobile phase compositions (pH, ionic strength, organic modifier and ion-pair agent concentrations). Dependences of capacity ratio values k' on all optimized components of the mobile phases were evaluated. Chromatographic conditions were optimized for the suitable chromatographic resolution (Rij > 1.25). Extraction recoveries for liquid-liquid and solid-phase extractions have been worked-out. Detection limits for catecholamines in urine were in the range of 0.3-0.6 ng/ml and 10-20 ng/ml for HVA, VMA using coulometric detector.

Profound decrement of mesolimbic dopaminergic neuronal activity during ethanol withdrawal syndrome in rats: electrophysiological and biochemical evidence.

Activity of the mesolimbic dopaminergic system was investigated in rats withdrawn from chronic ethanol administration by single-cell extracellular recordings from dopaminergic neurons of the ventrotegmental area, coupled with antidromic identification from the nucleus accumbens, and by microdialysis-technique experiments in the nucleus accumbens. Spontaneous firing rates, spikes per burst, and absolute burst firing but not the number of spontaneously active neurons were found drastically reduced; whereas absolute and relative refractory periods increased in rats withdrawn from chronic ethanol treatment as compared with chronic saline-treated controls. Consistently, dopamine outflow in the nucleus accumbens and its acid metabolites were reduced after abruptly stopping chronic ethanol administration. All these changes, as well as ethanol-withdrawal behavioral signs, were reversed by ethanol administration. This reversal suggests that the abrupt cessation of chronic ethanol administration plays a causal role in the reduction of mesolimbic dopaminergic activity seen in the ethanol-withdrawal syndrome. Results indicate that during the ethanol-withdrawal syndrome the mesolimbic dopaminergic system is tonically reduced in activity, as indexed by electrophysiological and biochemical criteria. Considering the role of the mesolimbic dopaminergic system in the reinforcing properties of ethanol, the depressed activity of this system during the ethanol-withdrawal syndrome may be relevant to the dysphoric state associated with ethanol withdrawal in humans.

Assessment of dopamine and its metabolites in the intracellular and extracellular compartments of the rat striatum after peripheral administration of L-[11C]dopa.

Success in the synthesis of L-3,4-[beta-11C]dihydroxyphenylalanine (L-[11C]DOPA) and its application to positron emission tomography encouraged us to perform radioactive metabolite analyses in rats in an early phase after peripheral injection of L-[11C]DOPA. Following intravenous injection of [11C]DOPA, the radioactivity associated with DOPA and its metabolites was determined in the striatum after decapitation and in striatal extracellular fluid using in vivo brain microdialysis. Without pretreatment, 70-80% of 11C-radioactivity taken up into the striatum was associated with acidic metabolites of dopamine (DA) from 2 to 30 min after administration of L-[11C]DOPA with or without 300 micrograms/kg of unlabelled L-DOPA. In contrast, 80-90% of 11C-radioactivity in the striatum was associated with DOPA and DA after pretreatment with benserazide (25 mg/kg, i.p.) followed by administration of L-[11C]DOPA with or without unlabelled L-DOPA. The radioactivity in the DOPA fraction decreased with time (from 35% of 11C-radioactivity in the striatum at 5 min to 10% at 30 min), but that in the DA fraction increased (from 57% to 68%). The 11C-radioactivity in the extracellular fluid determined by brain microdialysis was less than 0.4% of that in the whole striatum and no radioactivity was present in the DA fraction. These results suggest that, in an early phase after administration of L-[11C]DOPA, [11C]DA is the main metabolite and is localized exclusively in the intracellular compartment within this time frame.

A kinetic study of the rate of formation of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the brain of the rat: implications for the origin of DOPAC.

The rates of disappearance of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the brain of rats treated with the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine were measured. Levels of dopamine. DOPAC and HVA in tissue were quantified in the striatum, nucleus accumbens and frontal cortex of the rat by means of high pressure liquid chromatography with electrochemical detection. Rats were sacrificed 1, 2 or 3 hr after the administration of alpha-methyl-p-tyrosine (200 mg/kg, i.p.). Levels of dopamine, DOPAC and HVA in tissue were logarithmically transformed, plotted against the duration of inhibition of tyrosine hydroxylase and the rate constant of the decline of dopamine and of its metabolites calculated. The rate constant of decline of DOPAC in each of brain studied was found to be greater than that for dopamine. The rate constant for the decline of dopamine was found to be greater in areas of the brain presenting the largest DOPAC/dopamine tissue ratios (frontal cortex greater than nucleus accumbens greater than striatum). The present results suggest that a substantial amount of DOPAC in brain derives from a newly formed pool of dopamine.

Modification of reversed-phase separations of small molecules using non-ionic surfactants and mixed ionic-non-ionic surfactants.

The effects of non-ionic surfactants, Tween 20 and Tween 60, on reversed-phase separations of small molecules have been examined. Tween compounds were found to partition irreversibly into the ODS material used, markedly decreasing capacity factors for the compounds tested. Compounds which could hydrogen bond were less affected. Ion pairing using either anionic or cationic surfactants was possible in the presence of the non-ionic surfactants. While reversed-phase effects predominate under these conditions, secondary effects on retention order were observed and attributed to hydrogen bonding. Primary amines were retained longer than the corresponding secondary amine while catechols were retained longer than the corresponding methoxyphenols.

Simultaneous determination of homovanillic acid, 3,4-dihydroxyphenylacetic acid and 5-hydroxyindoleacetic acid in brain tissue, using a double-column procedure.

A double-column procedure for simultaneous determination of homovanillic acid (HVA), 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindoleacetic acid (5-HIAA) in brain tissue was described. These compounds extracted with perchloric acid were adsorbed on Sephadex G-10 in the column. HVA and DOPAC were desorbed from Sephadex G-10 with 0.01 N HCl and then retained on QAE-Sephadex A-25 (acetate form) placed beneath the Sephadex G-10 column. HVA and DOPAC were eluted with small volume of 0.2 mol/l NaCl. 5-HIAA remaining on the Sephadex G-10 was eluted with 0.05 mol/l phosphate buffer at pH 7.5. Each substance was determined fluorometrically. The recovery rates of HVA, DOPAC and 5-HIAA were more than 80, 80 and 85%, respectively. Effects of oxypertine on dopamine (DA) neurons in the rat brain were investigated using the method described here. Oxypertine caused marked increases in the levels of HVA and DOPAC in cortex and striatum of the rat brain, without any obvious change in the level of 5-HIAA after the intraperitoneal administration of 10 mg/kg oxypertine. These findings suggest that the favorable antipsychotic action of oxypertine may be due not only to a marked reduction in brain norepinephrine level, but to a blockade of DA receptors.

Conjugates of dopamine and serotonin in ventriculocisternal perfusates of the cat.

The in vivo formation of acid-hydrolyzable conjugates of dopamine and of serotonin (presumably dopamine-O-sulfate and serotonin-O-sulfate) in ventriculocisternal perfusions of the cat is described. Small amounts of these amine conjugates were detected under quiescent conditions and during evoked release of the parent amines. The amounts of conjugated dopamine in perfusate were increased during and immediately after the period in which release of dopamine was evoked, but were not affected by inhibition of monoamine oxidase. In contrast, the efflux of serotonin conjugate during the evoked release of serotonin was not increased unless monoamine oxidase was inhibited. The data suggest that conjugation of amines in the CNS may be of functional importance in their disposition either under conditions of augmented release or during inhibition of oxidative deamination.

Capillary gas-chromatographic determination of urinary homovanillic acid and vanillylmandelic acid.

This relatively simple, rapid method for quantification of homovanillic acid (HVA) and vanillylmandelic acid (VMA) involves solvent extraction and capillary gas chromatography. With use of this extraction method, the overall analytical recovery from an aqueous solution is 92.5% for HVA and 79.2% for VMA. 3,4-Dihydroxybenzoic acid is the internal standard. Minimal detectable quantities with this method are less than 1 mg/g of creatinine. Capillary gas chromatography produces a chromatogram that is superior to that of packed-column gas chromatography. We also report quantitative results for urinary HVA and VMA in neuroblastoma patients and in normal individuals, demonstrating the diagnostic value of this method.

Urinary 4-hydroxy-3-methoxymandelic (vanillylmandelic) acid, 4-hydroxy-3-methoxyphenylacetic (homovanillic) acid, and 5-hydroxy-3-indoleacetic acid determined by liquid chromatography with electrochemical detection.

We describe a simple liquid-chromatographic assay of urinary 4-hydroxy-3-methoxymandelic (vanillylmandelic) acid, 4-hydroxy-3-methoxyphenylacetic (homovanillic) acid, and 5-hydroxy-3-indoleacetic acid with electrochemical detection, with direct injection of the sample. The first two analytes are measured simultaneously; 5-hydroxy-3-indoleacetic acid is measured separately. Chromatographic conditions for assay of the three were: column temperature, 65 and 60 degrees C; mobile phase, potassium phosphate buffer (0.2 mol/L, pH 3.0) for 6 min, then potassium phosphate buffer plus acetonitrile (9/1 by vol) for 20 min; flow rate, 0.7 mL/min; oxidation potential, 600 and 450 mV vs an Ag/AgCl reference electrode; and sensitivity, 40 and 160 nA at full scale. Values so obtained agreed well with those obtained for samples that were first solvent-extracted.

Determination of methionine-enkephalin, norepinephrine, dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and 3-methoxy-4-hydroxyphenylacetic acid (HVA) in brain by high-pressure liquid chromatography with electrochemical detector.

High-pressure liquid chromatography with electrochemical detection is utilized in a procedure for the determination of methionine-enkephalin (MET-ENK), norepinephrine (NE), dopamine (DA), 3, 4-dihydroxyphenylacetic acid (DOPAC) and 3-methoxy-4-hydroxyphenylacetic acid (HVA) in same brain tissue samples. The high degree of selectivity and sensitivity (picomole limits for each component) makes this technique directly applicable to extremely small samples. After optimal conditions had been established, the procedure was employed in the determination of MET-ENK, NE, DA, DOPAC and HVA contents in whole brains, 7 discrete brain areas of mice and rats.

Reverse-phase chromatography of polar biological substances: separation of catechol compounds by high-performance liquid chromatography.

Catecholamines and their metabolites have been separated isocratically by reverse-phase chromatography with aqueous (no organic solvent admixed) eluents. Unlike ion-exchange or ion-pair chromatography, mixtures of both acidic and basic substances can be separated in a single chromatographic run, because the retention is governed by hydrophobic interactions between the nonpolar moiety of the solute molecules and the octadecyl-silica stationary phase. The relative retention values strongly depend on the pH of the eluent, which governs the degree of dissociation of ionogenic solutes. The reproducibility of the results and the stability and efficiency of the chromatographic systems make this approach particularly attractive for use in clinical analysis.