Determination of 2,3-dimercaptosuccinic acid in mice blood and tissues by HPLC with fluorescence detection

2,3-Dimercaptosuccinic acid (DMSA) is an orally effective chelating agent for the treatment of heavy metal poisoning. The increasing therapeutic use of DMSA has stimulated the need for sensitive and selective methods for its determination in biological samples, as well as study on pharmacokinetics and tissue distribution. According to the previously reported method, an improved method was established for the determination of DMSA in mice blood and tissues, in which oxidized DMSA was reduced by the disulfide-reducing agent, dithiothreitol (DTT), and DMSA was converted to a highly fluorescent and stable derivative by reaction with monobromobimane (mBBr) in alkaline solution. Acetonitrile was used for deproteinization and dichloromethane was used for condensation and purification, which significantly shortened the amount of time used to process the sample. Meanwhile isocratical elution was performed and excellent separation of the DMSA derivative was obtained, this enabled a run finish within 20 min. The limits of quantitation were 0.025 μg/ml in brain and 0.1 μg/ml in blood, lung, heart, intestine, liver, spleen and kidney, respectively. The calibration curves were linear in all samples (r² > 0.992) with a range of 0.025–1.6 μg/ml for brain homogenate and 0.1–6.4 μg/ml for blood and homogenates of lung, heart, intestine, liver, spleen and kidney, respectively. Therefore, the method is simple, rapid and sensitive, and it could be applicable to the studies in an animal model to evaluate the distribution of DMSA in blood and tissues.     

Determination of lycopene in tissues and plasma of rats by normal-phase high-performance liquid chromatography with photometric detection

An analytical method for the determination of lycopene in tissues and plasma of rats is described. The method was validated for the determination of lycopene in liver and plasma with respect to selectivity, linearity, accuracy, recovery and precision. Following precipitation of proteins with water–ethanol plasma was extracted with hexane; tissues were extracted with acetone followed by precipitation of proteins with water–ethanol and extraction of lycopene with hexane. Separation and quantification of geometrical isomers of lycopene was achieved by normal-phase HPLC with UV/VIS detection at 471 nm. The method proved to be selective and specific for lycopene in plasma and liver. Detector response was linear in the range from 2 ng/g to 10 μg/g liver and 0.5 ng/ml to 2 μg/ml plasma, respectively. Average recoveries ranged from 96 to 101% in spiked liver samples and from 91 to 94% in spiked plasma samples. Intra-day variability (C.V.) was ≤6% and ≤5% in liver and plasma, respectively. Inter-day precision was ≤9% for liver samples and ≤6% for plasma samples. The procedures were successfully applied to the sample analysis of pharmacokinetic and metabolism studies.     

Rapid determination of zearalenone in edible oils by HPLC with fluorescence detection

A fast, cost-efficient, sensitive and accurate assay method for zearalenone in edible oils is described, as an alternative to gel permeation chromatography (GPC). Oil samples were extracted with an alkaline mixture of methanol and water (methanol +10 g/l aqueous ammonium carbaminate solution, pH 9; 9 + 1, v+v). The pH of the extract was neutralized with hydrochloric acid and then concentrated to dryness. The residue was dissolved with HPLC solvent, and zearalenone was determined by high-performance liquid chromatography with fluorometric detection (HPLC-FLD). The method was successfully validated for two matrices, maize oil and rapeseed oil. The recovery rate was 87%, and the coefficient of variation was 2.8% in a rapeseed oil sample contaminated with 27 μg zearalenone/kg. At a signal-to-noise ratio of 3:1, the method detection limit was 10 μg/kg, which was considered to be adequate in view of the present European Union maximum level of 400 μg/kg.     

Sensitive determination of ranitidine in rabbit plasma by HPLC with fluorescence detection

A sensitive high-performance liquid chromatographic method for determination of ranitidine (RAN) in rabbit plasma is described. The method is based on liquid-liquid extraction, labeling with dansyl chloride and monitoring with fluorescence detector at 338nm (ex)/523nm (em). Plasma samples were extracted with diethyl ether alkalinized with 1M sodium hydroxide. Ephedrine HCl (EPH-HCl) was used as internal standard. Both, RAN and EPH were completely derivatized after heating at 60 degrees C for 10min in sodium bicarbonate solution (pH 9.5). The derivatized samples were analyzed by HPLC using Agilent Zorbax Extended C18 column (150mmx4.6mm i.d.) and mobile phase consists of 48% acetonitrile and 52% sodium acetate solution (0.02M, pH 4.6). The linearity of the method was in the range of 0.025-10microg/ml. The limits of detection (LOD) and quantification (LOQ) were 7.5+/-0.18 and 22.5+/-0.12ng/ml, respectively. Ranitidine recovery was 97.5+/-1.1% (n=6; R.S.D.=1.8%). The method was applied on plasma collected from rabbits at different time intervals after oral administration of 5mg/kg ranitidine HCl.     

Expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in maize tissues

The activity of the enzyme 3-hydroxy-3-methlglutaryl-coenzyme A reductase (HMGR, EC 1.1.1.34) is highly expressed in 4-day-old etiolated seedlings of normal (cv. DeKalb XL72AA), dwarf ( d ₅) and albino ( lw ₃) maize ( Zea mays L.). HMGR activity of maize seedlings appeared to be exclusively associated with the microsomal rather than the plastidic fraction of maize cells. Maize tissues with high meristematic activity such as germinating seeds, leaf bases, root tips and the site of origin of lateral roots contained high levels of microsomal HMGR activity. The activity of HMGR extracted from leaf tips of normal, dwarf and albino maize seedlings is regulated by light. Microsomal HMGR activity from leaf tips of 4-day-old maize seedlings was inhibited significantly following exposure to strong light (600 μmol m⁻² s⁻¹) for more than 10 h. By comparison, microsomal HMGR activity from leaf bases and root tips of maize was not inhibited by exposure to strong light. These results suggest that the microsomal HMGR which is highly expressed in maize may be related to sterol biosynthesis and membrane biogenesis rather than plastidic-associated isoprenoid synthesis and that light may regulate HMGR activity indirectly by increasing cell differentiation.     

Regulation of 3-hydroxyl-3-methylglutaryl-coenzyme A reductase activity in rat uterine tissues

Initiation of uterine DNA synthesis and mitosis in response to estrogen appears to depend upon the stimulation of protein synthesis. 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase could have a key function in controlling uterine mitosis through its control of mevalonic acid and cholesterol synthesis as the rate-limiting enzyme in their synthetic pathways. These studies were initiated to examine the kinetics of the uterine increases in HMG-CoA reductase activity in response to estradiol. In the uterus of the ovariectomized mature rat, estradiol increased levels of enzyme activity in both the luminal epithelium and stroma-myometrium up to 12 h after estradiol treatment. Levels of HMG-CoA reductase activity decreased after 12 h in the luminal epithelium and further increased in the stroma-myometrium. Previous studies have shown that estradiol does not increase DNA synthesis and mitosis in the stroma-myometrium of the uterus of the ovariectomized mature rat. Since estradiol increased HMG-CoA reductase activity in both the luminal epithelium and stroma-myometrium, we conclude that even though increased HMG-CoA reductase activity may be a prerequisite for increased DNA synthesis, increases in uterine HMG-CoA reductase activity are not necessarily followed by increased DNA synthesis.     

Quantification of nortriptyline in plasma by HPLC and fluorescence detection

A simple, sensitive and specific high-performance liquid chromatography method has been developed for the determination of nortriptyline (NT) in plasma samples. The assay involved derivatization with 9H-fluoren-9-ylmethyl chloroformate (Fmoc-Cl) and isocratic reversed-phase (C₁₈) chromatography with fluorescence detection. The developed method required only 100 μl of plasma sample, deproteinized and derivatized in one step. Calibration curves were lineal over the concentration range of 5–5000 ng/ml. The derivatization reaction was performed at room temperature in 20 min and the obtained NT derivative was stable for at least 48 h at room temperature. The within-day and between-day relative standard deviation was below 8%. The limit of detection (LOD) was 2 ng/ml, and the lower limit of quantification (LLOQ) was established at 10 ng/ml. The method was applied on plasma collected from rats, at different time intervals, after intravenous administration of 0.5 mg of NT.     

Regulation of ribonucleotide reductase activity in mammalian cells

Mammalian ribonucleotide reductase catalyzes the rate-limiting for the de novo synthesis 2'-deoxyribonucleoside 5'-triphosphates. There is some suggestion that this step may also be the rate-limiting step of DNA synthesis. It is apparent that the level of the enzyme, ribonucleotide reductase, varies through the cell cycle and is highest in those tissues with the greatest proliferation rate. This increase in activity is associated with increased protein synthesis. The purified enzyme has been shown to be subject to strict allosteric regulation by the various nucleoside triphosphates and it has been proposed that allosteric regulation plays an important role in the level of ribonucleotide reductase activity which is expressed. All experimental data relating to this point, however, do not support the role of deoxyribonucleoside triphosphates as a major factor in determining cellular reductase activity during normal cell division. Several naturally occurring factors have been isolated from cells which lower ribonucleotide reductase activity in vitro. These factors have been found in tissues of low growth fraction and appear to be absent or low in tissues or high growth fraction such as tumor, regenerating liver and embryonic tissues. The expression of intracellular ribonucleotide reductase activity is therefore controlled at various levels and by various factors and the prevailing mode of regulation may vary throughout the cell cycle transverse and also in the various types of cells.     

高效液相色谱-荧光检测法分析酱油中的氨基甲酸乙酯

建立了高效液相色谱-荧光法检测酱油中氨基甲酸乙酯的分析方法.首先用乙酸乙酯萃取酱油中的氨基甲酸乙酯,再经减压旋转蒸发浓缩得待测样品.其次确定了衍生化反应条件:取1.0 mL待测样品,加入200 μL 0.02 mol/L占吨醇,再加入100 μL 1.5 mol/L盐酸于暗处反应30min.实验结果表明:该方法标准曲线良好,线性范围为10～200 μg/L,相关系数为R2=0.988,氨基甲酸乙酯的出峰时间为14.882 min,平均回收率为100.12％.最低检测限为5μg/L,比已经发表的最低检测限(20 μg/L)要低.最后对市售酱油S1～S6样品中的氨基甲酸乙酯含量进行了检测,表明市售酱油S1～S6中氨基甲酸乙酯的含量为31.25～114.78μg/L.本文所建立的方法具有简便快捷、灵敏度高的特点,可满足对酱油中氨基甲酸乙酯含量检测的需要.     

Down-regulation of mammalian 3-hydroxy-3-methylglutaryl coenzyme A reductase activity with highly purified liposomal cholesterol.

Chinese hamster ovary-215 cells (CHO-215) cannot synthesize C27 and C28 sterols because of a defect in the reaction that decarboxylates 4-carboxysterols [Plemenitas, A., Havel, C.M. & Watson, J.A. (1990) J. Biol. Chem. 265, 17012-17017]. Thus, CHO-215 cell growth is dependent on an exogenous metabolically functional source of cholesterol. We used CHO-215 cells to (a) determine whether highly purified (> 99.5%) cholesterol, in egg lecithin liposomes, could down-regulate derepressed 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity and if so (b) determine whether the loss in reductase catalytic activity correlated kinetically with the synthesis and accumulation of detectable oxycholesterol derivatives. Liposomal cholesterol (26-39 microM) supported maximum CHO-215 growth and initiated suppression of HMG-CoA reductase activity at concentrations greater than 50 microM. Maximum suppression (50-60%) of reductase activity was achieved with 181.3 microM liposomal cholesterol in 6 h. Also, regulatory concentrations of highly purified liposomal [3H]cholesterol were not converted (biologically or chemically) to detectable levels of oxy[3H]cholesterol derivatives during 3-6 h incubations. Lastly, a broad-spectrum cytochrome P450 inhibitor (miconazole) had no effect on liposomal cholesterol-mediated suppression of HMG-CoA reductase activity. These observations established that (a) highly purified cholesterol, incorporated into egg lecithin liposomes, can signal the down-regulation of derepressed mammalian cell HMG-CoA reductase activity and (b) if oxycholesterol synthesis was required for liposomal cholesterol-mediated down-regulation, the products had to be more potent than 24-, 25-, or 26-/27-hydroxycholesterol.     

Regulation of ribonucleotide reductase activity in intact mammalian cells

An intact cell assay system based upon Tween-80 permeabilization was used to investigate the regulation of ribonucleotide reductase activity in Chinese hamster ovary cells. Models used to explain the regulation of the enzyme have been based upon work carried out with cell-free extracts, although there is concern that the properties of such a complex enzyme would be modified by extraction procedures. We have used the intact cell assay system to evaluate, within whole cells, the current model of ribonucleotide reductase regulation. While some of the results agree with the proposals of the model, others do not. Most significantly, it was found that ribonucleotide reductase within the intact cell could simultaneously bind the nucleoside triphosphate activators for both CDP and ADP reductions. According to the model based upon studies with cell-free preparations, the binding of one of these nucleotides should exclude the binding of others. Also, studies on intracellular enzyme activity in the presence of combinations of nucleotide effectors indicate that GTP and perhaps dCTP should be included in a model for ribonucleotide reductase regulation. For example, GTP has the unique ability to modify through activation both ADP and CDP reductions, and synergistic effects were obtained for the reduction of CDP by various combinations of ATP and dCTP. In general, studies with intact cells suggest that the in vivo regulation of ribonucleotide reductase is more complex than predicted from enzyme work with cell-free preparations. A possible mechanism for the in vivo regulation of ribonucleotide reductase, which combines observations of enzyme activity in intact cells and recent reports of independent substrate-binding subunits in mammalian cells is discussed.     

Determination of closantel residues in plasma and tissues by high-performance liquid chromatography with fluorescence detection

The influence of the pH of the mobile phase with some modifiers on the chromatographic behavior and fluorescence properties of closantel have been investigated. At acidic pH values (2–6), the benzamide moiety of the closantel forms a six-membered ring by hydrogen bonding and possesses a native fluorescence. Using the fluorescence emission of closantel at λ_ex=335 nm, λ_em=510 nm, and pH 2.5 of the mobile phase, a linear calibration curve was estimated over a concentration range of about two orders of magnitude with a correlation coefficient larger than 0.992. The limit of the fluorescence detection was 10 μg/kg. This value was at least 10 times lower than that using UV detection. The method was applied to the determination of closantel in plasma and tissue samples, purified by a solid-phase extraction with C₁₈ cartridges.     

Inactive 3-hydroxy-3-methylglutaryl-coenzyme A reductase in broken cell preparations of various mammalian tissues and cell cultures.

Preincubation of broken cell preparations from a variety of tissues and cell cultures resulted in an apparent increase in the level of 3-hydroxy-3-methylglutaryl-CoA reductase activity. However, apparent activation of the reductase in mouse liver, hepatomas and primary liver cell cultures was attributed largely to the loss, during the preincubation period, of an interfering enzyme, 3-hydroxy-3-methylglutaryl-CoA lyase. Among non hepatic cells and tissues (which did not contain appreciable lyase activity) the proportion of latent reductase was high in sonicates of fetal brain and in L cells and was independent of the level of total enzyme activity present. Activation of the reductase was blocked by hydroxymethylglutaryl-CoA and NADPH as well as by KF so that activation did not occur under the conditions of the enzyme assay. The enzyme was activated slowly at 4 degrees C, so that partial activation of the latent form occurred during isolation of the microsomal fraction by differential centrifugation. The reductase present in sonicates of cells with either a high or low proportion of the latent enzyme was inactivated by incubation with ATP and Mg2+. Suppression of reductase activity in L cell cultures by treatment with 25-hydroxycholesterol and an age-related decline in brain enzyme activity did not involve reversible conversion of the reductase to an inactive form.     

Quantitative analysis of polymeric proanthocyanidins in birch leaves with normal-phase HPLC

The proanthocyanidin composition and content in the leaves of nine birch species (Betula albosinensis, B. ermanii B. maximowicziana, B. nana, B. papyrifera, B. pendula, B. platyphylla, B. pubescens, and B. pubescens ssp. czerepanovii) were studied with different methods including colorimetric assay, HPLC coupled with PAD or ESI/MS and NMR. Total proanthocyanidin content was determined using the acid butanol assay. A normal phase-HPLC method was applied for the analysis of polymeric proanthocyanidins. The content of polymeric proanthocyanidins was estimated from a late eluting peak in the chromatogram. With this HPLC method, quantitative analysis of polymeric proanthocyanidins could be performed directly from leaf extracts: no additional purification or preparation steps were required. It was shown that birch leaves contained mainly polymeric proanthocyanidins with a degree of polymerisation greater than 10. Total proanthocyanidin content (expressed as dry weight) was found to vary from 44mg/g (B. papyrifera) to 145mg/g (B. nana), and polymeric proanthocyanidin content from 39mg/g (B. pendula) to 119 mg/g (B. nana).     

Determination of intoplicine, a new antitumour drug, in human whole blood and plasma by normal-phase high-performance liquid chromatography with fluorescence detection

Intoplicine, a benzopyrido-indole derivative, is a novel anticancer agent currently under phase I clinical evaluation. A selective, sensitive normal-phase high-performance liquid chromatographic (HPLC) assay with fluorescence detection, suitable for the determination of intoplicine in human plasma and whole blood, is described. The sample pretreatment involves a protein precipitation step with 2-propanol. The reported assay was validated, and the stability of the analyte in plasma, in whole blood and in the extraction fluid was investigated. The method has been implemented in a pharmacokinetic phase I clinical trial with intoplicine given as a 24-h intravenous infusion.     

Determination of long-chain fatty acids in bryophyte plants extracts by HPLC with fluorescence detection and identification with MS

A sensitive method for the determination of long-chain fatty acids (LCFAs) (>C20) using 1-[2-(p-toluenesulfonate)-ethyl]-2-phenylimidazole-[4,5-f]-9,10-phenanthrene (TSPP) as tagging reagent with fluorescence detection and identification with post-column APCI/MS has been developed. The LCFAs in bryophyte plant samples were obtained based on distillation extraction with 1:1 (v/v) chloroform/methanol as extracting solvent. TSPP could easily and quickly label LCFAs at 90 degrees C in the presence of K2CO3 catalyst in DMF. Eleven free LCFAs from the extracts of bryophyte plants were sensitively determined. Maximal labeling yields close to 100% were observed with a five-fold excess of molar reagent. Separation of the derivatized fatty acids exhibited a good baseline resolution in combination with a gradient elution on a reversed-phase Eclipse XDB-C8 column. Calculated detection limits from 1.0 pmol injection, at a signal-to-noise ratio of 3, were 26.19-76.67 fmol. Excellent linear responses were observed with coefficients of >0.9996. Good compositional data were obtained from the analysis of the extracted LCFAs containing as little as 0.2g of bryophyte plant samples. Therefore, the facile TSPP derivatization coupled with HPLC/APCI/MS analysis allowed the development of a highly sensitive method for the quantitation of trace levels of LCFAs from biological and natural environmental samples.     

Metabolism of androstenedione by human ovarian tissues in vitro with particular reference to reductase and aromatase activity

The ability of granulosa and theca cells of the human ovarian follicle at different stages of development, as well as stromal and luteal tissues from human ovaries to metabolize androstenedione (delta 4) to testosterone (T), dihydrotestosterone (DHT), estrone (E1) and estradiol (E2) with or without exposure to additional amounts of folicle-stimulating hormone was investigated by in vitro experiments. The results show that all the aforementioned ovarian tissues metabolized delta 4 to DHT. Indeed, with the exception of estrogen-secreting granulosa cells from large antral follicle (greater than 10 mm diameter) and possibly also luteal tissue from mid-luteal phase ovaries, the various ovarian tissues preferentially metabolized delta 4 to DHT instead of E (E1 + E2). Although thecal tissue is a major source of delta 4 in human ovaries it is concluded that the granulosa cells do not interact with the theca for the synthesis of E as the follicle enlarges from 1 to 10 mm in diameter. Indeed, excessive thecal delta 4 during this growth phase probably inhibits normal follicular development. However, as the follicle enlarges beyond 10 mm in diameter, and as the granulosa cells begin to preferentially metabolize delta 4 to E, the two cell-types of the follicle may increasingly interact to enhance the follicular output of E.