Theoretical study on the chemical fate of adducts formed through free radical addition reactions to carotenoids

It is well known that free radicals are responsible for oxidative stress and cause numerous health disorders. As a result, the study of molecules that can scavenge free radicals is significant. One of the most important classes of free radical scavengers are carotenoids (CAR). In this work, the effectiveness of the CAR in terms of the radical adduct formation (RAF) reaction is studied using density functional theory calculations (in polar and non-polar environments). The reactions between four CAR [β-carotene (BC), zeaxanthin (ZEA), canthaxanthin (CANTA) and astaxanthin (ASTA)] with eight different radicals (^?OH, ^?OOH, ^?CH₃, ^?O–CH₃, ^?OO–CH₃, ^?SH, ^?O–CH₂–CH=CH₂, and ^?OO–CH₂–CH=CH₂), as well as substantial further reactions involved in the radical chain propagation, are analyzed. According to our results, the RAF reactions are controlled to a larger extent by the nature of the free radical than by the particular CAR they are reacting with. Thermochemistry calculations predict that each CAR molecule is able to scavenge at least two free radicals, which would lead to the termination of the radical chain process. Epoxy and diepoxy CAR species can be formed, being epoxy molecules as good free radical scavengers as their parent CAR. ASTA and CANTA are predicted to be less reactive, when reacting through RAF mechanism, than BC and ZEA.     

高效液相色谱法测定血清中头孢噻肟浓度

用固相萃取法处理样品,以扑热息痛为内标物、甲醇－醋酸钠／醋酸缓冲溶液作流动相,采用反相高效液相色谱法测定血清中头孢噻肟的浓度。头孢噻肟和内标物的平均回收率分别为９６．７％和９７．７％,头孢噻肟血清浓度在１０ｍｇ／Ｌ至１５０ｍｇ／Ｌ范围内有良好线性关系,最低检测浓度为２ｍｇ／Ｌ,日内和日间相对标准偏差分别在３．０％和４．１％以内。结果表明方法准确、简便。     

Mitochondrial porin incorporation into black lipid membranes: ionic and gating contribution to the total current

We present a new ac device useful for simultaneous measurements of ionic charge movement (conductance) and gating charge displacement (capacitance) in mitochondrial porin channels incorporated in two kinds of black lipid membranes (BLMs), made up of phosphatidylinositol (charged surface) and oxidized cholesterol (neutral surface). In particular, we investigated the conductance/capacitance variations during the process of porin incorporation (VDAC) at different porin concentrations. While conductance variations are present throughout the porin concentration range investigated, a threshold value seems to be necessary in order to detect a significant capacitance variation. A clear steady state in both conductance and capacitance is reached for the phosphatidylinositol bilayer, while for the oxidized cholesterol membranes, the steady state is reached only for the conductance. The dependence of capacitance characteristics on the membrane applied voltage V(m) is investigated before porin incorporation and at the ionic current steady state. The results obtained confirm that before porin incorporation, there is a small dependence on V(m)(2), while afterwards we find evidence of a dual exponential voltage dependence (a result similar to that found for conductance). Finally, we investigated the capacitance dependence on the radius of the hole separating the two compartments of the cell used in the measurements. In this study, performed only with oxidized cholesterol, the radius was varied from 200 to 1050 microm. We observed a significant variation in the specific capacitance in particular for smaller radii. The results were interpreted by a simple geometrical model taking into account the influence of the torus.     

Molecularly Imprinted Polymers as Specific Adsorbents for Zearalenone Produced by Precipitation Polymerization and Applied to Mycotoxin Production

In this work, molecularly imprinted polymers (MIPs) using the mycotoxin zearalenone (ZEA) as template were first synthesized via precipitation polymerization and then applied as specific adsorbents in molecularly imprinted solid-phase extraction (MISPE) cartridges. The MISPE procedure was optimized with 3 mL of acetonitrile for preconditioning, 1 mL of acetonitrile:H₂O (60:40) for loading, 1 mL of acetonitrile:H₂O (30:70), and 3 mL of methanol:acetic acid (95:5) for elution. The obtained MIPs showed high selectivity of 96.9% towards ZEA, and low cross-reactivity (1-20%) to other Fusarium mycotoxins including deoxynivalenol, nivalenol, HT-2 toxin and T-2 toxin. The cross-reactivity to fumonisin B₁ amounted to 61%. The MISPE was applied for enrichment of ZEA, which was produced by Fusarium graminearum strains. An enrichment factor above 50 was reached. Recoveries of 1 µg/mL were between 90.8% and 99.6%. A small amount of ZEA was produced by 9 F. graminearum strains with a maximum of 13 µg, then purified by the developed MISPE and analyzed by LC-MS/MS.     

Effects of Ca2+, Zn2+ and Cd2+ on uridine diphosphate-glucuronyltransferase and beta-glucuronidase activities in rat liver microsomes

The effect of various metals on uridine diphosphate (UDP)-glucuronyltransferase and beta-glucuronidase activities in rat liver microsomes was investigated. The presence of Mn2+, Cd2+, Zn2+, V5+, Ni2+, Co2+, Cu+ or Ca2+ (20 microM) in the enzyme reaction mixture did not cause a significant alteration of UDP-glucuronyltransferase activity in hepatic microsomes. Of these metals, Zn2+ and Cd2+ (20 microM) caused a remarkable increase in hepatic microsomal beta-glucuronidase activity. Appreciable effects of Zn2+ and Cd2+ on beta-glucuronidase activity were seen at 5.0 microM, and the effects were saturated at 50 microM. Ca2+ (5.0-50 microM) and/or the Ca2(+)-binding protein regucalcin (2.0 microM) did not have an appreciable effect on UDP-glucuronyltransferase and beta-glucuronidase activities in hepatic microsomes. Thus, Zn2+ and Cd2+ uniquely increased beta-glucuronidase activity. The Zn2(+)- and Cd2(+)-induced increase in beta-glucuronidase activity was completely reversed by the presence of an SH group-protecting reagent (dithiothreitol). The response of the microsomal enzyme to Zn2+ and Cd2+ (20 microM) was no longer seen after treatment with 0.2% Triton X-100 [polyoxyethylene(10)octylphenyl ether], indicating that the stimulation by these metals is dependent on membrane association. The present study suggests that, of various metals tested, Zn2+ and Cd2+ can uniquely increase hepatic microsomal beta-glucuronidase activity and that their effect is based on binding to membranous SH groups, beside the enzyme protein.     

Preparation and mechanical properties of bacterial cellulose nanocomposites loaded with silica nanoparticles

Bacterial cellulose (BC), which is produced by Gluconacetobacter xylinus (Ga. xylinus) in culture, is made up of a three-dimensional network of ribbon-shaped bundles of cellulose microfibrils. In the current studies, we used two processes to prepare nanocomposites of BC filled with silica particles. In Process I, Ga. xylinus was incubated in medium containing silica sol Snowtex 0 (ST 0, pH 2–4) or Snowtex 20 (ST 20, pH 9.5–10.0). The elastic modulus at 20 °C was improved by keeping the amount of silica in the nanocomposites below 4% when ST 20 was used and below 8.7% when ST 0 was used. This process allowed incorporation of 50% silica in BC. Inclusion of higher amounts of silica reduced the modulus at 20 °C and the strength of the nanocomposites below that of BC. X-ray diffraction measurements revealed that the silica particles disturb the formation of ribbon-shaped fibrils and affect the preferential orientation of the ( ) plane. We also produced BC-silica nanocomposites by Process II, wherein the BC hydrogel was immersed in different concentrations of silica sols, allowing silica particles to diffuse into the BC hydrogel and lodge in the spaces between the ribbon-shaped fibrils. This method increased the modulus at 20°C and the strength compared to the BC matrix, but it was difficult to load the BC with more than 10% silica in this way.     

免疫亲和柱净化-柱后光化学衍生-高效液相色谱法同时检测粮谷中的黄曲霉毒素、玉米赤霉烯酮和赭曲霉毒素A

建立了同时检测粮谷中黄曲霉毒素(B1、B2、G1和G2)、玉米赤霉烯酮和赭曲霉毒素A的免疫亲和柱净化-柱后光化学衍生-高效液相色谱方法。样品经过甲醇-水(体积比为80∶20)提取,通过免疫亲和柱富集和净化,采用Waters Nova-Pak色谱柱(3.9 mm i.d.×150 mm,4 μm),以甲醇、乙腈和1%的磷酸溶液为流动相,梯度洗脱,柱后光化学衍生、改变波长荧光检测。黄曲霉毒素(B1、B2、G1和G2)、玉米赤霉烯酮和赭曲霉毒素A检出限分别为0.24,4.0和0.5 μg/kg,标准曲线的线性范围分别为0.24~6.0,4.0~100.0和0.5~40.0 μg/L;在小麦、玉米、黑麦样品中,平均加标回收率为70.8% ~94.0%,相对标准偏差为2.79% ~9.38%。     

Interaction of hexadecylbetainate chloride with biological relevant lipids

The present work investigates the interaction of hexadecylbetainate chloride (C(16)BC), a glycine betaine-based ester with palmitoyl-oleoyl-phosphatidylcholine (POPC), sphingomyelin (SM), and cholesterol (CHOL), three biological relevant lipids present in the outer leaflet of the mammalian plasma membrane. The binding affinity and the mixing behavior between the lipids and C(16)BC are discussed based on experimental (isothermal titration calorimetry (ITC) and Langmuir film balance) and molecular modeling studies. The results show that the interaction between C(16)BC and each lipid is thermodynamically favorable and does not affect the integrity of the lipid vesicles. The primary adsorption of C(16)BC into the lipid film is mainly governed by a hydrophobic effect. Once C(16)BC is inserted in the lipid film, the polar component of the interaction energy between C(16)BC and the lipid becomes predominant. Presence of CHOL increases the affinity of C(16)BC for membrane. This result can be explained by the optimal matching between C(16)BC and CHOL within the film rather by a change of membrane fluidity due to the presence of CHOL. The interaction between C(16)BC and SM is also favorable and gives rise to highly stable monolayers probably due to hydrogen bonds between their hydrophilic groups. The interaction of C(16)BC with POPC is less favorable but does not destabilize the mixed monolayer from a thermodynamic point of view. Interestingly, for all the monolayers investigated, the exclusion surface pressures are above the presumed lateral pressure of the plasma membranes suggesting that C(16)BC would be able to penetrate into mammalian plasma membranes in vivo. These results may serve as a useful basis in understanding the interaction of C(16)BC with real membranes.     

Proteomic approach to the identification of cell membrane proteins

The expression of plasma membrane proteins in human monocyte-derived U937 cells was examined by cell disruption and isolation of microsomal fractions. Two alternative procedures for cell disruption, Dounce homogenization and nitrogen cavitation, were compared. Cell homogenization and sequential centrifugation resulted in an approximately fivefold enrichment of plasma membrane proteins in the microsomal fraction. However, identification of 30 such apparently enriched proteins by two-dimensional (2-D) electrophoresis, proteolytic digestion, and mass spectrometry revealed that only eight were plasma membrane proteins, the remaining 22 being contaminants. In contrast, nitrogen cavitation followed by sequential centrifugation and solubilization of proteins with sodium dodecyl sulfate (SDS) and 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS) detergent yielded subcellular fractions, including microsomes, that showed little overlap in constituent proteins as revealed by 2-D electrophoresis. These results highlight the importance of obtaining pure plasma membranes and complete solubilization of membrane proteins for proteomic analysis.     

Up-regulation of Bax and endonuclease G, and down-modulation of Bcl-XL involved in cardiotoxin III-induced apoptosis in K562 cells

Cardiotoxin III (CTX III), a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, has been reported to have anticancer activity. CTX III-induced K562 cell apoptosis was confirmed by DNA fragmentation (DNA ladder, sub-G1 formation) and phosphatidylserine (PS) externalization with an IC(50) value of 1.7 microg/ml at 48 h. A mechanistic analysis demonstrated that CTX III-induced apoptotic cell death was accompanied by up-regulation of both Bax and endonuclease G (Endo G), and downregulation of Bcl-X(L). CTX III had no effect on the levels of Bcl-2, Bid, XIAP survivin, and AIF proteins. CTX III treatment caused loss of the mitochondrial membrane potential (DeltaPsim), release of mitochondrial cytochrome c to the cytosol, and activation of both caspase-9 and -3. CTX III-induced apoptosis was significantly blocked by the broad-spectrum caspase inhibitor Z-VAD-FMK. However, CTX III did not generate reactive oxygen species (ROS) and antioxidants, including N-acetylcysteine and catalase, did not block CTX III-induced apoptosis in K562 cells. Modulation of Bax, Bcl-XL, and the Endo G proteins, release of mitochondrial cytochome c, and activation of caspase-3 and -9 all are involved in the CTX III-triggered apoptotic process in human leukemia K562 cells.     

High-performance liquid chromatography assays for desmethoxyyangonin, methysticin, kavain and their microsomal metabolites

Three novel, simple and reproducible high-performance liquid chromatography quantitative assays with UV detection were developed and validated for three major kavalactones--desmethoxyyangonin, methysticin and kavain--in rat liver microsomes using diazepam as an internal standard; liquid-liquid extraction was used for sample preparation and analysis was performed on a Shimadzu 10A high-performance liquid chromatography system. The analysis was carried out in reversed-phase mode with a Luna C(18) column (150 x 2.00 mm, 3 microm) at 40 degrees C. The limit of quantitation was 0.1 microg/mL using 0.25 mL of microsomal solution. The assays were linear over the range 0.1-10 microg/mL for desmethoxyyangonin, methysticin and kavain. Quality control samples exhibited good accuracy and precision with relative standard deviations lower than 15% and recoveries between 85 and 105%. The assays exhibited satisfactory performance with high sensitivity for quantifying desmethoxyyangonin, methysticin and kavain in rat liver microsomes and were successfully used to determine the three kavalactones and their microsomal metabolites.     

Chiral gas chromatographic assay with flame ionization detection for amphetamine enantiomers in microsomal incubates

A chiral assay for amphetamine enantiomers in rat liver microsomal incubates is based on derivatization with (S)-(-)-N-(trifluoroacetyl)-prolyl chloride (S-TFPC), capillary chromatographic separation of the diastereomeric amide derivatives, and detection by a flame ionization detector. The method is capable of detecting low levels of S- or R-amphetamine. The assay is linear from 5 to 250 micrograms/mL for each enantiomer, and the limit of detection is 0.5 microgram/mL. The analytical method affords the average recoveries of 77.53 +/- 5.22% for R-amphetamine and 74.47 +/- 3.08% for S-amphetamine. The method allows the study of the metabolic depletion of S- and R-amphetamine in rat liver microsomal incubates. The time-dependent concentration of amphetamine enantiomers in rat liver microsomes was determined, and the stereoselectivity of amphetamine phase I metabolism was observed.     

Studies on toxicological mechanisms of gas-phase cigarette smoke and protection effects of GTP

Gas-phase cigarette smoke (GPCS) was able to induce lipid peroxidation in lecithin liposomes, rat liver microsomes, and rat lung cells (RLC), and change the membrane fluidity of RLCs. Lipid free radicals were trapped in a GPCS-treated microsomal suspension by using 4-POBN as the spin trap. In addition, it was found that GPCS-peroxidized liposomes in appropriate degree of lipid peroxidation had the ability to increase the generation of superoxide anions in rat peritoneal neutrophils (RPN). Effects of green tea polyphenols (GTP) on the GPCS-induced damages were investigated The results showed that GTP was capable of inhibiting the GPCS-induced damages.     

Evaluation of the suitability of low hazard surfactants for the separation of phenols and carotenoids from red‐flesh orange juice and olive mill wastewater using cloud point extraction

Natural antioxidants derived from plant sources attract considerable scientific interest. While classic extraction methods consume high volumes of toxic organic solvents, cloud point extraction requires surfactant not exceeding 15% of the waste volume. In preliminary tests, the suitability of various low hazard surfactants (Span 20, PEG 400, Tween 80 and 20) was explored for separation of phenols and carotenoids from olive mill wastewater and red‐flesh orange juice. Tween 80 showed the highest recovery and further applied to the next experiments. The most appropriate surfactant concentrations were 5% (for olive mill wastewater) and 7% (for orange juice) as indicated by recovery % and the rest cloud point extraction parameters (analyte concentration, concentration factor, and phase volume ratio). A double step CPE with 5% + 5% of Tween 80 recovered up to 94.4% of the total phenols from olive mill wastewater, while a 7% + 7% of Tween 80 recovered up to 72.4% of the total carotenoids from orange juice. Evaluation of the final effects and extraction efficiency of single and double step cloud point extraction shows that double step scheme seems to be preferable in both cases. Finally, phenols and carotenoids recovered by Tween 80 maintained high antiradical activity (DPPH test).     

Oxidation-reduction potential of soluble and membrane-bound rabbit liver microsomal cytochrome P-450 LM2

The redox midpoint potentials of rabbit liver microsomal cytochromes P-450 and of soluble and membrane-bound rabbit liver microsomal cytochrome P-450 LM2 were determined using EPR-spectroscopy and absorption difference spectrometry with NADPH or dithionite as reductants. Using EPR, a redox midpoint potential of -0.36 V was obtained both for the low spin and the high spin components of microsomal cytochrome P-450. Spectrophotometrical determinations yielded very similar values: -0.37 V and -0.34 V for the low and high spin signals, respectively. Soluble cytochrome P-450 LM2 had a midpoint potential of -0.32 V. This redox potential was not significantly affected by incorporation of the protein into an artificial membrane structure or, furthermore, by the presence of cytochrome b5 the same membrane.     

Local unitary transformation method for large-scale two-component relativistic calculations: case for a one-electron Dirac Hamiltonian

An accurate and efficient scheme for two-component relativistic calculations at the spin-free infinite-order Douglas-Kroll-Hess (IODKH) level is presented. The present scheme, termed local unitary transformation (LUT), is based on the locality of the relativistic effect. Numerical assessments of the LUT scheme were performed in diatomic molecules such as HX and X(2) (X = F, Cl, Br, I, and At) and hydrogen halide clusters, (HX)(n) (X = F, Cl, Br, and I). Total energies obtained by the LUT method agree well with conventional IODKH results. The computational costs of the LUT method are drastically lower than those of conventional methods since in the former there is linear-scaling with respect to the system size and a small prefactor.     

Use of A New HPLC Method in Rat Liver Microsomal Testosterone Monooxygenation and Its Application to Study the Sex Dependent Expression of Several Hydroxylases

Abstract

An HPLC method described by Mancilla and Gil [Analytical Letters 17, (B9), 1984, 873-886] has been applied to study the sex dependent expression of several rat liver testosterone hydroxylases. A sample clean up procedure has been developed using SEP-PACK C- 18 cartridges which retained testosterone and its microsomal oxidative products. Undesired components were not retained or selectivly eluted with organic solvents. The clean steroid sample was eluted with a mixture of n-hexane and 2-propanol. HPLC of testosterone microsomal oxidation products was performed by normal phase In a Lichrosorb diol column using an Isocratic mixture of n-hexane and 2-propanol. The main five testosterone metabolites produced by male and female rat liver microsomes were determined In only 24 min. The turnover rates for testosterone oxidation were similar in male and female microsomes, but significant differences were observed in the rate of production of different metabolites. Male microsomes catalized mainly oxidation at positions 2 α, and 7 α; whereas female microsomes produced mainly 7 α OHT and androstenedione. These results might be explained by the different contribution of some cytochrome P-450 isozymes in microsomes from the different sexes. This method provides a useful tool to study the P-450 isozymic contributions to microsomal activities in different tissues and might facilitate the comparison of P-450 isozymes purified in different laboratories.     

Development of a rapid and sensitive high-performance liquid chromatographic method to determine CYP2D6 phenotype in human liver microsomes

Dextromethorphan is a probe substrate to determine CYP2D6 phenotype. The conversion of dextromethorphan to dextrorphan by CYP2D6 accounts for approximately 60% of total metabolism. Most analytical methods utilize complicated labor- and time-intensive sample processing methods with several liquid-liquid extraction (LLE) steps. Our goal was to develop a non-LLE based rapid and sensitive HPLC method, to measure dextromethorphan metabolism in human liver microsomes. A solid-phase filtration based reverse-phase HPLC method with fluorescence detection was developed and validated. Human liver (n = 6) microsomal incubations were carried out with dextromethorphan, under optimum conditions. The analytes were separated by one-step centrifugal filtration with Nanosep separation units. The filtrate was injected ( 50 microL) into a Waters Alliance 2690 HPLC system. Metabolic incubations were also conducted to determine levels using LLE for comparisons. The Nanosep separation step reduced the extraction time from 3h to 40 min. The limit of quantitation was 23.8 nM (9.7 ng/mL), recovery was approximately 98%, the mean precision values were <10% RSD for the controls (80, 320 and 640 nM) and mean percentage error was <5%. Michaelis-Menten parameters were determined to distinguish CYP2D6 phenotypes. A rapid and sensitive HPLC method is reported, which may be suitable for automation and allows phenotyping of human liver microsomes.     

Controlling the water content of never dried and reswollen bacterial cellulose by the addition of water-soluble polymers to the culture medium

For the modification of medically useful biomaterials from bacterially synthesized cellulose, fleeces of Acetobacter xylinum have been produced in the presence of 0.5, 1.0, and 2.0% (m/v) carboxymethylcellulose (CMC), methylcellulose (MC), and poly(vinyl alcohol) (PVA), respectively, in the Hestrin-Schramm culture medium. The incorporation of the water-soluble polymers into cellulose and their influence on the structure, crystal modifications, and material properties are described. With IR and solid-state ¹³C NMR spectroscopy of the fleeces, the presence of the cellulose ethers and an increase in the amorphous parts of the cellulose modifications (NMR results) have been detected. The incorporation is represented by a higher product yield, too. As demonstrated by scanning electron microscopy, a porelike cellulose network structure forms in the presence of CMC and MC. This modified structure increases the water retention ability (expressed as the water content), the ion absorption capacity, and the remaining nitrogen-containing residues from the culture medium or bacteria cells. The water content of bacterial cellulose (BC) in the never dried state and the freeze-dried, reswollen state can be controlled by the CMC concentration in the culture solution. The freeze-dried, reswollen BC-CMC (2.0%) contains 96% water after centrifugation, whereas standard BC has only 73%. About 98% water is included in a BC-MC composite in the wet state, and about 93% is included in the reswollen state synthesized in the presence of 0.5, 1.0, or 2.0% MC. These biomaterial composites can be stored in the dried state and reswollen before use, reaching a higher water absorption than pure, never dried BC. The copper ion capacity of BC-CMC composites increases proportionally with the added amount of CMC. BC-CMC (0.5%) can absorb 3 times more copper ions than original BC. In the case of 0.5 and 1.0% PVA additions to the culture solution, this polymer cannot be detected in the cellulose fleeces after they are washed. Nevertheless the presence of PVA in the culture medium effects a decreased product yield, a retention of nitrogen-containing residues in the material during purification, a reduced water absorption ability, and a slightly higher copper ion capacity in comparison with original BC. The water content of freeze-dried, reswollen BC-PVA (0.5%) is only 62%. © 2003 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 463–470, 2004