Luciana Leone

Improved FIA-ABTS method for antioxidant capacity determination in different biological samples.

In order to evaluate the actual antioxidant features of foods, beverages and also plasma from patients, a number of assays have been developed in the last few years to determine the so called total antioxidant activity (TAA), intended as the cumulative capacity of a biological sample to scavenge free radicals. Most of the assays partially failed in obtaining a good reproducibility when using plasma because it is composed of a large number of substances, some of which are present at very high concentrations and possess masking features. For these reasons we have improved the widely known ABTS method by means of a FIA system where both temperature and dispersion of sample and reagent were strictly controlled. We found that temperature may be a critical aspect in the measurement of plasma TAA whilst its influence may be less important in the assay of non-complex biological samples. We demonstrated that also the reaction time may be critical, depending on the nature of the substance employed. Data confirmed the high TAA of a methylsalicylate-containing mouthrinse as well as the negligible TAA offered by the chlorhexidine containing one. White wines (Verdicchio) also displayed interesting TAA values. The improved method was useful to screen rapidly, without dilution, with very limited handling of the sample and with high repeatability the TAA of plasma in addition to chemical products, beverages and non-complex biological mixtures.

Alterations in the Oxidation Products, Antioxidant Markers, Antioxidant Capacity and Lipid Patterns in Plasma of Patients Affected by Papillon-Lefèvre Syndrome

Papillon-Lefèvre syndrome (PLS) is an uncommon disease. Less than 300 cases have been described. PLS is characterized by the association between palmar plantar hyperkeratosis (PPK) and severe precocious periodontitis that results in the premature loss of both the primary and secondary dentitions. It is known that periodontitis (PE), the destructive phase of periodontal disease, is a multifactor phenomenon involving a variety of molecular species, among them free radicals and reactive oxygen species (ROS). Antioxidants have been shown to play a critical role in modulating ROS-induced damages during PE. We wondered if patients belonging to a family group with different grades of PLS severity may present altered plasma concentrations of oxidation products as well as of lipophilic antioxidants, like Coenzyme Q or vitamin E, which are molecules that possess well-known antioxidant properties and could play a role in PE processes. We also wondered about the actual plasma total antioxidant capacity of these subjects as well as a complete identification of their plasma fatty acids features, which have been never investigated before. The results we obtained indicate an impairment in the antioxidant capacity of the subjects characterized by abnormally high hydroperoxide levels and, in some cases, by altered CoQ and vitamin E contents. Moreover, an essential fatty acid deficiency (EFAD) was registered on the basis of the peculiar plasma fatty acid patterns found (i.e. low PUFA, high MUFA and high Δ-9 desaturase activity). This finding would support the hypothesis by Gutteridge and co-workers (Free Radic. Res. 1998, 28: 109–114) that conditions exist in which some forms of oxidative stress can lead to changes in fatty acids patterns characteristic of EFAD.

Coenzymes Q9 and Q10, vitamin E and peroxidation in rat synaptic and non-synaptic occipital cerebral cortex mitochondria during ageing.

Abstract Great attention has been devoted both to ageing phenomena at the mitochondrial level and to the antioxidant status of membrane structures. These kinds of investigations are difficult to perform in the brain because of its heterogeneity. It is known that synaptic heavy mitochondria (HM) may represent an aged mitochondrial population characterized by a partial impairment of their typical mitochondrial function. We arranged a novel system requiring no extraction procedure, very limited handling of the samples and their direct injection into the HPLC apparatus, to carry out, for the first time, a systematic and concomitant determination of vitamin E, Coenzyme Q[9] (CoQ[9]) and Coenzyme Q[10] (CoQ[10]) contents in rat brain mitochondria. The trends found for CoQ[9] and CoQ[10] levels in synaptic and nonsynaptic occipital cerebral cortex mitochondria during rat ageing are consistent with previous data. Hydroperoxides (HP) differed with age and it was confirmed that in the HM fraction the summation of contributions results in an oxidatively jeopardized subpopulation. We found that vitamin E seems to increase with age, at least in nonsynaptic free (FM) and synaptic light (LM) mitochondria, while it was inclined to remain substantially constant in HM.

High-performance liquid chromatography-EC assay of mitochondrial coenzyme Q9, coenzyme Q9H2, coenzyme Q10, coenzyme Q10H2, and vitamin E with a simplified on-line solid-phase extraction.

Publisher Summary Coenzyme Q (CoQ), the essential hydrophobic molecule that links flavoproteins and cytochromes in the mitochondrial respiratory chain, as well as in other extramitochondrial redox chains, also possesses antioxidant properties. The redox state of CoQ homologues in mitochondrial and other membranes is important for the evaluation of cell welfare; unfortunately, because of their chemical features, these homologues are very sensitive and unstable compounds, especially in their reduced form, which displays the highest antioxidant potentiality. All the valid analytical methods require a large extraction procedure, and sometimes, complicated and time-consuming high-performance liquid chromatography (HPLC) analysis. HPLC method makes it possible to carry out, for the first time, a systematic and simultaneous determination of coenzyme Q9, coenzyme Q9H2, coenzyme Q10, coenzyme Q10H2, and vitamin E contents from rat liver mitochondria. The described method allows the simultaneous detection of vitamin E, CoQ9H2, CoQ9, CoQ10H2, and CoQ10 with good precision in rat liver mitochondria. The length of the assay is reasonably short and is performed with a simple isocratic HPLC separation. This simple coulometric technique enables the analyst to use only different electrodes for the simultaneous determination of reduced and oxidized CoQn instead of the concomitant use of different kinds of detectors (e.g., diode array and amperometric detectors)

Antiangiogenic, antitumoural and antimetastatic effects of two distamycin A derivatives with anti-HIV-1 Tat activity in a Kaposi's sarcoma-like murine model

The antiangiogenic, antitumoural and antimetastatic effects of two novel sulphonic derivatives of distamycin A, PNU145156E and PNU153429, were studied in a Kaposi’s sarcoma-like tumour model obtained by injecting nude mice with cells releasing extracellular HIV-Tat protein, derived from a tumour which developed in a BK virus/tat transgenic mouse. Both PNU145156E and PNU153429 were administered intraperitoneally every fourth day for three weeks at doses of 100 or 50 mg/kg of body weight respectively, starting one day after injecting the tumour cells. Both drugs delayed tumour growth in nude mice, preventing neovascularization induced by the Tat protein. PNU153429 also significantly reduced the number and size of spontaneous tumour metastases. Both effects on tumour growth and metastases were augmented by treating simultaneously nude mice with 7.5 mg/kg of body weight of minocycline given per os daily for four weeks starting four days after injecting the tumour cells. Neither acute nor chronic toxic side-effects were observed during the life span of treated nude mice. Due to their antiangiogenic and anti-Tat effects, these drugs are promising for the treatment of Kaposi’s sarcoma in AIDS patients.

Coenzyme Q homologs and vitamin E in synaptic and non-synaptic occipital cerebral cortex mitochondria in the ageing rat

The coenzyme Q8 (CoQ8) and alpha-tocopherol contents of different mitochondrial fractions were investigated from occipital cerebral cortices of different ages. The highest CoQ8 and vitamin E concentrations were found in non-synaptic free mitochondria (FM) fractions. In several cases heavy mitochondria (HM) fractions displayed the lowest values. Occipital cerebral cortex mitochondria contained higher CoQ9 and lower CoQ10 amounts than those typical for other brain regions.

Bioavailability study of two different formulations of azithromycin

Abstract In a single-dose, balanced, two-period crossover study, we evaluated the bioavailability of two different commercial formulations of azithromycin in 18 healthy male volunteers. Both the test formulation (500-mg tablet) and the reference formulation (500-mg capsule) were administered to each subject. The two treatment phases were separated by a 15-day washout period. Blood samples were drawn before and up to 48 hours after dosing; we used high-performance liquid chromatography to determine drug plasma concentrations. We calculated the area under the plasma concentration—time curve (AUC), maximum plasma concentration (C max ), and time of maximum plasma concentration (t max ) for each subject. Logarithmic transformation of the AUC and C max was used for the statistical analysis and to assess the bioavailability of the two formulations. Analysis of variance (ANOVA) for t max , ln(C max ), and ln(AUC) provided the appropriate intrasubject variance estimate to evaluate the 90% confidence interval for the difference between study variables after administration of the test and reference formulations. The ANOVA showed no significant effect in the model other than a subject effect. The intrasubject coefficient of variation indicated that the two formulations were equivalent with regard to the rate and extent of absorption.