Alpha-tocopherol supplementation decreases electronegative low-density lipoprotein concentration [LDL(-)] in haemodialysis patients

Background. Oxidative stress is a significant contributor to cardiovascular diseases (CVD) in haemodialysis (HD) patients, predisposing to the generation of oxidized low-density lipoprotein (oxLDL) or electronegatively charged LDL subfraction. Antioxidant therapy such as alpha-tocopherol acts as a scavenger of lipid peroxyl radicals attenuating the oxidative stress, which decreases the formation of oxLDL. The present study was designed to investigate the influence of the alpha-tocopherol supplementation on the concentration of electronegative low-density lipoprotein [LDL(-)], a minimally oxidized LDL, which we have previously described to be high in HD patients. Methods. Blood samples were collected before and after 120 days of supplementation by alpha-tocopherol (400 UI/day) in 19 stable HD patients (50 +/- 7.8 years; 9 males). The concentrations of LDL(-) in blood plasma [using an anti-LDL- human monoclonal antibody (mAb)] and the anti-LDL(-) IgG auto-antibodies were determined by ELISA. Calculation of body mass index (BMI) and measurements of waist circumference (WC), triceps skin folds (TSF) and arm muscle area (AMA) were performed. Results. The plasma alpha-tocopherol levels increased from 7.9 mu M (0.32-18.4) to 14.2 mu M (1.22-23.8) after the supplementation (P = 0.02). The mean concentration of LDL(-) was reduced from 570.9 mu g/mL (225.6-1241.0) to 169.1 mu g/mL (63.6-621.1) (P < 0.001). The anti-LDL(-) IgG auto-antibodies did not change significantly after the supplementation. The alpha-tocopherol supplementation also reduced the total cholesterol and LDL-C levels in these patients, from 176 +/- 42.3 mg/dL to 120 +/- 35.7 mg/dL (P < 0.05) and 115.5 +/- 21.4 mg/dL to 98.5 +/- 23.01 mg/dL (P < 0.001), respectively. Conclusion. The oral administration of alpha-tocopherol in HD patients resulted in a significant decrease in the LDL(-), total cholesterol and LDL-C levels. This effect may favour a reduction in cardiovascular risk in these patients, but a larger study is required to confirm an effect in this clinical setting.

Sterilization by pure oxygen plasma and by oxygen-hydrogen peroxide plasma: An efficacy study

Plasma is an innovative sterilization method characterized by a low toxicity to operators and patients, and also by its operation at temperatures close to room temperatures. The use of different parameters for this method of sterilization and the corresponding results were analyzed in this study. A low-pressure inductive discharge was used to study the plasma sterilization processes. Oxygen and a mixture of oxygen and hydrogen peroxide were used as plasma source gases. The efficacy of the processes using different combinations of parameters such as plasma-generation method, type of gas, pressure, gas flow rate, temperature, power, and exposure time was evaluated. Two phases were developed for the processes, one using pure oxygen and the other a mixture of gases. Bacillus subtilis var. niger ATCC 9372 (Bacillus atrophaeus) spores inoculated on glass coverslips were used as biological indicators to evaluate the efficacy of the processes. All cycles were carried out in triplicate for different sublethal exposure times to calculate the D value by the enumeration method. The pour-plate technique was used to quantify the spores. D values of between 8 and 3 min were obtained. Best results were achieved at high power levels (350 and 40oW) using pure oxygen, showing that plasma sterilization is a promising alternative to other sterilization methods. (c) 2007 Elsevier B.V. All rights reserved.

Enantioselective determination of chloroquine and its n-dealkylated metabolites in plasma using liquid-phase microextraction and LC-MS

A selective method using three-phase liquid-phase microextraction (LPME) in conjunction with LC-MS-MS was devised for the enantioselective determination of chloroquine and its n-dealkylated metabolites in plasma samples. After alkalinization of the samples, the analytes were extracted into n-octanol immobilized in the pores of a polypropylene hollow fiber membrane and back extracted into the acidic acceptor phase (0.1 M TFA) filled into the lumen of the hollow fiber. Following LPME, the analytes were resolved on a Chirobiotic V column using methanol/ACN/glacial aceti acid/diethylamine (90:10:0.5:0.5 by volume) as the mobile phase. The MS detection was carried out using multiple reaction monitoring with ESI in the positive ion mode. The optimized LPME method yielded extraction recoveries ranging from 28 to 66%. The method was linear over 5 - 500 ng/mL and precision (RSD) and accuracy (relative error) values were below 15% for all analytes. The developed method was applied to the determination of the analytes in rat plasma samples after oral administration of the racemic drug.

Liquid-phase microextraction combined with high-performance liquid chromatography for the enantioselective analysis of mefloquine in plasma samples

A simple and rapid method, which involves liquid-phase microextraction (LPME) followed by HPLC analysis using Chiralpak AD column and UV detection, was developed for the enantioselective determination of mefloquine in plasma samples. Several factors that influence the efficiency of three-phase LPME were investigated and optimized. Under the optimal extraction conditions, the mean recoveries were 33.2 and 35.0% for (-)-(SR-)-mefloquine and (+)-(RS)-mefloquine, respectively. The method was linear over 50-1500 ng/ml range. Within-day and between-day assay precision and accuracy were below 15% for both enantiomers at concentrations of 150, 600 and 1200 ng/ml. Furthermore, no racemization or degradation were seen with the method described. (C) 2007 Elsevier B.V. All rights reserved.

Enantioselective analysis of zopiclone and its metabolites in plasma by liquid chromatography/tandem mass spectrometry

A high-performance liquid chromatographic method with triple-quadrupole mass spectrometry detection (LC-MS-MS) was developed and validated for the first time for the simultaneous quantification of zopiclone and its metabolites in rat plasma samples. The analytes were isolated from rat plasma by liquid-liquid extraction and separated using a chiral stationary phase based on an amylose derivative, Chiralpak ADR-H column, and ethanol-methanol-acetonitrile (50:45:5, v/v/v) plus 0.025% diethylamine as the mobile phase, at a flow-rate of 1.0 mL min(-1). Moclobemide was used as the internal standard. The developed method was linear over the concentration range of 7.5-500 ng mL(-1). The mean absolute recoveries were 74.6 and 75.7; 61.6 and 56.9; 72.5, and 70.7 for zopiclone enantiomers, for N-desmethyl zopiclone enantiomers and for zopiclone-N-oxide enantiomers, respectively, and 75.9 for the internal standard. Precision and accuracy were within acceptable levels of confidence (<15%). The method application in a pilot study of zopiclone kinetic disposition in rats showed that the levels of (+)-(S)-zopiclone were always higher than those of (-)-R-zopiclone. Higher concentrations were also observed for (+)-(S)-N-desmethyl zopiclone and (+)-(S)-N-oxide zopiclone, confirming the stereoselective disposition of zopiclone.

Enantioselective analysis of mirtazapine and its two major metabolites in human plasma by liquid chromatography-mass spectrometry after three-phase liquid-phase microextraction

A three-phase liquid-phase microextraction (LPME) method using porous polypropylene hollow fibre membrane with a sealed end was developed for the extraction of mirtazapine (MRT) and its two major metabolites, 8-hydroxymirtazapine (8-OHM) and demethylmirtazapine (DMR), from human plasma. The analytes were extracted from 1.0 mL of plasma, previously diluted and alkalinized with 3.0 mL 0.5 mol L-1 pH 8 phosphate buffer solution and supplemented with 15% sodium chloride (NaCl), using n-hexyl ether as organic solvent and 0.01 moL L-1 acetic acid solution as the acceptor phase. Haloperidol was used as internal standard. The chromatographic analyses were carried out on a chiral column, using acetonitrile-methanol-ethanol (98:1:1, v/v/v) plus 0.2% diethylamine as mobile phase, at a flow rate of 1.0 mL min(-1). Multi-reaction monitoring (MRM) detection was performed by mass spectrometry (MS-MS) using a triple-stage quadrupole and electrospray ionization interface operating in the positive ion mode. The mean recoveries were in 18.3-45.5% range with linear responses over the 1.25-125 ng mL(-1) concentration range for all enantiomers evaluated. The quantification limit (LOQ) was 1.25 ng mL(-1). Within-day and between-day assay precision and accuracy (2.5, 50 and 100 ng mL(-1)) showed relative standard deviation and the relative error lower than 11.9% for all enantiomers evaluated. Finally, the method was successfully used for the determination of mirtazapine and its metabolite enantiomers in plasma samples obtained after single drug administration of mirtazapine to a healthy volunteer. (c) 2007 Elsevier B.V. All rights reserved.

Determination of beta-artemether and its main metabolite dihydroartemisinin in plasma employing liquid-phase microextraction prior to liquid chromatographic-tandem mass spectrometric analysis

A method for the determination of artemether (ART) and its main metabolite dihydroartemisinin (DHA) in plasma employing liquid-phase microextraction (LPME) for sample preparation prior to liquid chromatography-tandem mass spectrometry (LC-MS-MS) was developed. The analytes were extracted from 1 nil, of plasma utilizing a two-phase LPME procedure with artemisinin as internal standard. Using the optimized LPME conditions, mean absolute recovery rates of 25 and 32% for DHA and ART, respectively, were achieved using toluene-n-octanol (1:1, viv) as organic phase with an extraction time of 30 min. After extraction, the analytes were resolved within 5 min using a mobile phase consisting of methanol-ammonium acetate (10 mmol L(-1) pH 5.0, 80:20. v/v) on a laboratory-made column based on poly(methyltetradecylsiloxane) attached to a zirconized-silica support. MS-MS detection was employed using an electrospray interface in the positive ion mode. The method developed was linear over the range of 5-1000 ng mL(-1) for both analytes. Precision and accuracy were within acceptable levels of confidence (<15%). The assay was applied to the determination of these analytes in plasma from rats treated with ART. The two-phase LPME procedure is affordable and the solvent consumption was very low compared to the traditional methods of sample preparation. (C) 2010 Elsevier B.V. All rights reserved.

LC-MS-MS Identification and Determination of the Flavone-C-Glucoside Vicenin-2 in Rat Plasma Samples Following Intraperitoneal Administration of Lychnophora Extract

The flavone C-glucoside, vicenin-2, in semi-purified extracts of the leaves of Lychnophora ericoides was quantified in rat plasma samples using a method based on reversed-phase high performance liquid chromatography coupled to tandem mass spectrometry. Vicenin-2 was analyzed on a LiChrospher (R) RP18 column using an isocratic mobile phase consisting of a mixture of methanol: water (30:70, v/v) plus 2.0% glacial acetic acid at a flow rate of 0.8 mL min(-1). Genistein was used as internal standard. The mass spectrometer was operated in positive ionization mode and analytes were quantified by multiple reaction monitoring at m/z 595 > 457 for vicenin-2 and m/z 271 > 153 for internal standard. Prior to the analysis, each rat plasma sample was acidified with 200 mu L of 50 mmol L(-1) acetic acid solution and extracted by solid-phase extraction using a C18 cartridge. The absolute recoveries were reproducible and the coefficients of variation values were lower than 5.2%. The method was linear over the 12.5 - 1500 ng mL(-1) concentration range and the quantification limit was 12.5 ng mL(-1). Within-day and between-day assay precision and accuracy were studied at three concentration levels (40, 400 and 800 ng mL(-1)) and were lower than 15%. The developed and validated method seems to be suitable for analysis of vicenin-2 in plasma samples obtained from rats that receive a single i.p. dose of 200 mg kg(-1) vicenin-2 extract.

Enantioselective analysis of praziquantel and trans-4-hydroxypraziquantel in human plasma by chiral LC-MS/MS: Application to pharmacokinetics

A simple enantioselective method for the determination of praziquantel (PZQ) and trans-4-hydroxypraziquantel (4-OHPZQ) in human plasma was developed and validated by high-performance liquid chromatography/mass spectrometry. The plasma samples were prepared by liquid-liquid extraction using a mixture of methyl-tert-butylether/dichloromethane (2:1, v/v) as extraction solvent. The direct resolution of PZQ and 4-OHPZQ enantiomers was performed on a Chiralpak AD column using hexane-isopropanol (75:25, v/v) as the mobile phase. Diazepam was used as internal standard. The method described here is simple and reproducible. The quantitation limit of 1.25 ng/ml for each PZQ enantiomer and of 12.5 ng/ml for each 4-OHPZQ enantiomer permits the use of the method in studies investigating the kinetic disposition of a single dose of 1.5g racemic PZQ. Enantioselectivity in the kinetic disposition of PZQ and 4-OHPZQ was observed in the clinical study. with the demonstration of a higher proportion of the (+)-(S)-PZQ and (-)-(R)-4-OHPZQ enantiomers in plasma. (C) 2009 Elsevier B.V. All rights reserved.

Simultaneous determination of multibenzodiazepines by HPLC/UV: Investigation of liquid-liquid and solid-phase extractions in human plasma

A method for simultaneous determination of seven benzodiazepines (BZPs) (flunitrazepam, clonazepam, oxazepam, lorazepam, chlordiazepoxide, nordiazepam and diazepam using N-desalkylflurazepam as internal standard) in human plasma using liquid-liquid and solid-phase extractions followed by high-performance liquid chromatography (HPLC) is described. The analytes were separated employing a LC-18 DB column (250 mm x 4.6 mm, 5 mu m) at 35 degrees C under isocratic conditions using 5 mM KH(2)PO(4) buffer solution pH 6.0: methanol: diethyl ether (55:40:5, v/v/v) as mobile phase at a flow rate of 0.8 mL min(-1). UV detection was carried out at 245 nm. Employing LLE, the best conditions were achieved with double extraction of 0.5 mL, plasma using ethyl acetate and Na(2)HPO(4) pH 9.5 for pH adjusting. Employing SPE, the best conditions were achieved with 0.5 mL plasma plus 3 mL 0.1 M borate buffer pH 9.5, which were then passed through a C18 cartridge previously conditioned, washed for 3 times with these solvents: 3 mL 0.1 M borate buffer pH 9.5,4 mL Milli-Q water and 1 mL acetonitrile 5%, finally the BZPs elution was carried with diethyl ether: n-hexane: methanol (50:30:20). In both methods the solvent was evaporated at 40 degrees C under nitrogen flow. The validation parameters obtained in LLE were linearity range of 50-1200 ng mL(-1) plasma (r >= 0.9927), limits of quantification of 50 ng mL(-1) plasma, within-day and between-day CV% and E% for precision and accuracy lower than 15%, and recovery above 65% for all BZPs. In SPE, the parameter obtained were linearity range of 30-1200 ng mL(-1) plasma (r >= 0.9900), limits of quantification of 30 ng mL(-1) plasma, within-day and between-day CV% and E% for precision and accuracy lower than 15% and recovery above 55% for all BZPs. These extracting procedures followed by HPLC analysis showed their suitable applicability in order to examine one or more BZPs in human plasma. Moreover, it could be suggested that these procedures might be employed in various analytical applications, in special for toxicological/forensic analysis. (c) 2008 Elsevier B.V. All rights reserved.

Fluorescent indication that nitric oxide formation in NTS neurons is modulated by glutamate and GABA

Nitric oxide (NO) in NTS plays an important role in regulating autonomic function to the cardiovascular system. Using the fluorescent dye DAF-2 DA, we evaluated the NO concentration in NTS. Brainstem slices of rats were loaded with DAF-2 DA, washed, fixed in paraformaldehyde and examined under fluorescent light. In different experimental groups, NTS slices were pre-incubated with 1 mM L-NAME (a non-selective NOS inhibitor), 1 MM D-NAME (an inactive enantiomere of L-NAME), 1 mM kynurenic acid (a nonselective ionotropic receptors antagonist) or 20 mu M bicuculline (a selective GABA(A) receptors antagonist) before and during DAF-2 DA loading. Images were acquired using a confocal microscope and the intensity of fluorescence was quantified in three antero-posterior NTS regions. In addition, slices previously loaded with DAF-2 DA were incubated with NeuN or GFAP antibody. A semi-quantitative analysis of the fluorescence intensity showed that the basal NO concentration was similar in all antero-posterior aspects of the NTS (rostral intermediate, 15.5 +/- 0.8 AU: caudal intermediate, 13.2 +/- 1.4 AU; caudal commissural, 13.8 +/- 1.4 AU, n = 10). In addition, the inhibition of NOS and the antagonism of glutamatergic receptors decreased the NO fluorescence in the NTS. On the other hand, D-NAME did not affect the NO fluorescence and the antagonism of GABAA receptors increased the NO fluorescence in the NTS. It is important to note that the fluorescence for NO was detected mainly in neurons. These data show that the fluorescence observed after NTS loading with DAF-2 DA is a result of NO present in the NTS and support the concept that NTS neurons have basal NO production which is modulated by L-glutamate and GABA. (C) 2009 Elsevier Inc. All rights reserved.

Two-step liquid-phase microextraction and high-performance liquid chromatography for the simultaneous analysis of the enantiomers of mefloquine and its main metabolite carboxymefloquine in plasma

A method for the simultaneous analysis of the enantiomers of mefloquine (MQ) and its main metabolite carboxymefloquine (CMQ) in plasma is described for the first time. The assay involves two-step liquid-phase micro-extraction (LPME) and enantioselective high-performance liquid chromatography. In the first LPME step, the enantiomers of MQ were extracted from an alkalinized sample through a thin layer of di-n-hexyl ether immobilized in the pores of the hollow fiber and into 0.01 M perchloric acid as acceptor solution. In the second LPME step, the same sample was acidified to enable the extraction of CMQ using the same organic solvent and 0.05 M sodium hydroxide as acceptor phase. The analytes were resolved on a Chirobiotic T column in the polar-organic mode of elution and detected at 285 nm. The recovery rates from 1 mL of plasma were in the range 35-38%. The method presented limits of quantification of 50 ng/mL for all analytes and was linear up to 1,500 and 3,000 ng/mL for the enantiomers of MQ and CMQ, respectively. The plasmatic concentrations of (+)-(RS)-MQ were higher than those of (-)-(SR)-MQ after oral administration of the racemic drug to rats.

Relationship between Rheological Properties and One-Step W/O/W Multiple Emulsion Formation

Formation of a normal (not temporary) W/O/W multiple emulsion via the one-step method as a result of the simultaneous occurrence of catastrophic and transitional phase inversion processes has been recently reported. Critical features of this process include the emulsification temperature (corresponding to the ultralow surface tension point), the use of a specific nonionic surfactant blend and the surfactant blend/oil phase ratio, and the addition of the surfactant blend to the oil phase. The purpose of this study was to investigate physicochemical properties in an effort to gain a mechanistic understanding of the formation of these emulsions. Bulk, surface, and interfacial theological properties of adsorbed nonionic surfactant (CremophorRH40 and Span80) films were investigated under conditions known to affect W/O/W emulsion formation. Bulk viscosity results demonstrated that CremophorRH40 has a higher mobility in oil compared than in water, explaining the significance of the solvent phase. In addition, the bulk viscosity profile of aqueous solutions containing CremophorRH40 indicated a phase transition at around 78 +/- 2 degrees C, which is in agreement with cubic phase formation in the Winsor III region. The similarity in the interfacial elasticity values of CremophorRH40 and Span80 indicated that canola oil has a major effect on surface activity, showing the significance of vegetable oil. The highest interfacial shear elasticity and viscosity were observed when both surfactants were added to the oil phase, indicating the importance of the microstructural arrangement. CremophorRH40/Span80 complexes tended to desorb from the solution/solution interface with increasing temperature, indicating surfactant phase formation as is theoretically predicted in the Winsor III region. Together these interfacial and bulk rheology data demonstrate that one-step W/O/W emulsions form as a result of the simultaneous occurrence of phase-transition processes in the Winsor III region and explain the critical formulation and processing parameters necessary to achieve the formation of these normal W/O/W emulsions.

Influence of Phase Inversion on the Formation and Stability of One-Step Multiple Emulsions

A novel method of preparation of water-in-oil-in-micelle-containing water (W/O/W(m)) Multiple emulsions using the one-step emulsification method is reported. These multiple emulsions were normal (not temporary) and stable over a 60 day test period. Previously, reported multiple emulsion by the one-step method were abnormal systems that formed at the inversion point of simple emulsion (where there is an incompatibility in the Ostwald and Bancroft theories, and typically these are O/W/O systems). Pseudoternary phase diagrams and bidimensional process-composition (phase inversion) maps were constructed to assist in process and composition optimization. The surfactants used were PEG40 hydrogenated castor oil and sorbitan oleate, and mineral and vegetables oils were investigated. Physicochemical characterization studies showed experimentally, for the First time, the significance of the ultralow surface tension point oil multiple emulsion formation by one-step via phase inversion processes. Although the significance of ultralow surface tension has been speculated previously, to the best of our knowledge, this is the first experimental confirmation. The multiple emulsion system reported here was dependent not only upon the emulsification temperature, but also upon the component ratios, therefore both the emulsion phase inversion and the phase inversion temperature were considered to fully explain their formation. Accordingly, it is hypothesized that the formation of these normal multiple emulsions is not a result of a temporary incompatibility (at the inversion point) during simple emulsion preparation, as previously reported. Rather, these normal W/O/W(m) emulsions are a result of the simultaneous occurrence of catastrophic and transitional phase inversion processes. The formation of the primary emulsions (W/O) is in accordance with the Ostwald theory and the formation of the multiple emulsions (W/O/W(m)) is in agreement with the Bancroft theory.

A common matrix metalloproteinase (MMP)-2 polymorphism affects plasma MMP-2 levels in subjects environmentally exposed to mercury

Mercury (Hg) exposure is associated with disease conditions, including cardiovascular problems. Although the mechanisms implicated in these complications have not been precisely defined yet, matrix metalloproteinases (MMPs) may be involved. The gene encoding MMP-2 presents genetic polymorphisms which affect the expression and activity level of this enzyme. A common polymorphism of MMP-2 gene is the C(-1306)T (rs 243865), which is known to disrupt a Sp1-type promoter site (CCACC box), thus leading to lower promoter activity associated with the T allele. This study aimed at examining how this polymorphism affects the circulating MMP-2 levels and its endogenous inhibitor, the tissue inhibitor of metalloproteinase-2 (TIMP-2) in 210 subjects environmentally exposed to Hg. Total blood and plasma Hg concentrations were determined by inductively coupled plasma-mass spectrometry (ICP-MS). MMP-2 and TIMP-2 concentrations were measured in plasma samples by gelatin zymography and ELISA, respectively. Genotypes for the C(-1306)T polymorphism were determined by Taqman (R) Allele Discrimination assay. We found a positive association (p = 0.0057) between plasma Hg concentrations and MMP-2/TIMP-2 (an index of net MMP-2 activity). The C(-1306)T polymorphism modified MMP-2 concentrations (p = 0.0465) and MMP-2/TIMP-2 ratio (p = 0.0060) in subjects exposed to Hg, with higher MMP-2 levels been found in subjects carrying the C allele. These findings suggest a significant interaction between the C(-1306)T polymorphism and Hg exposure, possibly increasing the risk of developing diseases in subjects with the C allele. (C) 2011 Elsevier B.V. All rights reserved.