Protective effects of Mangifera indica L extract (Vimang), and its major component mangiferin, on iron-induced oxidative damage to rat serum and liver

In vivo preventive effects of a Mangifera indica L extract (Vimang) or its major component mangiferin on iron overload injury have been studied in rats given respectively, 50, 100, 250 mg kg(-1) body weight of Vimang, or 40 mg kg(-1) body weight of mangiferin, for 7 days prior to, and for 7 days following the administration of toxic amounts of iron-dextran. Both Vimang or mangiferin treatment prevented iron overload in serum as well as liver oxidative stress, decreased serum and liver lipid peroxidation, serum GPx activity, and increased serum and liver GSH, serum SOD and the animals overall antioxidant condition. Serum iron concentration was decreased although at higher doses, Vimang tended to increase it; percent tranferrin saturation, liver weight/body mass ratios, liver iron content was decreased. Treatment increased serum iron-binding capacity and decreased serum levels of aspartate-amine transferase (ASAT) and alanine-amine transferase (ALAT), as well as the number of abnormal Kupffer cells in iron-loaded livers. It is suggested that besides acting as antioxidants, Vimang extract or its mangiferin component decrease liver iron by increasing its excretion. Complementing earlier in vitro results from our group, it appears possible to support the hypothesis that Vimang and mangiferin present therapeutically useful effects in iron overload related diseases. (C) 2007 Elsevier Ltd. All rights reserved.

Hollow fiber-based liquid-phase microextraction (HF-LPME) of isradipine and its main metabolite followed by chiral HPLC analysis: application to an in vitro biotransformation study

An enantioselective liquid chromatographic method using two-phase hollow fiber liquid-phase microextraction (HF-LPME-HPLC) was developed for the determination of isradipine (ISR) enantiomers and its main metabolite (pyridine derivative of isradipine, PDI) in microsomal fractions isolated from rat liver. The analytes were extracted from 1 mL of microsomal medium using a two-phase HF-LPME procedure with hexyl acetate as the acceptor phase, 30 min of extraction, and sample agitation at 1,500 rpm. For the first time, ISR enantiomers and PDI were resolved. For this separation, a ChiralpakA (R) AD column with hexane/2-propanol/ethanol (94:04:02, v/v/v) as the mobile phase at a flow rate of 1.5 mL min(-1) was used. The column was kept at 23 A +/- 2 A degrees C. The drug and metabolite detection was performed at 325 nm and the internal standard oxybutynin was detected at 225 nm. The recoveries were 23% for PDI and 19% for each ISR enantiomer. The method presented quantification limits (LOQ) of 50 ng mL(-1) and was linear over the concentration range of 50-5,000 and 50-2,500 ng mL(-1) for PDI and each ISR enantiomer, respectively. The validated method was employed to an in vitro biotransformation study of ISR using rat liver microsomal fraction showing that (+)-(S)-ISR is preferentially biotransformed.

Enantioselective analysis of propranolol and 4-hydroxypropranolol by CE with application to biotransformation studies employing endophytic fungi

A CE method is described for the enantioselective analysis of propranolol (Prop) and 4-hydroxypropranolol (4-OH-Prop) in liquid Czapek medium with application in the study of the enantioselective biotransformation of Prop by endophytic fungi. The electrophoretic conditions previously optimized were as follows: an uncoated fused-silica capillary, 4%w/v carboxymethyl-beta-CD in 25 mmol/L triethylamine/phosphoric acid (H(3)PO(4)) buffer at pH 9 as running electrolyte and 17 kV of voltage. UV detection was carried out at 208 nm. Liquid-liquid extraction using diethyl ether: ethyl acetate (1:1 v/v) as extractor solvent was employed for sample preparation. The calibration curves were linear over the concentration range of 0.25-10.0 mu g/mL for each 4-OH-Prop enantiomer and 0.10-10.0 mu g/mL for each Prop enantiomer (r >= 0.995). Within-day and between-day relative standard deviations and relative errors for precision and accuracy were lower than 15% for all the enantiomers. Finally, the validated method was used to evaluate Prop biotransformation in its mammalian metabolite 4-OH-Prop by some selected endophytic fungi. The screening of five strains of endophytic fungi was performed and all of them could biotransform Prop to some extent. Specifically, Glomerella cingulata (VA1) biotransformed 47.8% of (-)-(S)-Prop to (-)-(S)-4-OH-Prop with no formation of (+)-(R)4-OH-Prop in 72 h of incubation.

Uncoupling and oxidative stress in liver mitochondria isolated from rats with acute iron overload

One hypothesis for the etiology of cell damage arising from iron overload is that its excess selectively affects mitochondria. Here we tested the effects of acute iron overload on liver mitochondria isolated from rats subjected to a single dose of i.p. 500 mg/kg iron-dextran. The treatment increased the levels of iron in mitochondria (from 21 +/- A 4 to 130 +/- A 7 nmol/mg protein) and caused both lipid peroxidation and glutathione oxidation. The mitochondria of iron-treated rats showed lower respiratory control ratio in association with higher resting respiration. The mitochondrial uncoupling elicited by iron-treatment did not affect the phosphorylation efficiency or the ATP levels, suggesting that uncoupling is a mitochondrial protective mechanism against acute iron overload. Therefore, the reactive oxygen species (ROS)/H(+) leak couple, functioning as a mitochondrial redox homeostatic mechanism could play a protective role in the acutely iron-loaded mitochondria.

Mercury speciation in seafood samples by LC-ICP-MS with a rapid ultrasound-assisted extraction procedure: Application to the determination of mercury in Brazilian seafood samples

This paper describes a simple method for mercury speciation in seafood samples by LC-ICP-MS with a fast sample preparation procedure. Prior to analysis, mercury species were extracted from food samples with a solution containing mercaptoethanol, L-cysteine and HCl and sonication for 15 min. Separation of mercury species was accomplished in less than 5 min on a C8 reverse phase column with a mobile phase containing 0.05%-v/v mercaptoethanol, 0.4% m/v L-cysteine and 0.06 mol L(-1) ammonium acetate. The method detection limits were found to be 0.25, 0.20 and 0.1 ng g(-1) for inorganic mercury, ethylmercury and methylmercury, respectively. Method accuracy is traceable to Certified Reference Materials (DOLT-3 and DORM-3) from the National Research Council Canada (NRCC). With the proposed method there is a considerable reduction of the time of sample preparation. Finally, the method was applied for the speciation of mercury in seafood samples purchased from the Brazilian market. (C) 2010 Elsevier Ltd. All rights reserved.

Simultaneous Analysis of Tramadol, O-Desmethyltramadol, and N-Desmethyltramadol Enantiomers in Rat Plasma by High-Performance Liquid Chromatography-Tandem Mass Spectrometry: Application to Pharmacokinetics

Tramadol (T) is available as a racemic mixture of (+)-trans-T and (-)-trans-T. The main metabolic pathways are O-demethylation and N-demethylation, producing trans-O-desmethyltramadol (M1) and trans-N-desmethyltramadol (M2) enantiomers, respectively. The analgesic effect of T is related to the opioid activity of (+)-trans-T and (+)-M1 and to the monoaminergic action of (+/-)-trans-T. This is the first study using tandem mass spectrometry as a detection system for the simultaneous analysis of trans-T, M1, and M2 enantiomers. The analytes were resolved on a Chiralpak (R) AD column using hexane: ethanol (95.5:4.5, v/v) plus 0.1% diethylamine as the mobile phase. The quantitation limits were 0.5 ng/ml for trans-T and M1 and 0.1 ng/ml for M2. The method developed and validated here was applied to a pharmacokinetic study in rats. Male Wistar rats (n = 6 at each time point) received a single oral dose of 20 mg/kg racemic trans-T. Blood samples were collected up to 12 h after drug administration. The kinetic disposition of trans-T and M2 was enantioselective (AUC((+)/(-)) ratio = 4.16 and 6.36, respectively). The direction and extent of enantioselectivity in the pharmacokinetics of trans-T and M2 in rats were comparable to data previously reported for healthy volunteers, suggesting that rats are a suitable model for enantioselective studies of trans-T pharmacokinetics. Chirality 23: 287-293, 2011. (C) 2010 Wiley-Liss, Inc.

Selection bias in gene extraction on the basis of microarray gene-expression data

In the context of cancer diagnosis and treatment, we consider the problem of constructing an accurate prediction rule on the basis of a relatively small number of tumor tissue samples of known type containing the expression data on very many (possibly thousands) genes. Recently, results have been presented in the literature suggesting that it is possible to construct a prediction rule from only a few genes such that it has a negligible prediction error rate. However, in these results the test error or the leave-one-out cross-validated error is calculated without allowance for the selection bias. There is no allowance because the rule is either tested on tissue samples that were used in the first instance to select the genes being used in the rule or because the cross-validation of the rule is not external to the selection process; that is, gene selection is not performed in training the rule at each stage of the cross-validation process. We describe how in practice the selection bias can be assessed and corrected for by either performing a cross-validation or applying the bootstrap external to the selection process. We recommend using 10-fold rather than leave-one-out cross-validation, and concerning the bootstrap, we suggest using the so-called. 632+ bootstrap error estimate designed to handle overfitted prediction rules. Using two published data sets, we demonstrate that when correction is made for the selection bias, the cross-validated error is no longer zero for a subset of only a few genes.

Chemical treatment of Escherichia coli. II. Direct extraction of recombinant protein from cytoplasmic inclusion bodies in intact cells

A method is presented for the direct extraction of the recombinant protein Long-R-3-IGF-I from inclusion bodies located in the cytoplasm of intact Escherichia coli cells. Chemical treatment with 6M urea, 3 mM EDTA, and 20 mM dithiothreitol (DTT) at pH 9.0 proved an effective combination for extracting recombinant protein from intact cells. Comparable levels of Long-R-3-IGF-I were recovered by direct extraction as achieved by in vitro dissolution following mechanical disruption. However, the purity of directly extracted recombinant protein was lower due to contamination by bacterial cell components. The kinetics of direct extraction are described using a first-order equation with the time constant of 3 min. Urea appears important for permeabilization of the cell and dissolution of the inclusion body. Conversely, EDTA is involved in permeabilization of the cell wall and DTT enhances protein release. pH proved to be important with lower levels of protein release achieved at low pH values (

The effect of protein binding on the hepatic first pass of O-acyl salicylate derivatives in the rat

In this work the in-situ perfused rat liver has been used to examine the effect of changing the protein content of the perfusate on the hepatic extraction of O-acyl esters of salicylic acid. The hepatic availability (F) of these solutes was studied at a flow-rate of 30 mt min(-1) with perfusate albumin concentrations of 0, 2, and 4% w/v. The hepatic availability of the esters was shown to decrease with increasing carbon-chain length in the O-acyl group; for all the esters the hepatic availability increased with increasing albumin concentration in the perfusate. The dispersion-model-derived efficiency number (R-N) Of the esters was shown to increase with increasing lipophilicity and decrease with increasing albumin concentration in the perfusate. The unbound fraction (f(u),) of the esters decreased with lipophilicity. R-N/f(u), for acetylsalicylic acid remained relatively constant as the albumin concentration was increased. However, R-N/f(u), for n-pentanoyl- and n-hexanoylsalicylic acids increased significantly as albumin concentration increased from 0% to 4%. Thus, for the more lipophilic solutes (n-pentanoyl- and n-hexanoylsalicylic acids) the presence of albumin apparently facilitates the uptake of unbound solute relative to acetylsalicylic acid.

Metabolite mean transit times in the liver as predicted by various models of hepatic elimination

Predicted area under curve (AUC), mean transit time (MTT) and normalized variance (CV2) data have been compared for parent compound and generated metabolite following an impulse input into the liver, Models studied were the well-stirred (tank) model, tube model, a distributed tube model, dispersion model (Danckwerts and mixed boundary conditions) and tanks-in-series model. It is well known that discrimination between models for a parent solute is greatest when the parent solute is highly extracted by the liver. With the metabolite, greatest model differences for MTT and CV2 occur when parent solute is poorly extracted. In all cases the predictions of the distributed tube, dispersion, and tasks-in-series models are between the predictions of the rank and tube models. The dispersion model with mixed boundary conditions yields identical predictions to those for the distributed tube model (assuming an inverse gaussian distribution of tube transit times). The dispersion model with Danckwerts boundary conditions and the tanks-in series models give similar predictions to the dispersion (mixed boundary conditions) and the distributed tube. The normalized variance for parent compound is dependent upon hepatocyte permeability only within a distinct range of permeability values. This range is similar for each model but the order of magnitude predicted for normalized variance is model dependent. Only for a one-compartment system is the MIT for generated metabolite equal to the sum of MTTs for the parent compound and preformed metabolite administered as parent.

Improved one-step solid-phase extraction method for morphine, morphine-3-glucuronide, and morphine-6-glucuronide from plasma and quantitation using high-performance liquid chromatography with electrochemical detection

This communication describes an improved one-step solid-phase extraction method for the recovery of morphine (M), morphine-3-glucuronide (M3G), and morphine-6-glucuronide (M6G) from human plasma with reduced coextraction of endogenous plasma constituents, compared to that of the authors' previously reported method. The magnitude of the peak caused by endogenous plasma components in the chromatogram that eluted immediately before the retention time of M3G has been reduced (similar to 80%) significantly (p < 0.01) while achieving high extraction efficiencies for the compounds of interest, viz morphine, M6G, and M3G (93.8 +/- 2.5, 91.7 +/- 1.7, and 93.1 +/- 2.2%, respectively). Furthermore, when the improved solid-phase extraction method was used, the extraction cartridge-derived late-eluting peak (retention time 90 to 100 minutes) reported in our previous method, was no longer present in the plasma extracts. Therefore the combined effect of reducing the recovery of the endogenous components of plasma that chromatographed just before the retention time of M3G and the removal of the late-eluting, extraction cartridge-derived peak has resulted in a decrease in the chromatographic run-time to 20 minutes, thereby increasing the sample throughput by up to 100%.

Functional and structural characterization of isolated perfused stingray liver including effects of ischaemia/reperfusion

The morphological and functional characteristics of stingray liver were studied, including the effect of ischaemia/reperfusion. With an isolated perfused model, it was shown that the stingray liver was more resistant than the rat liver to ischaemia/reperfusion injury; this was consistent with the differing partial oxygen tensions usually present in the two species. This study confirmed that whereas stingray hepatocytes form tubules with central bile canaliculi as in other fish, the stingray liver has portal triads and a lobular architecture as in mammals. Apoptosis of hepatocytes, demonstrated in the normal liver, was only marginally enhanced by ischaemia/reperfusion. Resulting apoptotic bodies were phagocytized by macrophage-like cells in hepatocyte tubules. In contrast to rat liver, the stingray liver showed no necrosis after ischaemia-reperfusion. (C) 1998 W.B. Saunders Company Limited.