用高效液相色谱—电化学检测法快速、灵敏分析生物样本中的单胺类递质及其代谢产物

本实验用反相离子对色谱-电化学检测分析法,同时分离并测定生物样本中结构极为相似的单胺类递质及其代谢产物。实验采用等比洗脱,在30min内至少可测定九个这类化合物。生物样品的预处理简单,因而平均回收率能达到90％以上。十个化合物(包括3,4-二羟基苄胺)保留时间变异系数为0.73±0.18％(均值±标准差,下同);峰面积变异系数为1.11±0.45％;在50—100pg至40ng范围内,相夫系数为0.996±0.006。本文对我们所用的色谱分析条件进行了讨论。     

HPLC法测定大鼠血浆中EGCG的血药浓度

目的:建立快速、高效、灵敏的HPLC法测定大鼠血浆中的EGCG血药浓度。方法:以睾丸酮为内标物,血浆经盐酸和乙酸乙酯等去除蛋白。采用Agilent 20RBAX SB-C18色谱柱,乙腈-0.3%乙酸为流动相,流速1.0ml/min,检测波长为280nm,柱温40℃。结果:EGCG和内标的出峰时间分别为12.21min和15.4min。EGCG在0.05μg/ml-100μg/ml范围内线性关系良好(R2=0.9920),高、中、低3种浓度下的日内、日间精密度RSD均低于15%,相对回收率与绝对回收率均在100±15(%)范围内,稳定性好。结论:本方法灵敏、准确、高效,适用于大鼠血浆中EGCG的血药浓度检测。     

高效液相色谱法测定大鼠血清染料木素浓度

目的:建立大鼠血清中染料木素浓度的HPLC测定方法.方法:大鼠血清以叔丁基甲醚萃取,萃取物用氮气吹干后,用甲醇溶解用于色谱分析.色谱条件:采用Thermo C18柱(250ram×4.6mm,5μm);以乙腈-0.02mol/L磷酸二氢钾(35:65,pH=4.3)为流动相;流速为1.0mL/min;检测波长为260hm;柱温为40℃;进样量为10μL.结果:染料木素最低检测浓度为0.01mg/L;标准曲线线性范围为0.01～10.00μg/mL(r=0.9998);相对回收率为(101.31±3.47)%;日内RSD与日间RSD均小于10.00%.结论:该方法简便、快速、灵敏度高,重现性及稳定性较好,适用于大鼠血清染料木素浓度测定和药代动力学的研究.     

高效液相色谱法测定三种虫草发酵液与菌丝体核苷化合物含量

本项研究测定了三种虫草(江西虫草、古尼虫草、戴氏虫草)发酵液与菌丝体中核苷类化合物的含量,并建立了定量分析的方法体系。其中,采用液体发酵的方法获得虫草菌丝体及其发酵液,应用超声破碎法提取虫草菌丝体中的核苷类化合物,最后应用高效液相色谱法-二极管阵列检测器进行色谱分析,并以保留时间和吸收光谱双指标作为定性依据。实验取得了预期结果:优化了5种核苷化合物:腺苷、虫草素、尿苷、肌苷、鸟苷的色谱分离条件,分析了3种虫草中核苷类化合物的含量,精密度、稳定性、重现性和回收率实验表明这一方法可高效地定量分析虫草中的核苷类化合物。实验表明,HPLC-DAD法对虫草中的核苷类化合物的定量分析是一种有效的方法,该方法可快速准确地检测虫草有效成分的含量,本研究同时测定了胞外部分的化合物含量,系首次对这三种虫草细胞内外成分同时进行测定,对虫草的品质评价体系及其质量控制标准的建立具有重要意义。     

高效液相色谱法分析吡咯喹啉醌

目的:建立一种通过高效液相色谱(HPLC)定量测定吡咯喹啉醌(PQQ)的分析方法。方法:将PQQ标准品及发酵制备的PQQ结晶粉末溶于10 mmol/L Na OH溶液,利用高效液相色谱仪进行测定。采用Waters XBridge C18(4.6 mm×150 mm,5μm)作为分离柱,用甲醇-水(用三氟乙酸调节p H值为1.0)梯度洗脱,流速为1.0 m L/min,于室温下检测PQQ,检测波长为365.8 nm。结果:检测得PQQ标准品的保留时间约为7.8 min,在0.031 25~1 mg/m L范围内线性关系良好,相关系数(r2)在0.9999以上,平均回收率为98.6%。结论:高效液相色谱法分析PQQ的灵敏度和准确度高,是一种可靠的PQQ定量分析方法。     

高效液相色谱测定发酵液中1，3－二羟基丙酮（DHA）方法的建立

摘要：本文建立了简单且准确的测定1,3-二羟基丙酮（DHA）的高效液相色谱（HPLC）方法。以Alltima C18（5μm,250×4.6mm）为分离柱,5％甲醇水溶液（0.05％H3PO4调pH至3.0）,流速为1mL/min,用紫外检测器在200nm处检测DHA。结果测得DHA标准样品的保留时间为6.2min,并测得DHA的线性范围为0.1～10.0 g/L。用HPLC法测得以甘油为底物发酵产DHA发酵液中DHA含量为6.2 g/L。并证明高效液相色谱法可有效应用于产DHA发酵过程中产物的检测。     

穿心莲体内血清指纹图谱建立

为了建立穿心莲药材含药血清指纹图谱,对大鼠血清进行一系列的考察,分辨给药后血中产生的药源性成分,从而分析其物质基础.本实验采用HPLC-DAD技术,筛选出穿心莲给药大鼠最佳取血时间,最佳血清处理方法以及最佳检测波长,建立穿心莲血清指纹图谱,初步分析其血中成分来源.通过筛选,确定血清药品制备方法为:给药后lh后,血清采用甲醇沉淀法,检测波长为275 nm.含药血清色谱图中有10个共有峰.本研究建立的方法,准确、可靠,可用于穿心莲血清指纹图谱的研究.     

利用色谱法快速检测分析啤酒腐败菌的新方法

啤酒在生产过程中很容易染菌,而传统的用于啤酒腐败菌检测的方法耗时长,不能满足实际需求.由于腐败菌污染的啤酒通过色谱检测,相应组分(生物胺、有机酸和风味物质)都会有特征峰的产生,所以本研究通过建立无腐败菌污染的啤酒中各组分的标准色谱图,再使啤酒强制染菌,对其组分进行色谱分析,并与标准色谱图进行比较,从而找出各组分对应的特征峰.未来,此方法可用于实际生产线上快速检测啤酒是否发生微生物污染.     

大黄药材蒽醌成分的HPLC指纹图谱研究

以大黄酚为参照物,利用高效液相色谱梯度洗脱,测定了10批大黄样品,建立以大黄药材蒽醌及衍生物成分为特征的指纹图谱,色谱柱为YMC-ODS-AQC18柱(250 mm×4.6 mm,5μm);流动相:乙腈(A)-0.2%磷酸水(B),检测波长270 nm,柱温25℃,流速1.0 mL/min,通过高压液相指纹色谱分析,鉴定了13个色谱峰的化学成分,找出24个共有峰,相似度均大于0.92。本文所建立的方法可作为大黄药材质量控制。     

龙血竭提取物有效成分剑叶龙血素A小鼠药动学研究

目的:选取中药广西龙血竭提取物(EDB)中有效成分之一的剑叶龙血素A(2,6-三甲氧基-4'-羟基二氢查耳酮)作为考察对象,研究其药动学规律,对龙血竭临床用药具有一定的指导意义.方法:建立实验小鼠血浆中剑叶龙血素A含量的高效液相色谱测定方法,测定给药后不同时间点血浆中剑叶龙血素A的浓度.通过药动学软件3P87进行数据处理,得出剑叶龙血素A的相关药动学参数.结果:剑叶龙血素A血药浓度高效液相色谱测定方法的线性范围为50-500ng/ml,高中低三种浓度回收率分别是114.80±2.86、103.60±4.38、101.80±2.12,最小检测浓度为10ng/ml,日内差为1.28%、日间差为2.26%、精密度(RSD)小于3%;剑叶龙血素A的药动学参数是:T(peak)为77.73min、C(max)为224.99ng/ml、T1/2Ke为72.91 min、T1/2Ka为40.94min、V/F(C)为1.019(mg/kg)/(ng/ml)、AUC为49553.47(ng/ml)*min.结论:建立了小鼠血浆中剑叶龙血素A高效液相色谱测定方法;EDB口服后剑叶龙血素A药动学为一室模型,有较大的吸收利用率,其消除半衰期为吸收半衰期的1.78倍,是吸收比消除快的药物.     

Kinetic evidence for rapid oxidation of (-)-epicatechin by human myeloperoxidase

Apocynin has been reported to require dimerization by myeloperoxidase (MPO) to inhibit leukocyte NADPH oxidase. (-)-Epicatechin, a dietary flavan-3-ol, has been identified as a ‘prodrug’ of apocynin-like metabolites that inhibit endothelial NADPH oxidase activity and elevate the cellular level of nitric oxide. Since (-)-epicatechin has tentatively been identified as substrate of MPO, we studied the one-electron oxidation of (-)-epicatechin by MPO. By using multi-mixing stopped-flow technique, we demonstrate that (-)-epicatechin is one of the most efficient electron donors for heme peroxidases investigated so far. Second order rate constants for the (-)-epicatechin-mediated conversion of MPO-compound I to compound II and compound II to resting enzyme were estimated to be 1.9 × 10⁷ and 4.5 × 10⁶ M⁻¹ s⁻¹, respectively (pH 7, 25 °C). The data indicate that (-)-epicatechin is capable of undergoing fast MPO-mediated one-electron oxidation.     

Cell clones and pattern formation: Genetic eye mosaics inDrosophila melanogaster

Summary Genetic eye mosaics ofDrosophila melanogaster have been studied by means of anatomical techniques. Using different cell markers it was found that the ommatidia at the boundaries between phenotypes are composed of cells belonging to different clones. Therefore, the formation of an individual ommatidium does not obey a mechanism based on a common clonal origin of its constituent elements. A statistical analysis of mosaic ommatidia shows that there is a significant tendency for the receptor cellsR2-R5 on the one hand and the receptor cellsR1, R6 andR7 on the other to belong to the same cell clone. The implications of these findings are discussed.     

Tyrosyl Radicals in Dehaloperoxidase: HOW NATURE DEALS WITH EVOLVING AN OXYGEN-BINDING GLOBIN TO A BIOLOGICALLY RELEVANT PEROXIDASE*

Dehaloperoxidase (DHP) from Amphitrite ornata, having been shown to catalyze the hydrogen peroxide-dependent oxidation of trihalophenols to dihaloquinones, is the first oxygen binding globin that possesses a biologically relevant peroxidase activity. The catalytically competent species in DHP appears to be Compound ES, a reactive intermediate that contains both a ferryl heme and a tyrosyl radical. By simulating the EPR spectra of DHP activated by H₂O₂, Thompson et al. (Thompson, M. K., Franzen, S., Ghiladi, R. A., Reeder, B. J., and Svistunenko, D. A. (2010) J. Am. Chem. Soc. 132, 17501–17510) proposed that two different radicals, depending on the pH, are formed, one located on either Tyr-34 or Tyr-28 and the other on Tyr-38. To provide additional support for these simulation-based assignments and to deduce the role(s) that tyrosyl radicals play in DHP, stopped-flow UV-visible and rapid-freeze-quench EPR spectroscopic methods were employed to study radical formation in DHP when three tyrosine residues, Tyr-28, Tyr-34, and Tyr-38, were replaced either individually or in combination with phenylalanines. The results indicate that radicals form on all three tyrosines in DHP. Evidence for the formation of DHP Compound I in several tyrosine mutants was obtained. Variants that formed Compound I showed an increase in the catalytic rate for substrate oxidation but also an increase in heme bleaching, suggesting that the tyrosines are necessary for protecting the enzyme from oxidizing itself. This protective role of tyrosines is likely an evolutionary adaptation allowing DHP to avoid self-inflicted damage in the oxidative environment.     

复方利多海浮膏对混合痔患者外剥内扎术后肛缘疼痛的临床效果

目的：探讨复方利多海浮膏用于治疗混合痔外剥内扎术后肛缘疼痛和水肿的临床疗效。方法：选择2012 年7月-2014 年12 月在我院接受混合痔外剥内扎术治疗的患者70 例,随机分为治疗组和对照组,每组各35 例。治疗组采用复方利多海浮膏外敷换 药治疗,对照组采用黄连膏换药治疗。观察并比较两组患者治疗前后肛缘疼痛和水肿的变化情况。结果：两组患者术前疼痛评分 及肛缘水肿得分差异无统计学意义（P＞0.05）;治疗组患者治疗后1 天、2 天、3 天、5 天的疼痛评分和肛缘水肿得分均显著低于对 照组,差异有统计学意义（P＜0.05）;治疗组患者治疗总有效率显著优于对照组,差异有统计学意义（P＜0.05）。结论：复方利多海 浮膏外敷换药用于治疗混合痔外剥内扎术后肛缘疼痛和水肿方面的疗效更好。     

Redox properties of the couples compound I/compound II and compound II/native enzyme of human myeloperoxidase

Myeloperoxidase (MPO) is an important component of the neutrophil's antimicrobial armory and has been implicated in promoting tissue damage in numerous inflammatory diseases. For the first time the standard reduction potential of the redox couple compound II/native enzyme has been determined to be (0.97+/-0.01)V at pH 7.0 and 25 degrees C. This was achieved by rapid mixing of preformed compound II with either tyrosine or nitrite by using the sequential-mixing stopped-flow technique and measuring spectrophotometrically the concentrations of the reacting species and products at equilibrium. Using the recently determined standard reduction potential for the couple compound I/native enzyme (1.16 V), the reduction potential of the couple compound I/compound II was calculated to be 1.35 V at pH 7 and 25 degrees C. These data reveal substantial differences between the two known heme peroxidase superfamilies and reflect the dramatic differences observed in the oxidisability of substrates by the MPO redox intermediates compound I and compound II.     

Influence of the magnetic fields on frog sciatic nerve

The constant magnetic field (1000–7120 gauss) was applied to previously stimulated frog sciatic nerve. The following was observed : a) There is no instantaneous effect of either parallel or perpendicular magnetic field on compound action potential amplitude. b) Parallel magnetic field of 1000–7120 gauss does not change the amplitude of compound action potential significantly with time. c) When perpendicular magnetic field was applied to the nerve, an increase in the amplitude of compound action potential was observed, which can mean that the nerve exhibits some sort of magnetic anisotropy.     

On the identity and reactivity patterns of the "second oxidant" of the T252A mutant of cytochrome P450cam in the oxidation of 5-methylenenylcamphor

Density functional calculations show that in the absence of Compound I, the primary oxidant species of P450, the precursor species, Compound 0 (FeOOH), can effect double bond activation of 5-methylenylcamphor (1). The mechanism is initiated by homolytic cleavage of the O–O bond and formation of an OH radical bound to the Compound II species by hydrogen bonding interactions. Subsequently, the so-formed OH radical can either activate the double bond of 1 or attack the meso position of the heme en route to heme degradation. The calculations show that double bond activation is preferred over attack on the heme. Past the double bond activation, the intermediate can either lead to epoxidation or to a glycol formation. The glycol formation is predicted to be preferred, although in the P450_cam pocket the competition may be closer. Therefore, in the absence of Compound I, Compound 0 will be capable of epoxidizing double bonds. Previous studies [E. Derat, D. Kumar, H. Hirao, S. Shaik, J. Am. Chem. Soc. 128 (2006) 473–484] showed that in the case of a substrate that can undergo only C–H activation, the bound OH prefers heme degradation over hydrogen abstraction. Since the epoxidation barrier for Compound I is much smaller than that of Compound 0 (12.8 vs. 18.9 kcal/mol), when Compound I is present in the cycle, Compound 0 will be silent. As such, our mechanism explains lucidly why T252A P450_cam can epoxidize olefins like 5-methylenylcamphor but is ineffective in camphor hydroxylation [S. Jin, T.M. Makris, T. A. Bryson, S.G. Sligar, J.H. Dawson, J. Am. Chem. Soc. 125 (2003) 3406–3407]. Our calculations show that the glycol formation is a marker reaction of Compound 0 with 5-methylenylcamphor. If this product can be found in T252A P450_cam or in similar mutants of other P450 isozymes, this will constitute a more definitive proof for the action of Cpd 0 in P450 enzymes.     

Spectrophotometric investigations with hexa-coordinate ferric lignin peroxidase: does water retention at the active site influence catalysis?

Native lignin peroxidase (LIP) can adopt either a stable penta- or hexa-coordinate state. We have examined catalysis with hexa-coordinate ferric LIP as the starting material, using rapid scanning spectrophotometry. Initial two-electron oxidation of hexa-coordinate native LIP by H(2)O(2) (Compound I formation) was accompanied by a shifting isosbestic point (419-->416 nm), consistent with displacement of a resident water molecule, prior to the reaction of the ferric iron with H(2)O(2). The Compound I species derived from a hexa-coordinate ferric state shows an unusual peak at 520 nm, which may be due to water retention in the vicinity of the heme active site. Compound I reduction by veratryl alcohol showed saturation kinetics, which contrasts with the situation observed when Compound I is derived from a penta-coordinate ferric state. The data inferred that water can interfere with heme access by electron donors, altering the mechanism of Compound I reduction.     

Myeloperoxidase-catalyzed taurine chlorination: initial versus equilibrium rate

Myeloperoxidase (MPO) catalyzes the two-electron oxidation of chloride, thereby producing hypochlorous acid (HOCl). Taurine (2-aminoethane-sulfonic acid, Tau) is thought to act as a trap of HOCl forming the long-lived oxidant monochlorotaurine [(N-Cl)-Tau], which participates in pathogen defense. Here, we amend and extend previous studies by following initial and equilibrium rate of formation of (N-Cl)-Tau mediated by MPO at pH 4.0-7.0, varying H(2)O(2) concentration. Initial rate studies show no saturation of the active site under assay conditions (i.e. [H(2)O(2)] > or = 2000 [MPO]). Deceleration of Tau chlorination under equilibrium is quantitatively described by the redox equilibrium established by H(2)O(2)-mediated reduction of compound I to compound II. At equilibrium regime the maximum chlorination rate is obtained at [H(2)O(2)] and pH values around 0.4mM and pH 5. The proposed mechanism includes known acid-base and binding equilibria taking place at the working conditions. Kinetic data ruled out the currently accepted mechanism in which a proton participates in the molecular step (MPO-I+Cl(-)) leading to the formation of the chlorinating agent. Results support the formation of a chlorinating compound I-Cl(-) complex (MPO-I-Cl) and/or of ClO(-), through the former or even independently of it. ClO(-) diffuses away and rapidly protonates to HOCl outside the heme pocket. Smaller substrates will be chlorinated inside the enzyme by MPO-I-Cl and outside by HOCl, whereas bulkier ones can only react with the latter.