Determination of Total Monoamines in Rat Brain via Nanotubes Based Human Monoamine Oxidase B Biosensor

A specific and sensitive electrochemical biosensor with human monoamine oxidase B (hMAO B) as biological receptor and a MnO₂ modified nanomaterial based transducer system has been developed and optimised. Best results for the biosensor were achieved when using enzyme immobilisation with a dialysis membrane (regenerated cellulose, molecular weight cut‐off 14000) and a 20 % (m/m) MnO₂ modified multi‐walled carbon nanotubes (MWCNTs, ratio of fluid to solid compounds of 1 : 0.7 (m/m)) paste electrode smoothed with a glassy carbon paste (GCP, ratio 1 : 3.6 (m/m)) containing the same mediator concentration. The biosensor was operated in a flow injection analysis (FIA) system with Sørensen phosphate buffer (33 mM, pH 7.5) and amperometric detection at a fixed potential of +400 mV vs. Ag/AgCl. The developed sensor underwent validation using phenylethylamine (PEA) as standard substrate showing linearity between 5.0 and 400 µM PEA and limits of detection and quantification of 1.5 and 5.0 µM PEA, respectively. The sensor was successfully tested for the determination of total monoamines in rat brain calculated as PEA equivalents showing a result of 1.2 µg/g brain tissue (n=3, relative standard deviation 4.4 %).     

Simultaneous HPLC Analysis of Catecholamines and Indoleamines in Mouse Brain Tissue Following Acetate Extraction and Treatment with Ascorbate Oxidase

Abstract

A refined HPLC Method for the determination of monoamine levels in six brain regions is presented. Analyses are made for the olfactory tubercles, prefrontal cortex, septum, striatum, amygdala and hypothalamus of adult male ICR mice. This system permits the simultaneous analysis of norepinephrine, dopamine, serotonin and their major metabolites during a single run of approximately twenty-five minutes without prior clean-up of samples.     

高效液相色谱法测定酶转化液中D-苯丙氨酸

采用HPLC法测定了酶转化液中的D_苯丙氨酸、N_氨甲酰_D_苯丙氨酸和苄基海因。流动相为乙腈-20mmol·L-1 磷酸二氢钾(体积比25∶75), 用磷酸调pH为5.0, 利用DAD检测器, 检测波长为202 nm, 流速为1.0mL·min-1。用外标法进行定量分析, 三者的线性范围分别是1.82~466 mg·L-1(r=0.999)、2.01~516 mg·L-1(r=0.999)、1.94~496 mg·L-1(r=0.999), 回收率为99.9%~100.1%、99.8%~100.0%、99.5%~ 100.4%,RSD(n=6)分别为1.8%、1.6%、1.0%, 检出限为18.7、2.41、64.6 pg。实验结果表明该法简便、快速、结果可靠。     

Specific Differential Spectrophotdmetric Determination of Ascorbic Acid in Plants Using Ascorbate Oxidase

Abstract

A specific enzymic-differential spectrophatometric method is described and evaluated for the determination of ascorbic acid in vegetables and medicinal plants. It is based on the absorbance measurement at 593 nm of the complex of ferrous ion with 2,4,6-tris (pyridyl)-S-triazine, which is produced by reduction of ferric ion by ascorbic acid, versus a blank sample treated with ascorbate oxidase. The absorbance difference is linearly related to ascorbic acid from 10 to 100 ug/ml. A standard deviation of ±0.5 ug/ml (n=5) and a mean recovery of 101.47. (98.3–103.3%.) from spiked plant extracts, were found. The method was used to determine ascorbic acid in various plants of the Greek flora. Acacia cvanophvlla was found to be the richest source tested for ascorbic acid (89.6 mq/100 q of leaves). The method is very simple and can be used in routine analysis.     

微柱液相色谱-电化学法检测鼠纹状体单胺类物质的含量

单胺类神经递质 ( Monoamines) ,包括儿茶酚胺 ( Catecholamine)和 5 -羟色胺 ( Serotonin) ,在中枢神经系统中起重要作用 .对其含量的准确测定在基础研究和临床诊断中均具有重要的意义 .微透析是 2 0世纪 80年代发展起来的活体取样技术 [1] ,为脑功能的研究提供了极为有力的手段 ,本实验室在此方面进行了多年的研究 [2 ,3] .微透析与液相色谱电化学检测联用[4 ] ,可测定神经细胞外液递质的含量及释放量 ,据此可对一系列重要的生理、病理和药理现象做出解释 .但使用常规高效液相色谱柱 ( =4.6mm i.d.)尚存在不足之处 .首先 ,透析液中神经…     

酒精处理后鼠脑内单胺氧化酶活性及单胺神经递质代谢变化的测定

应用高效液相色谱法测定了慢性酒精处理后胎鼠脑中单胺氧化酶(MAO)的活性,以及多巴胺(DA)、5-羟色胺(5-HT)和它们的代谢产物的含量。在怀孕的最后一周内对SD母鼠灌胃酒精,对照组用生理盐水处理,分娩后取胎鼠脑组织进行检测。胎鼠脑线粒体MAO的活性随酒精染毒剂量的增加而递减,呈现出剂量反应关系;与盐水对照组相比,酒精暴露可改变胎鼠全脑中DA和5-HT的水平,但统计结果无显著性差异。其中,HVA和5-HIAA的含量在4.0g/kg酒精处理后显著减少(P0.05)。慢性酒精暴露后MAO活性的下降及DA、5-HT的代谢变化可能在中枢神经系统的发育障碍中发挥了重要作用。     

HPLC Determination of Lovastatin in Rat Tissue

A simple HPLC method has been developed and validated for the determination of lovastatin in rat tissues. Samples were prepared by a simple protein precipitation. Separation was carried out on a C18 column with a mobile phase of acetonitrile:0.05 M ammonium acetate, a flow rate of 1.0 mL min⁻¹ and with detection at 238 nm. There was no interference from endogenous tissue compounds. The calibration curve was linear from 0.0175 to 7.0 μg mL⁻¹ with a limit of detection of 0.006 μg mL⁻¹. The method was used to measure the concentration of lovastatin in rat tissue after a single oral dose. The highest level was observed in the liver, then in kidney, heart and spleen; the lowest level was found in the brain. These results suggest that lovastatin distributes rapidly into all tissues and particularly the liver.     

Electrochemical Monitoring of Propagative Fluctuation of Ascorbate in the Live Rat Brain during Spreading Depolarization

Spreading depolarization (SD) occurs frequently in the injured brain, and its neurochemical effects are detrimental to brain function. We report the first observation that the release of ascorbate, an important neurochemical in the brain, is closely accompanied with SD. Ascorbate was monitored with carbon nanotube (CNT)‐sheathed carbon fiber microelectrodes (CFEs). This system features high selectivity and temporal/spatial resolution. With our sensor, we observed a significant increase in the concentration of ascorbate in response to SD induction. Mechanistic studies show a contrasting behavior; with a SD specific inhibitor, release was completely suppressed, whereas with inhibition of commonly employed glutamate transporters, ascorbate release was increased, demonstrating a powerful means of discriminating ascorbate release between disputed pathways. Most importantly, we observed the propagative nature of ascorbate release following SD.     

高效液相色谱—电化学检测法测定大鼠脊髓灌流液单胺类递质及其代谢产物

用反相离子对高效液相色谱－电化学检测法分离并同时测定了大鼠脊髓灌流液中部分单胺类神经递质及其代谢产物。选择了较为理想的色谱条件，使生物样品的检测限达到８０～６７０ｐｇ，单胺类递质代谢产物的回收率保持在８３％～１０６％范围内。     

CZE Determination of Quinolinic Acid in Rat Brain Tissue and Plasma

In this paper, a capillary zone electrophoretic method for the determination of the excitotoxic quinolinic acid in rat brain tissue (cerebellum, cortex, hippocampus, striatum) and plasma samples is described. Optimum separation of the excitotoxic quinolinic acid was achieved with a 14.4 mM boric acid/5.6 mM sodium tetraborate electrolyte solution at pH 8.84. The applied voltage was 30 kV and the capillary temperature was kept constant at 25 °C. The regression equations revealed a good linear correlation between the peak area and the concentration. The method was linear over the concentration range of 0.50 to 600 nM. All correlation coefficients were higher or equal to 0.9998. To optimize the analysis conditions, the effects of electrolyte solution pH, the concentration, and the use of methanol as an organic modifier were systematically studied. The amount of quinolinic acid in the rat brain tissue and plasma under control conditions were found to be: cerebellum 30.2 ± 1.7 nM (mean ± standard deviation); cortex 5.6 ± 0.7 nM; hippocampus 64.2 ± 9.4 nM; striatum 4.3 ± 0.6 nM, and plasma 40.1 ± 2.3 nM. The limits of detection and quantification were 0.47 nM (signal/noise = 3) and 1.58 nM, respectively. The method was successfully applied to quantify quinolinic acid in the rat brain striata under two neurotoxicity models with good repeatability (RSD < 10%) and recovery (98–102%). The proposed analytical method could be useful to clarify the role of quinolinic acid in neurodegenerative entities such as Alzheimer’s and Huntington’s diseases.     

Simple Determination of Steroid Sulfatase Activity in Enzyme Preparation

Abstract

Simple one tube method for the determination of steroid sulfatase activity in an enzyme preparation or commercially available preparation is described. Principle of the reaction is as follows; dehydroepi-androsterone sulfate (DHEA-S) as substrate is hydrolyzed by the catalysis of sulfatase and DHEA released is determined by the enzymic method using 3β-hydroxysteroid oxidase.     

Enzymatic Oxidation and Separation of Various Saccharides with Immobilized Glucose Oxidase

Glucose oxidase from Aspergillus niger, the specific enzyme for β-d-glucose oxidation, can also oxidize other related saccharides at very slow or negligible rates. The present study aimed to compare the kinetics of d-glucose oxidation using immobilized glucose oxidase on bead cellulose for the oxidation of related saccharides using the same biocatalyst. The significant differences were observed between the reaction rates for d-glucose and other saccharides examined. As a result, k _cat/K _M ratio for d-glucose was determined to be 42 times higher than d-mannose, 61.6 times higher than d-galactose, 279 times higher than d-xylose, and 254 times higher than for d-fructose and d-cellobiose. On the basis of these differences, the ability of immobilized glucose oxidase to remove d-glucose from d-cellobiose, d-glucose from d-xylose, and d-xylose from d-lyxose was examined. Immobilized catalase on Eupergit and mixed with immobilized glucose oxidase on bead cellulose or co-immobilized with glucose oxidase on bead cellulose was used for elimination of hydrogen peroxide from the reaction mixture. The accelerated elimination of d-glucose and d-xylose in the presence of co-immobilized catalase was observed. The co-immobilized glucose oxidase and catalase were able to decrease d-glucose or d-xylose content to 0–0.005% of their initial concentrations, while a minimum decrease of low oxidized saccharides d-xylose, d-cellobiose, and d-lyxose, respectively, was observed.     

Efficient Artificial Electron Acceptors for Monoamino Oxidase in Bioelectrooxidation of Monoamines: Phenothiazine Dyes

Catalytic properties of monoamino oxidase (MAO) in a homogeneous reaction of oxidation and a heterogeneous reaction of bioelectrocatalytic oxidation of benzylamine by derivatives of phenothiazine and phenoxazine (artificial electron acceptors) is studied. The efficiency of electroenzymic catalysis involving Methylene Blue and MAO immobilized on the electrode is comparable to that of homogeneous enzymic catalysis. When immobilized in a film of polymer electrolyte Eastman AQ-29, Methylene Blue and Toluidine Blue efficiently carry electrons from the reduced redox center of immobilized MAO onto a glassy-carbon electrode. Dependence of the oxidation current of these compounds on the concentration of the benzylamine substrate is studied and it is shown that the enzymic reaction is the limiting stage in a bioelectrocatalytic process. This conclusion gives grounds for using the dependence of the anodic current on the monoamine concentration as a calibration plot when assaying biogenic amines.     

石墨炉原子吸收光谱法测定大白鼠脑组织中痕量铝

采用具有横向加热石墨炉原子化器系统和纵向塞曼效应背景校正的原子吸收光谱仪测定了大白鼠脑组织中痕量铝,并探讨了相关试验条件。采用全自动进样器进行标准在线稀释绘制工作曲线,同时进行在线添加基体改进剂和在线加标回收率试验。方法的特征质量为26.2 pg,检出限为11.6 pg,相对标准偏差为3.8%,回收率为91.6%~98.2%。     

高效液相色谱法测定动物脑和脊髓内微透析样品中的递质氨基酸和单胺类递质及其代谢产物

测定了猴、兔的尾核、海马和小脑深核以及猫脊髓背角微透析样品中递质氨基酸和单胺类递质及其代谢产物的含量。浓度与响应的线性关系、检测极限和重复性均与以前的报道一致。对应用常规液相色谱方法分析脑内微透析样品中的上述化合物时应注意的问题作了扼要的讨论。     

High-Throughput Method for the Simultaneous Determination of Doxorubicin Metabolites in Rat Urine after Treatment with Different Drug Nanoformulations

Doxorubicin (DOX) is one of the most effective cytotoxic agents against malignant diseases. However, the clinical application of DOX is limited, due to dose-related toxicity. The development of DOX nanoformulations that significantly reduce its toxicity and affect the metabolic pathway of the drug requires improved methods for the quantitative determination of DOX metabolites with high specificity and sensitivity. This study aimed to develop a high-throughput method based on high-performance liquid chromatography with fluorescence detection (HPLC-FD) for the quantification of DOX and its metabolites in the urine of laboratory animals after treatment with different DOX nanoformulations. The developed method was validated by examining its specificity and selectivity, linearity, accuracy, precision, limit of detection, and limit of quantification. The DOX and its metabolites, doxorubicinol (DOXol) and doxorubicinone (DOXon), were successfully separated and quantified using idarubicin (IDA) as an internal standard (IS). The linearity was obtained over a concentration range of 0.05–1.6 μg/mL. The lowest limit of detection and limit of quantitation were obtained for DOXon at 5.0 ng/mL and 15.0 ng/mL, respectively. For each level of quality control (QC) samples, the inter- and intra-assay precision was less than 5%. The accuracy was in the range of 95.08–104.69%, indicating acceptable accuracy and precision of the developed method. The method was applied to the quantitative determination of DOX and its metabolites in the urine of rats treated by novel nanoformulated poly(lactic-co-glycolic acid) (DOX-PLGA), and compared with a commercially available DOX solution for injection (DOX-IN) and liposomal-DOX (DOX-MY).     

HPLC Determination of DCJW in Rat Plasma and Its Application to Pharmacokinetics Studies

A high-performance liquid chromatography–UV method for determining DCJW concentration in rat plasma was developed. The method described was applied to a pharmacokinetics study of intramuscular injection in rats. The plasma samples were deproteinized with acetonitrile in a one-step extraction. The HPLC assay was carried out using a VP-ODS column and the mobile phase consisting of acetonitrile–water (80:20, v/v) was used at a flow rate of 1.0 mL min⁻¹ for the effective eluting DCJW. The detection of the analyte peak area was achieved by setting a UV detector at 314 nm with no interfering plasma peak. The method was fully validated with the following validation parameters: linearity range 0.06–10 μg mL⁻¹ (r > 0.999); absolute recoveries of DCJW were 97.44–103.46% from rat plasma; limit of quantification, 0.06 μg mL⁻¹ and limit of detection, 0.02 μg mL⁻¹. The method was further used to determine the concentration–time profiles of DCJW in the rat plasma following intramuscular injection of DCJW solution at a dose of 1.2 mg kg⁻¹. Maximum plasma concentration (C _max) and area under the plasma concentration–time curve (AUC) for DCJW were 140.20 ng mL⁻¹ and 2405.28 ng h mL⁻¹.     

气相色谱/质谱联用测定大鼠脑部的神经甾体

应用气相色谱 质谱联用技术建立了大鼠脑部神经甾体的测定方法。游离型甾体和甾体硫酸酯分两步萃取。第一步用乙酸乙酯提取游离型甾体,第二步用氯仿/2 丁醇提取甾体硫酸酯,然后经固相萃取纯化。甾体硫酸酯进行溶剂解形成游离型甾体。游离型甾体和甾体硫酸酯分别经七氟丁酸酐衍生化后进行气相色谱 质谱分析。经初步研究雄性大鼠脑部游离型神经甾体孕烯醇酮(PREG)、黄体酮(PROG)、别孕烯醇酮(AP)和脱氢表雄酮(DHEA)的含量分别为（8.53±1.11） ng/g ,（ 7.01±2.60） ng/g ,（ 1.     

高效液相色谱法测定大鼠心肌组织中次黄嘌呤,肌苷和腺苷含量

研究了一种快速、简便测定大鼠心肌组织中次黄嘌呤（ＨＸ）、肌苷（Ｉ）和腺苷（ＡＤＯ）的高效液相色谱法。采用ＷａｔｅｒｓμＢｏｎｄａｐａｋＣ_（１８）色谱柱，含７％甲醇的２ｍｍｏｌ／Ｌ磷酸钠缓冲液（ｐＨ＝６．０）为流动相，等比洗脱，２５４ｎｍ处紫外检测，１２ｍｉｎ内即可分析一个样品。本法生物样本预处理简单，因而平均回收率达９５％以上。ＨＸ在５．０～５００ｎｇ，Ｉ和ＡＤＯ在１０～１０００ｎｇ范围内，线性关系良好，相对标准偏差分别低于３．４０％（日内）和５．２０％（日间），并测定了３０份心肌组织中次黄嘌呤、肌苷和腺苷的含量。对所用的色谱分析条件进行了讨论。     

Fluorometric Determination of Tryptophan and Its Brain Indoleamine Metabolites by Ion-Pair HPLC

Abstract

The reverse phase HPLC separation of tryptophan, serotonin, 5-hydroxyindoleacetic acid, tryptamine and indoleacetic acid using two solvent systems (one of them containing an ion-pairing reagent) is reported. The two low concentration eluents employed are either formic acid/water or 0.005 mol/L solution of 1-pentane sulfonn ic acid. In both cases chromatographic separation was achieved through a linear gradient elution using methanol/water (7/3) as the high concentration eluent. We also describe the variation of retention volumes of these compounds as a function of the pH of the mobile phase. pH values ranged from 1.5 to 4.0 and were obtained by adding either formic acid or NaOH respectively to the low concentration eluent. Tryptophan and its metabolites were detected fluorometrically. All compounds show a linear response in the pg to μg range. The chromatographic separation achieved allows a concurrent measurement of tryptophan and its main indoleamine metabolites which coupled to the high sensitivity of fluorometric detection permits a direct estimation of these metabolic pathways in brain tissue.