Evaluation of antioxidant properties of major dietary polyphenols and their protective effect on 3T3-L1 preadipocytes and red blood cells exposed to oxidative stress.

Obesity has been associated with a marked risk of metabolic diseases and requires therapeutic strategies. Changes in redox status with increased oxidative stress in adipose tissue have been linked with obesity-related disorders. Thus, the biological effect of antioxidants such as polyphenols is of high interest. We aimed to measure antioxidant capacities of 28 polyphenols representative of main dietary phenolic acids, flavonoids, stilbenes and curcuminoids. Then, 14 molecules were selected for the evaluation of their effect on 3T3-L1 preadipocytes and human red blood cells exposed to oxidative stress. Analysis of reducing and free radical-scavenging capacities of compounds revealed antioxidant properties related to their structure, with higher activities for flavonoids such as quercetin and epicatechin. Their effects on preadipocytes' viability also depended on their structure, dose and time of exposure. Interestingly, most of the compounds exhibited a protective effect on preadipocytes exposed to oxidative stress, by reversing H2O2-induced anti-proliferative action and reactive oxygen species production. Polyphenols also exerted an anti-inflammatory effect on preadipocytes exposed to H2O2 by reducing IL-6 secretion. Importantly, such antioxidant and anti-inflammatory effects were observed in co-exposition (polyphenol and prooxidant during 24 h) or pretreatment (polyphenol during 24 h, then prooxidant for 24 h) conditions. Moreover, compounds protected erythrocytes from AAPH radical-induced lysis. Finally, these results led to demonstrate that antioxidant and anti-inflammatory properties of polyphenols may depend on structure, dose, time of exposure and cell conditioning with oxidative stress. Such findings should be considered for a better understanding of polyphenols' benefits in strategies aiming to prevent obesity-related diseases.

Pharmacokinetics of folinic acid and 5-methyltetrahydrofolic metabolite after repeated oral administration of calcium folinate following methotrexate treatment.

The pharmacokinetic profiles of folinic acid (FA) and its active metabolite, 5-methyltetrahydrofolic acid, were studied after oral administration of decreasing doses of calcium folinate during 37 courses of high and intermediate dose methotrexate treatment in 25 lymphoma patients. FA was administered at a dose of 6 x 50 mg in 15 courses, 6 x 25 mg in seven courses, 6 x 15 mg in 10 courses and 6 x 7.5 mg in 5 courses. FA, 5-methyltetrahydrofolic acid, methotrexate and 70H-methotrexate were assayed simultaneously by high performance liquid chromatography. When FA was administered at doses between 50 and 15 mg, maximum concentrations of both the drug and its metabolite were always obtained after 1 to 2 h and remained stable. The same was true for the equilibrium concentration of the two products at doses over 15 mg. These findings suggest saturation of absorption and metabolism of folinic acid at doses over 15 mg.

Direct determination of mitoxantrone in plasma by high performance liquid chromatography using an automatic precolumn-switching system as sample clean-up procedure.

A high-performance liquid chromatography method which uses direct injection and a column-switching valve for determination of mitoxantrone in plasma is described. After addition of internal standard, plasma was deproteinized by adding 5-sulphosalicylic acid reagent. The supernatant was injected onto an enrichment precolumn flushed with washing solvent (methanol and water 5:95). Absorbed mitoxantrone was backflushed from the precolumn into an analytical column C18 Nucleosil 250 x 4 mm with a gradient elution (solvent A, ammonium formate buffer 1.6 M, pH 4.3; solvent B, acetonitrile and water 40:60; linear gradient from 45 to 55% of B for 30 min was programmed) at a flow rate of 1.3 mL/min. Detection was carried out at 665 nm. This method showed obvious advantages over conventional extraction procedures in terms of speed and facility of sample handling.

[Insufficiency fractures of the sacrum. 5 cases]. / Fractures de contrainte du sacrum. Cinq observations.

Five cases of insufficiency fracture of the sacrum in women aged over 60 are reported. The elements of diagnosis and causative factors are reviewed. In all cases the diagnosis was confirmed by technetium bone scintigraphy and computerized tomography of the sacrum. Bone tissue evaluation in search of a bone-wasting disease, the principal cause of this type of fracture, was conducted in all 5 patients, using photon absorptiometry and/or double energy computerized tomography and histomorphometry. These examinations provided detailed information on disorders of mineralization in the genesis of these insufficiency fractures.

Interactions between 7-hydroxymethotrexate and folinic acid in RAJI cells, in vitro.

HPLC analysis of plasma samples obtained from patients included in a high-dose methotrexate-folinic acid Rescue (HD-MTX-CF) protocol, allowed the simultaneous determination of MTX, CF and their respective plasma metabolites, 7-hydroxymethotrexate (7-OH-MTX) and 5-methyltetrahydrofolate (5-CH3-FH4). These 4 compounds interact at the cellular level to ensure the selective effectiveness of the HD-MTX-CF rescue protocol. An in vitro study has been investigated in RAJI cells to better describe the interference of CF on uptake, accumulation and metabolism of [3H]7-OH-MTX. Results about uptake and accumulation of CF were also obtained using [3H]CF, in the absence or the presence of unlabeled 7-OH-MTX. The rate of [3H]7-OH-MTX influx in RAJI cells (Km = 25.30 +/- 7.75 microM, n = 3) was competitively inhibited by the presence of 10 microM CF with a Ki value of 6.00 +/- 1.94 microM (n = 2). Intracellular 7-OH-MTX accumulation was decreased by approximately 30% when extracellular CF concentration was twice as high as that of 7-OH-MTX, and 70% when CF extracellular concentration was 5 times higher. The metabolism of 7-OH-MTX to its intracellular polyglutamyl derivatives was depressed by 90% when 10 microM CF were incubated for 2 h with equimolar [3H]7-OH-MTX, and it was completely abolished in the presence of 100 microM CF.

Direct determination of mitoxantrone and its mono- and dicarboxylic metabolites in plasma and urine by high-performance liquid chromatography.

The simultaneous isolation and determination of mitoxantrone (Novantrone) and its two known metabolites (the mono- and dicarboxylic metabolites) were carried out using a high-performance liquid chromatographic (HPLC) system equipped with an automatic pre-column-switching system that permits drug analysis by direct injection of biological samples. Plasma or urine samples were injected directly on to an enrichment pre-column flushed with methanol-water (5:95, v/v) as the mobile phase. The maximum amount of endogenous water-soluble components was removed from biological samples within 9 min. Drugs specifically adsorbed on the pre-column were back-flushed on to an analytical column (Nucleosil C18, 250 X 4.6 mm I.D.) with 1.6 M ammonium formate buffer (pH 4.0) (2.5% formic acid) containing 20% acetonitrile. Detection was effected at 655 nm. Chromatographic analysis was performed within 12 min. The detection limit of the method was about 4 ng/ml for urine and 10 ng/ml for plasma samples. The precision ranged from 3 to 11% depending on the amount of compound studied. This technique was applied to the monitoring of mitoxantrone in plasma and to the quantification of the unchanged compound and its two metabolites in urine from patients receiving 14 mg/m2 of mitoxantrone by intravenous infusion for 10 min.

Plasma kinetic study of folinic acid and 5-methyltetrahydrofolate in healthy volunteers and cancer patients by high-performance liquid chromatography.

A reversed-phase HPLC method is described for the simultaneous determination of folinic acid, MTX, and their plasma metabolites 5-CH3-FH4 and 7-OH-MTX respectively. In addition, this technique allows the separation of FA another naturally occurring folate, and of AMT, used as internal standard. Separation of these compounds was achieved on a Waters Spherical C18 column at a flow rate of 0.8 ml.min-1. Elution was carried out with 0.1 M sodium acetate buffer (pH 5.5) as solvent A and 7.5% acetonitrile 92.5% bidistilled water as solvent B. UV detection was performed at 280 nm. This method was applied in a pharmacokinetic study of folinic acid and its plasma metabolite 5-CH3-FH4 following two different protocols: (1) i.v. bolus injection of 50 mg calcium folinate in six healthy volunteers and (2) simultaneous i.v. bolus injections of 50 mg/m2 MTX and 50 mg/m2 folinic acid in four cancer patients. Mean apparent half-life values for folinic acid and its metabolite were 7.02 +/- 1.81 h and 3.90 +/- 0.86 respectively in the first protocol, 4.80 +/- 1.48 h and 4.74 +/- 1.47 h in the second protocol. MTX and 7-OH-MTX were also quantified in the second protocol and were found not to affect the pharmacokinetics of folinic acid and 5-CH3-FH4. Since in vitro studies on metabolism of folinic acid might be of great interest in trying to assess the mechanism of action of the folates and the potential interaction of MTX and 7-OH-MTX in this mechanism via the metabolism, the chromatographic method we describe here has been adapted for the separation of all the potential intracellular monoglutamyl metabolites of folinic acid.