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HPLC Analysis (hplc + analysis)
Selected AbstractsHPLC Analysis of Catechins, Theaflavins, and Alkaloids in Commercial Teas and Green Tea Dietary Supplements: Comparison of Water and 80% Ethanol/Water ExtractsJOURNAL OF FOOD SCIENCE, Issue 6 2006Mendel Friedman ABSTRACT:, To help meet the needs of consumers, producers of dietary tea supplements, and researchers for information on health-promoting tea compounds, we compared the following conditions for the extraction of tea leaves and green tea-containing dietary supplements: 80% ethanol/water at 60 °C for 15 min and boiled water for 5 min. The following 7 catechins, 4 theaflavins, and 3 alkaloids were separated in a 70-min single HPLC analysis: (,)-epigallocatechin, (,)-catechin, (+)-epicatechin, (,)-epigallocatechin-3-gallate, (,),gallocatechin-3-gallate, (,)-epicatechin-3-gallate, (,)-catechin-3-gallate, theaflavin, theaflavin-3-gallate, theaflavin-3,-gallate, theaflavin-3,3,-digallate, caffeine, theobromine, and theophylline. The following ranges of concentrations of flavonoids (catechins plus theaflavins) in the tea leaves extracted with 80% ethanol were observed (in mg/g): in 32 black teas, 19.8 to 115.1; in 24 green teas, 12.3 to 136.3; in 14 specialty teas, 4.9 to 118.5; in 7 herbal teas, 0 to 46.0. Total alkaloids in all teas ranged from 0 to 32.6 mg/g. Significantly greater amounts of flavonoids were extracted from the tea leaves with aqueous ethanol than with boiled water. Levels of tea catechins in 10 capsules sold as dietary supplements were about 50 to 75% lower than the amounts listed on the labels. Catechin content of 4 commercial green tea extracts ranged from 96 to 696 mg/g. The results make it possible to maximize the extraction of tea compounds to better relate the flavonoid and alkaloid content of teas and dietary tea supplements to their health-promoting effects. [source] CE- and HPLC-TOF-MS for the characterization of phenolic compounds in olive oilELECTROPHORESIS, Issue 5 2007Alegría Carrasco-Pancorbo Abstract We present an easy and rapid method for the analysis of phenolic compounds in extra-virgin olive oil by CZE coupled with ESI-TOF-MS. Optimum electrophoretic separation was obtained using a basic carbonate electrolyte. We thus achieved the determination of several important families (phenyl alcohols, phenyl acids, lignans, flavonoids, and secoiridoids) of the polar fraction of the olive oil. Furthermore, other "unknown" compounds were also identified. In addition to the CZE method, HPLC analyses were made, separating compounds belonging to the main families present in this polyphenolic fraction, as well as other new compounds. We compared the results obtained with both techniques and found it was possible to determine more than 45 compounds with both methods. The sensitivity, together with mass accuracy and true isotopic pattern of the TOF-MS, allowed the identification of a broad series of known and so far not described phenolic compounds present in extra-virgin olive oil. [source] Thrombin-mediated impairment of fibroblast growth factor-2 activityFEBS JOURNAL, Issue 12 2009Pierangela Totta Thrombin generation increases in several pathological conditions, including cancer, thromboembolism, diabetes and myeloproliferative syndromes. During tumor development, thrombin levels increase along with several other molecules, including cytokines and angiogenic factors. Under such conditions, it is reasonable to predict that thrombin may recognize new low-affinity substrates that usually are not recognized under low-expression levels conditions. In the present study, we hypothesized that fibroblast growth factor (FGF)-2 may be cleaved by thrombin and that such action may lead to an impairment of its biological activity. The evidence collected in the present study indicates that FGF-2-induced proliferation and chemotaxis/invasion of SK-MEL-110 human melanoma cells were significantly reduced when FGF-2 was pre-incubated with active thrombin. The inhibition of proliferation was not influenced by heparin. Phe-Pro-Arg-chloromethyl ketone, a specific inhibitor of the enzymatic activity of thrombin, abolished the thrombin-induced observed effects. Accordingly, both FGF-2-binding to cell membranes as well as FGF-2-induced extracellular signal-regulated kinase phosphorylation were decreased in the presence of thrombin. Finally, HPLC analyses demonstrated that FGF-2 is cleaved by thrombin at the peptide bond between residues Arg42 and Ile43 of the mature human FGF-2 sequence. The apparent kcat/Km of FGF-2 hydrolysis was 1.1 × 104 m,1·s,1, which is comparable to other known low-affinity thrombin substrates. Taken together, these results demonstrate that thrombin digests FGF-2 at the site Arg42-Ile43 and impairs FGF-2 activity in vitro, indicating that FGF-2 is a novel thrombin substrate. [source] Evidence for an amphipathicity independent cellular uptake of amphipathic cell-penetrating peptidesFEBS JOURNAL, Issue 19 2000Anne Scheller The cellular uptake of a peptide set derived from membrane-permeable ,-helical amphipathic peptides by stepwise alterations of structure forming propensity and charge was studied by confocal laser scanning microscopy (CLSM) combined with HPLC. For CLSM monitoring, an online protocol was employed that avoided bias of the uptake results by washout. Using this protocol, extensive fluorescence, approaching the intensity of the external peptide, was observed in the cytosol and nucleus within minutes in all cases, irrespective of the degree of amphipathicity. HPLC analyses of the cell lysates revealed the unmetabolized peptides to be the predominant source of the intracellular fluorescence. Significant amphipathicity-dependent differences became apparent only after washing the peptide-loaded cells, reflecting the effects of amphipathicity on resistance to wash out. Exposure of the cells to the peptides at 37 and 0 °C led to similar results, indicating the nonendocytic character of the uptake. With a view to practical applications , the results of the present study open the possibility of exploiting nonamphipathic peptides as vectors for translocating polar compounds into the cell interior, which would circumvent substantial obstacles currently connected with the use of amphipathic vector peptides, such as membrane toxicity and low solubility. Moreover, differences in the uptake of several members of the investigated peptide series into different cell types present a promising basis for the design of cell-type specific vector peptides. [source] Production of oxalates In Vitro by Microbes Isolated from Rock Surfaces with prehistoric paints in the Lower Pecos Region, TexasGEOARCHAEOLOGY: AN INTERNATIONAL JOURNAL, Issue 1 2008Darren Hess Calcium oxalate-rich rock coatings are ubiquitous on limestone inside dry rock shelters and under bluff overhangs along canyon walls in southwestern Texas. Prehistoric pictographs occur in more than 250 such sites, and the ancient paints are encapsulated within the natural rock coating. Previous studies suggest lichens were the source of the oxalate; however, we report here that microbes cultured and isolated from samples of the coating produce oxalate in vitro. Twenty different bacteria species have been identified in samples from eight different sites, with Bacillus the most common genus, represented by five species. HPLC analyses of inoculated R2B medium after eight months of bacterial growth revealed the presence of oxalate ions in the solid phase of the growth medium. © 2008 Wiley Periodicals, Inc. [source] Methotrexate induced differentiation in colon cancer cells is primarily due to purine deprivationJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2006R. Singh Abstract The folate antagonist methotrexate (MTX) inhibits synthesis of tetrahydrofolate (THF), pyrimidines and purines, and induces differentiation in several cell types. At 1 µM, MTX reduced proliferation and induced differentiation in HT29 colon cancer cells; the latter effect was augmented (P,<,0.001) by thymidine (100 µM) but was reversed (P,<,0.001) by the purines, hypoxanthine (Hx; 100 µM) and adenosine (100 µM). In contrast 5-fluoro-uracil (5-FU), a specific thymidylate synthase (TS) inhibitor, had no effect on differentiation, suggesting that MTX-induced differentiation is not due to a reduction in thymidine but to the inhibition of purine biosynthesis. Inhibition of cyclic AMP (cAMP) by RpcAMP (25 µM) further enhanced (P,<,0.001) MTX induced differentiation, whereas the cAMP activator forskolin (10 µM) reversed (P,<,0.001) MTX induced differentiation. These observations implicate a central role of adenosine and cAMP in MTX induced differentiation. By combining Western blot analysis with liquid chromatography-mass spectrometry (LC-MS)and HPLC analyses we also reveal both the expression and activity of key enzymes (i.e. methionine synthase (MS), s-adenosylhomocysteinase, cystathionine ,-synthase and ornithine decarboxylase) regulating methyl cycle, transsulfuration and polyamine pathways in HT29 colon cancer cells. At 1 µM, MTX induced differentiation was associated with a marked reduction in the intracellular concentrations of adenosine and, consequently, S-adenosylmethionine (SAM), S-adenosylhomocysteine, polyamines and glutathione (GSH). Importantly, the marked reduction in methionine that accompanied MS inhibition following MTX treatment was non-limiting with respect to SAM synthesis. Collectively, these findings indicate that the effects of MTX on cellular differentiation and single carbon metabolism are primarily due to the intracellular depletion of purines. J. Cell. Biochem. © 2006 Wiley-Liss, Inc. [source] Safety of Polyethylene Terephthalate Food Containers Evaluated by HPLC, Migration Test, and Estimated Daily IntakeJOURNAL OF FOOD SCIENCE, Issue 6 2008H.-J. Park ABSTRACT:, A comparative high-pressure liquid chromatography (HPLC) analysis of monomers, terephthalic acid (TPA), isophthalic acid (IPA), and dimethyl terephthalate (DMT) from polyethylene terephthalate (PET) food containers was conducted. Monomer linearities and sensitivities were calibrated between established and novel HPLC analyses. Safety of PET containers was evaluated with newly established detection methods for TPA, IPA, and DMT. Migration of the 3 monomers into food simulants (water, 4% acetic acid, 20% alcohol, and n-heptane) from 56 PET containers collected from open markets was monitored. Migrated monomers were not detected over 0.1 ppm of detection limit. The corresponding estimated daily intake was measured to confirm the safety of these publicly available PET containers and to permit comparison to the specific migration limit of the European Union. The estimated daily intake of 3 monomers migrating from PET was 0.0384 mg/kg each. This represented only 0.6% of the European Union's specific migration limit, confirming the safety of the examined containers. [source] Sol,gel microextraction phases for sample preconcentration in chromatographic analysisJOURNAL OF SEPARATION SCIENCE, JSS, Issue 19 2010Scott S. Segro Abstract Sol,gel technology provides a simple and reliable method for solid-phase microextraction (SPME) fiber preparation through in situ creation of surface-bonded organic,inorganic hybrid coatings characterized by enhanced thermal stability and solvent-resistance properties that are important for the coupling of SPME with GC and HPLC, respectively. The sol,gel coating technology has led to the development of an extensive array of sol,gel sorbent coatings for SPME. In this article, sol,gel microextraction coatings are reviewed, with particular attention on their synthesis, characterization, and applications in conjunction with GC and HPLC analyses. In addition, the development of sol,gel-coated stir bars, their inherent advantages, and applications are discussed. Next, the development and applications of sol,gel capillary microextraction (CME) in hyphenation with GC and HPLC is extensively reviewed. The newly emerging germania- and titania-based sol,gel microextraction phases look promising, especially in terms of pH and hot solvent stability. Finally, sol,gel monolithic beds for CME are reviewed. Such monolithic beds are in a position to greatly improve the extracting capabilities and enhanced sensitivity in CME. [source] Exogenous ethylene stimulates the long-term expression of genes related to anthocyanin biosynthesis in grape berriesPHYSIOLOGIA PLANTARUM, Issue 2 2003Ashraf El-Kereamy The treatment of grape berries (Vitis vinifera L. cv. Cabernet Sauvignon) with the ethylene-releasing compound, 2-chloroethylphosphonic acid (2-CEPA), at veraison is a method known to enhance grape skin colour. We observed that it produced a 6-fold increase, up to 30 pmol g,1 FW, of the cluster internal ethylene compared to untreated controls within the 24 h following treatment. This ethylene upsurge was associated with increased levels of chalcone synthase (CHS) and flavanone 3-hydroxylase (F3H) transcripts, which persisted over the following 20 days. Transcript levels of leucoanthocyanidin dioxygenase (LDOX) and UDP glucose-flavonoid 3- O -glucosyl transferase (UFGT) were similarly enhanced by 2-CEPA, although to a lesser extent. The effect on UFGT was confirmed at the protein level by an immunoblot analysis. The transcript accumulation of dihydroflavonol 4-reductase (DFR) was unaffected by 2-CEPA treatment. Examination of the levels of CHS, F3H and UFGT mRNAs in berries during bunch exposure to ethylene, revealed elevated levels of each transcript within the first 6 h of treatment when compared to nonethylene-treated controls. HPLC analyses of berry skin extracts showed that levels of each of the anthocyanins analysed (delphinidin, cyanidin, petunidin, peonidin and malvidin) increased over the 10 days following the ethylene burst, and decreased thereafter. However, anthocyanin levels at harvest were still higher in ethylene treated grapes than in controls. This data is the first evidence that ethylene triggers gene expression related to anthocyanin synthesis in grapes, and in addition, our results also confirm the existence of other regulatory modes in the anthocyanin biosynthetic pathway. [source] Identification of an arsenic tolerant double mutant with a thiol-mediated component and increased arsenic tolerance in phyA mutantsTHE PLANT JOURNAL, Issue 6 2007Dong-Yul Sung Summary A genetic screen was performed to isolate mutants showing increased arsenic tolerance using an Arabidopsis thaliana population of activation tagged lines. The most arsenic-resistant mutant shows increased arsenate and arsenite tolerance. Genetic analyses of the mutant indicate that the mutant contains two loci that contribute to arsenic tolerance, designated ars4 and ars5. The ars4ars5 double mutant contains a single T-DNA insertion, ars4, which co-segregates with arsenic tolerance and is inserted in the Phytochrome A (PHYA) gene, strongly reducing the expression of PHYA. When grown under far-red light conditions ars4ars5 shows the same elongated hypocotyl phenotype as the previously described strong phyA-211 allele. Three independent phyA alleles, ars4, phyA-211 and a new T-DNA insertion allele (phyA-t) show increased tolerance to arsenate, although to a lesser degree than the ars4ars5 double mutant. Analyses of the ars5 single mutant show that ars5 exhibits stronger arsenic tolerance than ars4, and that ars5 is not linked to ars4. Arsenic tolerance assays with phyB-9 and phot1/phot2 mutants show that these photoreceptor mutants do not exhibit phyA -like arsenic tolerance. Fluorescence HPLC analyses show that elevated levels of phytochelatins were not detected in ars4, ars5 or ars4ars5, however increases in the thiols cysteine, , -glutamylcysteine and glutathione were observed. Compared with wild type, the total thiol levels in ars4, ars5 and ars4ars5 mutants were increased up to 80% with combined buthionine sulfoximine and arsenic treatments, suggesting the enhancement of mechanisms that mediate thiol synthesis in the mutants. The presented findings show that PHYA negatively regulates a pathway conferring arsenic tolerance, and that an enhanced thiol synthesis mechanism contributes to the arsenic tolerance of ars4ars5. [source] Pharmacokinetics of luteolin and tetra-acetyl-luteolin assayed by HPLC in rats after oral administrationBIOMEDICAL CHROMATOGRAPHY, Issue 8 2010Xiujie Chen Abstract Accurate and reproducible HPLC methods were developed and validated for the determination of concentrations of luteolin (LT) and tetra-acetyl-luteolin (TALT) in rat plasma. HPLC analyses were performed on an Agilent TC-C18 column protected by a guard Agilent Zorbax Eclipse Plus. The mobile phase for LT was a binary mixture of acetonitrile,water (40:60, v/v) containing 0.5% phosphoric acid at a flow rate of 1.0,mL/min, and that for TALT was a binary mixture of methanol,water (70,:,30, v/v) containing 0.5% glacial acetic acid at the same flow rate. The UV detection wavelength for both analytes was set at 350,nm. The calibration curve was linear over the range of 40,1800,ng/mL, the lower limit of quantitation was 40,ng/mL and the lower limit of detection was 20,ng/mL for both LT and TALT. The intra- and inter-day precision (RSD) values for all samples were within 7.9%. The concentration,time curves of LT and TALT after oral administration (30,mg/kg) were both fitted to a two-compartment model. The pharmacokinetic characteristics of TALT were better than that of LT in the maximum plasma concentration (Cmax) and the area under the concentration,time curve (AUC). Copyright © 2010 John Wiley & Sons, Ltd. [source] FS01.3 Disperse (yes), orange (yes), 3 (no): what do we test in textile dye dermatitis?CONTACT DERMATITIS, Issue 3 2004Christophe J Le Coz Introduction:, Patients sensitized to para-phenylenediamine (PPD) have a high degree of patch test reactivity to Disperse Orange 3 (DO3), and a lesser one to Disperse Red 1 and Red 17. Two successive patients positive to PPD, Disperse Red 1 and 17, negative to DO3 were real eye-openers for our considerations about purity of our current allergen DO3. Materials and methods:, We realized comparative thin-layer chromatography (TLC), with DO3 from Chemotechnique®(DO3-Chem) and Trolab®(both extracted from petrolatum), and "pure" DO3 from two chemical providers. TLC clearly indicated that DO3-Chem was not DO3. HPLC analysis with pure DO3 from Chemotechnique® and comparison of structures by NMR with samples of DO3, revealed that DO3-Chem was Disperse Orange 31 (DO31). In addition, signals through the GERDA network allowed the collection of test materials and observations. Among other members, only 2 used DO3-Chem (from 2 different batches) that was DO31 too, according to TLC Results: According to their data, they observed no or a lower reactivity to DO3 than expected (4 patients DO3-Chem + among 23 PPD+ e.g.). Finally, the error was proved to be due to the provider of the dye to Chemotechnique®, who likely deleted the 1 of Disperse Orange 31 on his packaging. Discussion:, Chemical structure of DO31 indicates a possible in vivo hydrolysis into nitroaniline and a second compound, a substituted PPD derivative that clearly does not frequently react in PPD positive patients. Like drugs, patch tests are submitted to post-commercialization controls. In addition to allergens providers who should enhance their quality controls, dermato-allergologists have to be vigilant, and must active networks when they observe a rare bird. [source] Interplay of constitutively released nucleotides, nucleotide metabolism, and activity of P2Y receptorsDRUG DEVELOPMENT RESEARCH, Issue 2-3 2001Eduardo R. Lazarowski Abstract At least six mammalian P2Y receptors exist that are specifically activated by ATP, UTP, ADP or UDP. Although the existence of ectoenzymes that rapidly metabolize extracellular nucleotides is well established, the relative flux of ATP and UTP through their extracellular metabolic products remains undefined. In addition, the existence of basal nucleotide release and the contribution of resting levels of ATP and UTP to P2 receptor activation are poorly understood. In the absence of exogenous agonists, an apyrase-sensitive inositol phosphate accumulation was observed in resting 16HBE14o, human bronchial epithelial cells endogenously expressing P2Y receptors and in 1321N1 human astrocytoma cells expressing a recombinant P2Y2 receptor. To test whether nucleotide release may account for basal P2 receptor activities, the rates of extracellular accumulation and metabolism of endogenous ATP were examined with resting 16HBE14o,, C6 rat glioma, and 1321N1 cell cultures. Although extracellular ATP concentrations (1-5 nM) remained unchanged for up to 12 h, [,32P] ATP included in the medium (as a radiotracer) was completely degraded within 120 min, indicating that ATP release balanced ATP hydrolysis. The calculated basal rates of ATP release ranged from 20 to 200 fmol/min per million cells. HPLC analysis during steady state revealed that the gamma-phosphate of ATP was reversibly transferred to species further identified as UTP and GTP, implicating ecto-nucleoside diphosphokinase (NDPK)-catalyzed phosphorylation of endogenous UDP and GDP. At steady state, the final 32P-products of [,32P]ATP metabolism were 32P-orthophosphoric acid and a species further purified and identified as 32P-pyrophosphate. Constitutive nucleotide release balanced by the concerted activities of ecto-ATPase, ecto-ATP pyrophosphatase, and ecto-NDPK may determine the resting levels of extracellular nucleotides and therefore, the basal activity of P2 receptors. Drug Dev. Res. 53:66,71, 2001. © 2001 Wiley-Liss, Inc. [source] Sampling and analysis of microcystins: Implications for the development of standardized methodsENVIRONMENTAL TOXICOLOGY, Issue 2 2007Angeline R. Tillmanns Abstract Microcystins (MC), a group of cyanotoxins, have been found in lakes and rivers worldwide. One goal of MC research is to develop models which predict MC concentrations, but these efforts have been hampered by a lack of standardized methods necessary for comparing data across studies. Here, we investigate the effect of chemical analysis (HPLC-PDA and ELISA), sample collection (whole water, plankton tow and surface scum), and choice of normalizing parameter (volume, dry weight, and chlorophyll a) on reported MC concentrations. Samples were collected over three years from a temperate mesotrophic, shallow lake with episodic blooms of cyanobacteria. We found that microcystins were up to four times higher in lake samples when analyzed by ELISA relative to HPLC-PDA and that MC concentration measured by HPLC explained less than half of the variation in MC concentrations measured by ELISA. Also, samples collected by plankton tow gave consistently higher concentrations than whole water samples. An additional HPLC analysis of two chlorophyte cultures revealed the presence of compounds with a similar UV absorbance spectrum to MC-LR, suggesting that identifying MC based solely on UV absorbance is not valid. Our results document the discrepancy in MC concentrations that can arise by using different methods throughout all stages of sampling, analysis, and reporting of MC concentrations. © 2007 Wiley Periodicals, Inc. Environ Toxicol 22: 132,143, 2007. [source] Kinetics of Bis(p -nitrophenyl)phosphate (BNPP) Hydrolysis Reactions with Trivalent Lanthanide Complexes of N -Hydroxyethyl(ethylenediamine)- N,N,,N, -triacetate (HEDTA),EUROPEAN JOURNAL OF INORGANIC CHEMISTRY, Issue 8 2009C. Allen Chang Abstract Kinetic studies of hydrolysis reactions of BNPP [sodium bis(p -nitrophenyl)phosphate] with trivalent lanthanide (Ln3+) complexes of HEDTA [HEDTA = N -hydroxyethyl(ethylenediamine)- N,N,,N, -triacetate] were performed at pH 6.96,11.34 and 25 °C by a spectrophotometric method and by HPLC analysis. The reaction rates increase with increasing atomic number of lanthanide and solution pH from PrHEDTA to EuHEDTA and then decrease for heavier LnHEDTA complexes. Plots of pseudo-first-order rate constants (kobs) vs. pH could be fitted to the equation kobs = kLnL(OH)[LnL]T/{1,+,exp[,2.303(pH,,,pKh)]}, where kLnL(OH) is the rate constant for the reaction of LnHEDTA(OH), with BNPP, Kh is the hydrolysis constant of LnHEDTA, and [LnL]T is the total concentration of LnHEDTA. The pKh values obtained by the kinetic method are in the range 8.2,10.3 and are similar to those measured by potentiometric methods. At [LnL]T = 10,70 mM and pH 10.5, most of the observed pseudo-first-order rate constants could be fitted to a simple saturation kinetic model, kobs = k1K[LnHEDTA(OH),]/{1 + K[LnHEDTA(OH),]}, where K is the equilibrium constant for the formation for LnHEDTA(OH),BNPP and is in the range 2,147 M,1. The k1 values are in the range 1.12,×,10,5,2.71,×,10,3 s,1. The kobs data for TbHEDTA and HoHEDTA were fitted to a quadratic equation. It was observed that the dinuclear species are more reactive. ESI mass spectrometry confirmed that the reaction between BNPP and EuHEDTA is a simple hydrolysis but not a transesterification, presumably because the three inner-sphere coordinated water molecules are far away from the coordinated hydroxyethyl group. Hydrolysis is likely to occur by proton transfer from one inner-sphere coordinated water molecule to the deprotonated ethyl oxide group followed by nucleophilic attack of the resulting hydroxide ion on the bonded BNPP anion.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009) [source] Triacylglycerol migration and bloom in filled chocolates: Effects of low-temperature storageEUROPEAN JOURNAL OF LIPID SCIENCE AND TECHNOLOGY, Issue 3 2009Frédéric Depypere Abstract This study investigated the effect of storage temperature on triacylglycerol (TAG) migration, visual fat bloom and taste of industrially produced milk chocolates with a hazelnut-based filling. The chocolates were stored for up to 10,months at 18,°C, either directly after production or with the inclusion of a variable time at ,20 or 4,°C immediately after production and prior to further storage at 18,°C. TAG migration from the filling through the chocolate shell was quantified by HPLC analysis of chocolate sampled from the chocolates' surface. Both [OOO/SOS] and [LOO/SOS] were used as markers for oil migration. Compared to storage at 18,°C only, chilling or freezing of the chocolates for part of the storage time was found to reduce the amount of TAG migration. Effects on diffusion, capillary transport and TAG immobilization during the thermal treatment can be raised as possible reasons for this decrease. Furthermore, storage at ,20,°C decreased oil migration during subsequent storage at 18,°C. This suggests a crystallization effect during the storage at ,20,°C, leading to permanent (micro)structural changes. Although a thermal treatment at 4,°C compared to ,20,°C was less effective in retarding TAG migration, storage at low positive temperatures immediately after production appears already beneficial in the prevention of visual fat bloom. Adverse effects of the thermal treatments on the chocolates' taste were not observed. [source] Structured lipids from rice bran oil and stearic acid using immobilized lipase from Rhizomucor mieheiEUROPEAN JOURNAL OF LIPID SCIENCE AND TECHNOLOGY, Issue 1 2008Rajni Chopra Abstract The major objective of the present study was to prepare structured lipids rich in stearic acid from rice bran oil (RBO) using immobilized lipase (IM,60) from Rhizomucor miehei. The effects of incubation time and temperature, substrate molar ratio, and enzyme load on incorporation of stearic acid were studied. Acidolysis reactions were performed in hexane. Pancreatic lipase-catalyzed sn -2 positional analysis and tocopherol analyses were performed before and after enzymatic modification. The kinetics of the reaction was studied and maximum incorporation of stearic acid was observed at 6,h, at 37,°C, when the triacylglycerol and stearic acid molar ratio was maintained at 1,:,6 and the enzyme concentration was 10% of total substrates weight. Stearic acid in RBO after acidolysis was increased from 2.28 to 48.5%, with a simultaneous decrease in palmitic, oleic and linoleic acids. HPLC analysis of tocopherols and tocotrienols was carried out and their content in modified RBO was not significantly affected compared to that of native RBO. The oryzanol content of the modified RBO was reduced from 1.02 to 0.68%. Melting and crystallizing characteristics of the modified fat were studied using differential scanning calorimetry. The total solid fat content at 25,°C increased from 26.12 to 34.8% with an increase in stearic acid incorporation into RBO from 38 to 48%, but it was comparatively less than for cocoa butter and vanaspati. However, the modified RBO completely melted at 37,°C and was useful as plastic fat for various culinary purposes, bakery and confectionary applications. The results of the present study indicated that structured lipids prepared from RBO rich in stearic acid retained their beneficial nutraceuticals; in addition, they do not contain any trans fatty acids. [source] Dual metabolic pathway of 25-hydroxyvitamin D3 catalyzed by human CYP24FEBS JOURNAL, Issue 20 2000Toshiyuki Sakaki Human 25-hydroxyvitamin D3 (25(OH)D3) 24-hydroxylase (CYP24) cDNA was expressed in Escherichia coli, and its enzymatic and spectral properties were revealed. The reconstituted system containing the membrane fraction prepared from recombinant E. coli cells, adrenodoxin and adrenodoxin reductase was examined for the metabolism of 25(OH)D3, 1,,25(OH)2D3 and their related compounds. Human CYP24 demonstrated a remarkable metabolism consisting of both C-23 and C-24 hydroxylation pathways towards both 25(OH)D3 and 1,,25(OH)2D3, whereas rat CYP24 showed almost no C-23 hydroxylation pathway [Sakaki, T. Sawada, N. Nonaka, Y. Ohyama, Y. & Inouye, K. (1999) Eur. J. Biochem. 262, 43,48]. HPLC analysis and mass spectrometric analysis revealed that human CYP24 catalyzed all the steps of the C-23 hydroxylation pathway from 25(OH)D3 via 23S,25(OH)2D3, 23S,25,26(OH)3D3 and 25(OH)D3 -26,23-lactol to 25(OH)D3 -26,23-lactone in addition to the C-24 hydroxylation pathway from 25(OH)D3 via 24R,25(OH)2D3, 24-oxo-25(OH)D3, 24-oxo-23S,25(OH)2D3 to 24,25,26,27-tetranor-23(OH)D3. On 1,,25(OH)2D3 metabolism, similar results were observed. These results strongly suggest that the single enzyme human CYP24 is greatly responsible for the metabolism of both 25(OH)D3 and 1,,25(OH)2D3. We also succeeded in the coexpression of CYP24, adrenodoxin and NADPH-adrenodoxin reductase in E. coli. Addition of 25(OH)D3 to the recombinant E. coli cell culture yielded most of the metabolites in both the C-23 and C-24 hydroxylation pathways. Thus, the E. coli expression system for human CYP24 appears quite useful in predicting the metabolism of vitamin D analogs used as drugs. [source] Pigments and proteins in green bacterial chlorosomes studied by matrix-assisted laser desorption ionization mass spectrometryFEBS JOURNAL, Issue 2 2000Søren Persson We have used matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for mass determination of pigments and proteins in chlorosomes, the light-harvesting organelles from the photosynthetic green sulfur bacterium Chlorobium tepidum. By applying a small volume (1 µL) of a concentrated suspension of isolated chlorosomes directly to the target of the mass spectrometer we have been able to detect bacteriochlorophyll a and all the major homologs of bacteriochlorophyll c. The peak heights of the different bacteriochlorophyll c homologs in the MALDI spectra were proportional to peak areas obtained from HPLC analysis of the same sample. The same result was also obtained when whole cells of Chl. tepidum were applied to the target, indicating that MALDI-MS can provide a rapid method for obtaining a semiquantitative determination or finger-print of the bacteriochlorophyll homologs in a small amount of green bacterial cells. In addition to information on pigments, the MALDI spectra also contained peaks from chlorosome proteins. Thus we have been able with high precision to confirm the molecular masses of the chlorosome proteins CsmA and CsmE which have been previously determined by conventional biochemical and genetic methods, and demonstrate the presence of truncated versions of CsmA and CsmB. [source] Circulating EM66 is a highly sensitive marker for the diagnosis and follow-up of pheochromocytomaINTERNATIONAL JOURNAL OF CANCER, Issue 8 2006Johann Guillemot Abstract We have previously demonstrated that measurement of tissue concentration of the novel secretogranin II-derived peptide EM66 may help to discriminate between benign and malignant pheochromocytomas. The aim of the present study was to characterize EM66 in plasma and urine of healthy volunteers and pheochromocytoma patients, in order to further evaluate the usefulness of this peptide as a circulating marker for the management of the tumors. HPLC analysis of plasma and urine samples demonstrated that the EM66-immunoreactive material coeluted with the recombinant peptide. In healthy volunteers, plasma and urinary EM66 levels were, respectively, 2.6 (1.9,3.7) ng/ml and 2.9 (1.9,4.6) ng/ml. In patients with pheochromocytoma, plasma EM66 levels were 10-fold higher than those of healthy volunteers (26.9 (7.3,44) ng/ml), and returned to normal values after removal of the tumor. In contrast, urinary EM66 levels were not significantly different from those of healthy volunteers (3.2 (2.2,3.9) ng/ml). Measurement of total or free plasma metanephrines and 24 hr urinary metanephrines in our series of patients revealed that these tests, taken separately, are less sensitive than the EM66 determination. Pheochromocytes in primary culture secreted high levels of EM66, suggesting that the chromaffin tumor was actually responsible for the increased plasma peptide concentrations in the patients. These data indicate that EM66 is secreted in the general circulation and that elevated plasma EM66 levels are correlated with the occurrence of pheochromocytoma. Thus, EM66 is a sensitive plasma marker that should be considered as a complementary tool in the management of pheochromocytoma. © 2005 Wiley-Liss, Inc. [source] Abstracts: In vitro/in vivo and analytical evaluation of sunless tanning formulations containing different rheology modifiersINTERNATIONAL JOURNAL OF COSMETIC SCIENCE, Issue 5 2010O. V. Dueva-Koganov pp. 73,83 In vitro data suggest that different in vivo performances are expected for two dihydroxyacetone (DHA)-containing formulations with similar concentrations of DHA and excipients but different commercially available rheology modifiers: one with a cationic polymer-based rheology modifier (blend) [dimethylacrylamide/ethyltrimonium chloride methacrylate copolymer (and) propylene glycol dicaprylate/dicaprate (and) PPG-1 trideceth-6 (and) C10-11 isoparaffin]; and the other with a polyacrylamide-based rheology modifier (blend) [polyacrylamide (and) C13-14 isoparaffin (and) laureth-7]. Both rheology modifiers (blends) contained comparable levels of polymers and were used at 3% w/w (as supplied). Differences in color development were illustrated in vitro with respect to the yellow/red and lightness/chroma parameters, which were confirmed in the followup in vivo studies. The test article with the cationic polymer-based rheology modifier produced a more natural sunless tan, comparable to a desirable sun-induced tan, for all panelists, one that was more uniform and lasted longer compared with the sunless tan generated by the test article with the polyacrylamide-based rheology modifier. A method for HPLC analysis of DHA in sunless tanning formulations was established and utilized to confirm concentrations of DHA in test articles. [source] Modulation of expression of LDH isoenzymes in endothelial cells by laminin: Implications for angiogenesisJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2008V.B. Sameer Kumar Abstract Endothelial cell (EC) matrix interaction is critical in angiogenesis. Although matrix components can regulate the process of angiogenesis by acting as a reservoir of various cytokines, it is not clear if extracellular matrix (ECM) can modulate the production and activity of angiogenic cytokines. Investigations were therefore carried out to study the influence of the basement membrane (BM) protein, laminin (Ln) on the activity of vascular endothelial growth factor (VEGF), the major angiogenic cytokine, using isolated human umbilical vein ECs (HUVECs) in culture. Analysis of the biochemical markers of angiogenesis confirmed proangiogenic effect of Ln. The levels of VEGF protein and mRNA were not different in cells maintained on Ln, collagen I or polylysine substrata. Chorioallantoic membrane assay using VEGF isolated from cell extracts however revealed that Ln increased its angiogenic potency. Immunoblotting and HPLC analysis showed considerable reduction in poly adenosyl ribosylation of VEGF associated with a significant decrease in the levels of NAD+, in cells maintained on Ln substrata. Further, a shift in the isoenzymic pattern of LDH towards the B rich forms and an upregulation of LDH B gene were observed in cells maintained on Ln. Ln modulates expression of LDH gene through ,6,4 integrin mediated downstream signaling involving p38 mitogen activated protein kinases (MAPK) pathway. It thus appears that Ln can affect aerobic metabolism of ECs by modulating the expression of LDH isoenzymes resulting in a decrease in the level of NAD+ that can cause a reduction in the poly adenosyl ribosylation of VEGF altering its angiogenic potency. J. Cell. Biochem. 103: 1808,1825, 2008. © 2007 Wiley-Liss, Inc. [source] The rate of uptake of sex steroids from water by Tinca tinca is influenced by their affinity for sex steroid binding protein in plasmaJOURNAL OF FISH BIOLOGY, Issue 1 2005A. P. Scott Two experiments were carried out in which male and female tench Tinca tinca were placed in individual containers and tritiated steroids then added to the water. Water samples were collected over the next 6 or 7 h and the fish then sacrificed, bled and the gall bladder removed. Radioactivity was counted in all the samples. Over the course of the exposure period in the first experiment (7 h), radioactivity of 11-ketotestosterone (11-KT) in the water was depleted by 11%, 17,20,-dihydroxypregn-4-en-3-one (17,20ß-P) and 17,20,-dihydroxypregn-4-en-3-one (17,20,-P) by 28%, testosterone (T) by 56% and androstenedione (AD) by 68%. HPLC analysis of water samples at 3 h indicated that none of the steroids was extensively metabolized during the experiment. Females had a faster rate of uptake of AD than males. In the second experiment (6 h), radioactivity of cortisol in the water was depleted by 5%, 11-KT by 7%, 17-hydroxypregnen-4-ene (17-P) by 17%, 17,-oestradiol (E2) by 35%, T by 37% and AD by 44%. In both experiments, the amounts of radioactivity that were recovered from the gall bladder and plasma were positively correlated with the rate of disappearance of radioactivity from the water. The ability of the steroids to bind to sex steroid binding protein (SBP) of tench plasma was tested by incubating plasma with radioactive steroids and then separating bound and free with ice cold dextran-coated charcoal. When plasma at a final dilution of 1 : 60 (v/v) was incubated with 5 nM of each steroid, the percentage of radiolabel bound to SBP was: T 48% AD 44%, E2 30%, 17-P 17%, 11-KT 13·2%, 17,20,-P 10·3%, 17,20,-P 4·5% and cortisol 0%. Saturation analysis established dissociation constants (Kd; mean ± s.e.) of 3·4 ± 0·4, 2·2 ± 0·2, 4·0 ± 0·3. 9·0 ± 2·8 and 51·8 nM and binding capacities (Bmax) of 201 ± 29, 201 ± 33, 165 ± 3, 187 ± 15 and 13·4 nM for T, AD, E2,17-P and 17,20,-P respectively. The ability of steroids to displace tritiated T and AD from SBP was in the rank order AD > T > E2 > 17,20,P = 17,20,-P = 11-KT = 17-P > cortisol. Thus, the ability of tench plasma to bind certain steroids showed a relatively strong correlation with the ability of the fish to take up these steroids from water. Modelling of data for AD and 17,20,-P helped to show why and how plasma binding had a strong influence on the rate of uptake (and hence release) of the steroids. [source] Retention and Distribution of Polyphenols after Pan-Frying of French Fries in Oils Enriched with Olive Leaf ExtractJOURNAL OF FOOD SCIENCE, Issue 8 2007A. Chiou ABSTRACT:,Palm oil, olive oil, and sunflower oil were supplemented with an extract rich in polyphenols obtained from olive tree (Olea europaea) leaves at levels of 120 and 240 mg total polyphenols per kilogram of oil. Pan-frying of potatoes was performed in both the enriched and the nonsupplemented oils under domestic frying conditions. Total polyphenol content was estimated by the Folin,Ciocalteau assay, oleuropein was determined by HPLC analysis, while other individual polyphenols by GC/MS analysis. Fourteen polyphenol species were identified in the olive leaf extract, among which oleuropein predominated (1.25 g/kg olive leaves). All the enriched oils contained oleuropein before and after frying. Oleuropein as well as other polyphenol species were detected in all French fries cooked in enriched oils. Polyphenol intake by consuming French fries pan-fried in the enriched oils was calculated to be 6 to 31 times higher than that in the case of French fries fried in commercial oils, being dependent on the frying oil type. [source] HPLC Analysis of Catechins, Theaflavins, and Alkaloids in Commercial Teas and Green Tea Dietary Supplements: Comparison of Water and 80% Ethanol/Water ExtractsJOURNAL OF FOOD SCIENCE, Issue 6 2006Mendel Friedman ABSTRACT:, To help meet the needs of consumers, producers of dietary tea supplements, and researchers for information on health-promoting tea compounds, we compared the following conditions for the extraction of tea leaves and green tea-containing dietary supplements: 80% ethanol/water at 60 °C for 15 min and boiled water for 5 min. The following 7 catechins, 4 theaflavins, and 3 alkaloids were separated in a 70-min single HPLC analysis: (,)-epigallocatechin, (,)-catechin, (+)-epicatechin, (,)-epigallocatechin-3-gallate, (,),gallocatechin-3-gallate, (,)-epicatechin-3-gallate, (,)-catechin-3-gallate, theaflavin, theaflavin-3-gallate, theaflavin-3,-gallate, theaflavin-3,3,-digallate, caffeine, theobromine, and theophylline. The following ranges of concentrations of flavonoids (catechins plus theaflavins) in the tea leaves extracted with 80% ethanol were observed (in mg/g): in 32 black teas, 19.8 to 115.1; in 24 green teas, 12.3 to 136.3; in 14 specialty teas, 4.9 to 118.5; in 7 herbal teas, 0 to 46.0. Total alkaloids in all teas ranged from 0 to 32.6 mg/g. Significantly greater amounts of flavonoids were extracted from the tea leaves with aqueous ethanol than with boiled water. Levels of tea catechins in 10 capsules sold as dietary supplements were about 50 to 75% lower than the amounts listed on the labels. Catechin content of 4 commercial green tea extracts ranged from 96 to 696 mg/g. The results make it possible to maximize the extraction of tea compounds to better relate the flavonoid and alkaloid content of teas and dietary tea supplements to their health-promoting effects. [source] Effect of Heat Treatment on Antioxidant Capacity and Flavor Volatiles of MeadJOURNAL OF FOOD SCIENCE, Issue 2 2005Carol L. Wintersteen ABSTRACT: The objective of this study was to evaluate the effects of heat processing on the antioxidant capacity of mead (honey wine). Soy and buckwheat honey musts were subjected to 2 heat treatments and fermented into wine. Total phenolic concentration was determined. High-performance liquid chromatography (HPLC) phenolic profiling was performed on the methanol fraction of Amberlite extraction. Antioxidant capacity was evaluated using the oxygen radical absorbance capacity (ORAC) assay. Changes in volatile components were evaluated by headspace-solid phase microextraction/gas chromatography-mass spectrometry (H-SPME/GC-MS). ORAC values of experimental meads (3.62 mMTrolox equivalent) were comparable to those of commercial white wine (3.66 mMTrolox equivalent). No significant difference in antioxidant capacity due to heat treatment or honey type was observed. There was no difference in total phenolics between heat treatments in buckwheat mead; however, soy mead made from high-heated must had significantly greater phenolic concentration than the gently heated mead (,= 0.05). Linear regression analysis indicated a strong positive correlation between total phenolic concentration and antioxidant capacity by ORAC (r= 0.9077; P < 0.0001). HPLC analysis of phenolic profiles in the methanol fractions of Amberlite extraction of the meads indicated significantly higher levels of certain phenolics as a result of the high-heat process in buckwheat mead, but not in soy mead. Differences in volatile components that potentially impact flavor were noted between high and low heat treatments. Results of this study suggest dramatic heat treatments that are often avoided because their flavor impact in mead production have the potential to alter the antioxidant capacity of mead by changing phenolic profiles. [source] Synthesis and spectroscopic studies of optically active n -acetyl butenoates and n -acetyl-2-alkyl-pyrrolin-4-onesJOURNAL OF HETEROCYCLIC CHEMISTRY, Issue 1 2002Efstathios Gavrielatos The synthesis of a series of optically active N -acetyl butenoates 3,5 is described using a facile methodology. These butenoates undergo cyclization to the corresponding N -acetyl-2-alkyl-pyrrolin-4-ones 6,7 retaining their stereochemical integrity. The structure of the newly synthesized compounds has been elucidated through 1H- 13C NMR, IR spectroscopy and their enantiomeric excesses have been measured by chiral HPLC analysis. [source] Synthesis of [18F]3-[1-(3-fluoropropyl)-(S)-pyrrolidin-2-ylmethoxy]pyridine ([18F]NicFP): a potential ,4,2 nicotinic acetylcholine receptor radioligand for PETJOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, Issue 14 2003Filip Dumont Abstract Nicotinic acetylcholine receptors are widely distributed throughout the human brain and are believed to play a role in several neurological and psychiatric disorders. In order to identify an effective PET radioligand for in vivo assessment of the ,4,2 subtype of nicotinic receptor, we synthesized [18F]3-[1-(3-fluoropropyl)-(S)-pyrrolidin-2-ylmethoxy]pyridine (NicFP). The in vitro KD of NicFP was determined to be 1.1 nM, and the log P value obtained by HPLC analysis of the unlabelled standard was found to be 2.2. The radiosynthesis of [18F]NicFP was carried out by a nucleophilic substitution reaction of anhydrous [18F]fluoride and the corresponding mesylate precursor. After purification by HPLC, the radiochemical yield was determined to be 11.3±2.1% and the specific activity was 0.47±0.18 Ci/,mol (EOS, n = 3). The time of synthesis and purification was 99±2 min. The final product was prepared as a sterile saline solution suitable for in vivo use. Copyright © 2003 John Wiley & Sons, Ltd. [source] Comparative evaluation of 99mTc-ethylene bis-l-cysteine and 99mTc-ethylene bis-l-,-homocysteine during reversed phase HPLC analysis and electrophoresis at various pH conditionsJOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, Issue 4 2001K.O. Mang'era Ethylene bis- L -,-homocysteine (L,L -EH) differs from ethylene bis- L -cysteine (L,L -EC) in having an extra methylene group between each pair of amine and carboxyl groups. The objective of this study was to determine the effect of the extra methylene groups on the characteristics of the complex of these compounds with technetium-99m during analysis by reversed phase HPLC and by electrophoresis at various pH values. Up to pH 5.5, 99mTc- L,L -EH exhibits a substantially longer retention time during reversed phase HPLC than 99mTc- L,L -EC, suggesting a more lipophilic character for 99mTc- L,L -EH under these conditions. On the other hand, 99mTc- L,L -EH clearly possesses a higher negative charge in the pH range 3-6.5 as shown by the markedly greater migration towards the anode in electrophoresis experiments. A rational explanation for these seemingly opposing observations can not yet be offered. Copyright © 2001 John Wiley & Sons, Ltd. [source] Mass spectrometric characterization of covalent modification of human serum albumin by 4-hydroxy- trans -2-nonenalJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 9 2006Giancarlo Aldini Abstract Several pieces of evidence indicate that albumin modified by HNE is a promising biomarker of systemic oxidative stress and that HNE-modified albumin may contribute to the immune reactions triggered by lipid peroxidation-derived antigens. In this study, we found by HPLC analysis that HNE is rapidly quenched by human serum albumin (HSA) because of the covalent adduction to the different accessible nucleophilic residues of the protein, as demonstrated by electrospray ionization mass spectrometry (ESI-MS) direct infusion experiments (one to nine HNE adducts, depending on the molar ratio used, from 1 : 0.25 to 1 : 5 HSA : HNE). An LC-ESI-MS/MS approach was then applied to enzymatically digested HNE-modified albumin, which permitted the identification of 11 different HNE adducts, 8 Michael adducts (MA) and 3 Schiff bases (SB), involving nine nucleophilic sites, namely: His67 (MA), His146 (MA), His242 (MA), His288 (MA), His510 (MA), Lys 195 (SB), Lys 199 (MA, SB), Lys525 (MA, SB) and Cys34 (MA). The most reactive HNE-adduction site was found to be Cys34 (MA) followed by Lys199, which primarily reacts through the formation of a Schiff base, and His146, giving the corresponding HNE Michael adduct. These albumin modifications are suitable tags of HNE-adducted albumin and could be useful biomarkers of oxidative and carbonylation damage in humans. Copyright © 2006 John Wiley & Sons, Ltd. [source] | |||||||||||||||||||