超高效液相色谱-串联质谱法测定人尿中8-羟基脱氧鸟苷的含量——对吸烟与结直肠癌相关性的探讨

取经预处理的人尿(0.5 mL),加入同位素内标,经固相萃取柱纯化和真空离心干燥后用水0.2 mL溶解残渣。采用超高效液相色谱-串联质谱法测定DNA氧化损伤标志物8-羟基脱氧鸟苷(8-OHd G)的含量。按此法测定了65例正常人(吸烟及非吸烟者分别为21,44例)和61例直肠癌患者(吸收及非吸烟者分别为17,44例)尿液中8-OHd G的浓度水平。结果表明:吸烟正常人和吸烟结直肠癌患者的尿液8-OHd G浓度水平分别显著高于非吸烟正常人和非吸烟结直肠癌患者的;非吸烟结直肠癌患者和吸烟结直肠癌患者尿液8-OHd G浓度水平分别显著高于非吸烟正常人和吸烟正常人的。这表明吸烟作为一个风险因子,可能会促进DNA氧化损伤,进而诱导结直肠癌的发生。     

超高效液相色谱-串联质谱法测定氧化损伤标志物8-羟基脱氧鸟苷

发展了一种超高效液相色谱-串联质谱法(UPLC-MS/MS)检测脱氧核糖核酸(DNA)分子中8-羟基脱氧鸟苷(8-OHdG)的方法。DNA分子在酶解过程中,脱氧鸟苷(dG)易被氧化形成8-OHdG,从而使得8-OHdG的检测结果不准确。通过加入甲磺酸去铁铵作为保护剂,有效地避免了酶解过程造成的dG氧化。酶解液通过超滤膜(截留相对分子质量为3 000的分子)处理,有效去除大量蛋白分子后,直接进行UPLC-MS/MS测定。采用外标法定量,在17.6～1 400fmol范围内,8-OHdG的峰面积与其物质的量具有良好的线性关系,相关系数为0.999 8。利用本方法测定了小牛胸腺DNA中8-OHdG的含量(用比值8-OHdG/106dG表示)为12.9±2.35,与前人报道的检测结果一致。本方法也可以应用于评价各种氧化因素引起的DNA氧化损伤。     

羟基自由基引起的脱氧核糖核酸损伤研究

以鲱鱼精脱氧核糖核酸(Herring sperm DNA)为研究对象,利用紫外光(UV,200～275 nm,66.4 Lx)激发纳米TiO2发生光催化作用介导产生羟基自由基(Hydroxyl radical,.OH),探讨.OH引发DNA氧化损伤特性。采用凝胶电泳和高效液相色谱(HPLC)分析法跟踪DNA损伤历程;应用电子自旋共振(Electron spinresonance,ESR)及分光光度法跟踪损伤过程氧化物种及H2O2相对浓度的变化;运用生物标准样8-羟基脱氧鸟苷(8-Hydroxy-2’-deoxyguanosine,8-OHdG)为内标物,通过HPLC分析DNA损伤产物,研究DNA损伤机理。结果表明,较单纯UV辐照或暗光(Dark)催化条件,DNA浓度10 mg/L,TiO2浓度1.5 g/L、pH 7～8,紫外光激发纳米TiO2介导产生.OH引发DNA损伤程度最大;DNA损伤为.OH氧化历程,并伴随有深度氧化过程;DNA结构中鸟嘌呤最易氧化损伤,8-OHdG为DNA氧化损伤中间产物及鸟嘌呤氧化损伤的特异产物。     

新杂环化合物6,20-二硫杂-13,27-二氧杂三环[23,3,0,011,15]-3,8,17,22-四炔-二十八烷的合成及NMR结构鉴定

近年来,科学家先后从细菌分泌物中分离出几种含有环状"烯-二炔”的Calicheamicinγ1I[1～4]Esperamicin A1[5～7],Dynemicin A1[8]Neocarzinostatin 发色团[9～11],Kedarcidin发色团[12～13]等新型抗癌抗菌素,我们已做过评述[14].它们的"1,5-二炔-3-烯”部分经生物还原生成1,4-脱氢苯双自由基,从癌细胞DNA糖磷酯骨架上夺取氢原子,引起癌细胞DNA氧化断裂,杀灭癌细胞,因而有广泛的应用前景. 标题化合物是"烯-二炔”前体,以3,4-二(4′-甲磺酰氧-2′-丁炔基)四氢呋喃(1)为原料,在硫化钠作用下关环,得到了新杂环化合物(2).通过元素分析,红外光谱,质谱进行了分析,又进一步进行了1H NMR和13C NMR谱测定,并对其谱线进行归属,确证了结构.     

4-溴-5-甲基靛红的Baeyer-Villiger氧化反应

Baeyer-Viliger反应是由酮制备酯的重要方法^[1].氧化剂通常有两类:(1)有机过酸;(2)过硫酸钾/硫酸(Caro酸).由于靛红结构特殊,我们分别以单过邻苯二甲酸^[2]和过硫酸钾/硫酸^[3]为氧化剂氧化4-溴-5-甲基靛红(1).     

DNA氧化损伤评价模型的构建及其中药保护剂的筛选

香烟烟气含有上千种化合物,其中60多种证明具有致癌作用.另外香烟烟气含有高水平活性氧基团,例如超氧自由基、羟基自由基等,能够对细胞DNA造成氧化损伤~([1,2]).近年来研究表明:8-羟基脱氧鸟苷(8-OHdG)已成为检测DNA损伤的一种重要标志物.本研究以8-羟基脱氧鸟苷(8-OHdG)为DNA氧化损伤的标志物,构建了Fenton试剂(Fe~(2+)+H_2O_2)及吸烟产生的活性自由基对DNA的氧化损伤评价体系,并利用该体系筛选对DNA具有保护作用的中药单体.     

高效液相色谱法分离检测8-羟基喹啉和8-羟基喹啉铜

1　引　　言8 羟基喹啉铜 [Cu(HQ) 2 ]是一种杀菌剂 ,用于防治作物的丝核菌及木材的防腐或用于棉布、皮革、油毛毡等的杀菌。它的纯度分析一直是该杀菌剂开发的重要工作之一。JosephA .Akkara等试图用高效液相色谱法和气相色谱法直接测定[Cu(HQ) 2 ]均未获成功。目前 [Cu(HQ) 2 ]的含量测定多采用化学法 ,该法实际得到的是总铜量 ,由此推算 [Cu(HQ) 2 ]含量有一定误差。本文采用高效液相色谱法同时分离、测定 [Cu(HQ) 2 ]及 8 羟基喹啉 (HQ)。有关这方面的研究 ,国内外尚未见报道。该方法简便、可行 ,结果令人满意。2　实验部分2 .1…     

聚N-[5-(8-羟基喹啉)甲基]苯胺/硅杂化材料的制备与性能

以8-羟基喹啉、甲醛和苯胺为原料,制备了5-[(苯胺基)甲基-8-羟基喹啉,并以此为单体,以过硫酸铵作为氧化剂,采用化学氧化聚合法,在酸性水溶液中合成了聚N-[5-(8-羟基喹啉)甲基]苯胺.通过正硅酸乙酯表面修饰聚N-[5-(8-羟基喹啉)甲基]苯胺获得了具有荧光特性的聚N-[-5-(8-羟基喹啉)甲基]苯胺/硅杂化材料.该杂化材料不仅在480 nm附近发出较强的荧光,而且在强酸和弱酸电解质溶液中均表现出了较好的氧化-还原可逆性.     

手性金属配合物△,∧-[Ru(IP)2dppz]2+对含G:T错配的环丁烷嘧啶二聚体识别和部分构型修复的分子力学研究

环丁烷嘧啶二聚体(Cyclobutane Pyrimidine Dimer,CPD)是紫外线对DNA损伤导致皮肤癌的首要环节,XPC-hHR23B是最早作为对CPD的损伤识别剂的,但其识别效率很低.本文首次采用分子力学方法模拟了一种新的手性金属配合物△,∧-[Ru(IP)2dppz]2 对含G:T错配的CPD双螺旋DNA的识别作用.模拟结果显示:金属配合物[Ru(IP)2dppz]2 的两个手性异构体都对含G:T错配的CPD双螺旋DNA具有识别作用,识别的过程体现了很强的手性选择性、沟选择性和位点特异性.同时,我们发现:在∧-[Ru(IP)2dppz]2 插入到CPD后,形成CPD的两个T碱基由原来的敞口形状部分地转为近平行状,使其在构型上得到初步的修复.     

Fe3O4/葡聚糖/抗体磁性纳米生物探针的制备和层析检测

在免疫检测中 ,经常利用一些具有特殊物理化学性质的标记物对抗体 (或抗原 )进行偶联标记 ,在抗体与抗原识别后 ,通过对标记物的定性和定量检测而达到对抗原 (或抗体 )检测的目的 .传统的免疫标记物包括放射性同位素 [1] 、酶 [2 ] 、胶体金 [3] 和有机荧光染料分子 [4 ] 等 .近年来 ,随着纳米技术的发展 ,半导体荧光纳米晶 [5,6 ] 和磁性纳米晶 [7] 在免疫检测方面受到了广泛关注 .磁性纳米晶性能稳定 ,较易制备 ,可与多种分子复合使粒子表面功能化 ,并且由于磁纳米晶具有超顺磁性 ,为样品的分离、富集和提纯提供了很大方便 .这些优点使它…     

Capillary electrophoresis with end-column amperometric detection of urinary 8-hydroxy-2'-deoxyguanosine

Urinary 8-hydroxy-2'-deoxyguanosine (8OHdG) is an excellent marker of oxidative DNA damage. Until now, urinary 8OHdG has been measured by high-performance liquid chromatography with electrochemical detection. A simple and sensitive method for the analysis of urinary 8OHdG by capillary electrophoresis with end-column amperometric detection has been developed in our laboratory. A single-step solid-phase extraction procedure was optimized and used for extracting 8OHdG from human urine. To improve the sensitivity of this method, a new focusing technique based on a dynamic pH junction was used. The limit of detection was 20 nM (signal-to-noise ratio S/N = 3), the linear range was 50 nM-10 microM, and the correlation coefficient was better than 0.999. The relative standard deviation (RSD) was found to be 0.57% for migration time, and 4.79% for peak current. To show the usefulness of the method, the urinary concentration of 8OHdG in nine healthy persons and ten cancer patients was determined. The urinary concentration of 8OHdG in cancer patients was significantly higher than that in healthy persons.     

Assay of urinary 8‐hydroxy‐2′‐deoxyguanosine by capillary electrophoresis with spectrophotometric detection using a high‐sensitivity detection cell and solid‐phase extraction

A sensitive capillary electrophoretic method featuring spectrophotometric detection using a commercial Z‐cell was devised for the assay of 8‐hydroxy‐2′‐deoxyguanosine (8OHdG) in human urine. Solid‐phase extraction (SPE) based on hydrophilic‐lipophilic‐balanced RP sorbent was utilized for urine sample pretreatment and analyte preconcentration. The separation was carried out in conventional fused‐silica capillaries employing a Z‐cell with hydrodynamic sample injection (at 50 mbar for 12 s). The BGE (pH* 9.2, adjusted with 1 M NaOH) contained 0.15 M boric acid and 10% v/v ACN. The detection wavelength was 282 nm. The calibration curve for 8OHdG (measured in spiked urine) was linear in the range 10–1000 ng/mL; R² = 0.9993. The LOD was 3 ng/mL (11 nmol/L) of 8OHdG. Determination of the 8OHdG urinary levels was possible even in healthy individuals.     

Determination of urinary 8‐hydroxy‐2′‐deoxyguanosine by a combination of on‐line molecularly imprinted monolithic solid phase microextraction with high performance liquid chromatography–ultraviolet detection

8‐Hydroxy‐2′‐deoxyguanosine (8‐OHdG) is a sensitive biomarker for DNA oxidative damage. However, its determination in human urine is confounded by trace level and complex matrix. In this study, a new configuration of on‐line solid phase microextraction coupled to high performance liquid chromatography‐ultraviolet detection was established with molecularly imprinted monolithic column as extraction sorbent. The tailor made monolith exhibited high extraction efficiency with the enrichment factor 101.84 for 8‐OHdG owing to its special porous structure and inherent selectivity. Under optimal condition, appreciable sensitivity had been achieved for this incorporation with limit of detection 2.04 nmol/L (S/N = 3) and limit of quantification 7.12 nmol/L (S/N = 10), respectively. Precise determination with wide range linearity (0.007–5.00 μmol/L) afforded a practical alternative in urinary 8‐OHdG analysis and 107 different subjects had been successfully analyzed. This newly developed method embodied useful prospect for the investigation of DNA oxidative damage with less expense, convenient maintenance and ease of operation     

Molecularly imprinted monolith in-tube solid-phase microextraction coupled with HPLC/UV detection for determination of 8-hydroxy-2′-deoxyguanosine in urine

Urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) has been widely used as a biomarker of oxidative DNA damage. Measurements of 8-OHdG in urinary samples are challenging owing to the low level of 8-OHdG and the complex matrix. In this study, a novel molecularly imprinted polymer (MIP) monolithic column was synthesized with guanosine as a dummy template which was used as the medium for in-tube solid-phase microextraction (SPME). In-tube SPME coupled with HPLC/UV detection for extraction and determination of urinary 8-OHdG was developed. The synthesized MIP monolithic column exhibited high extraction efficiency owing to its greater phase ratio with convective mass transfer and inherent selectivity. The enrichment factor for 8-OHdG was found to be 76 and the limits of detection and quantification of the method for urinary samples were 3.2 nmol/L (signal-to-noise ratio 3) and 11 nmol/L (signal-to-noise ratio 10), respectively. The MIP^’s selectivity also made the sample preparation procedure and chromatographic separation much easier. The linear range of the proposed method was from 0.010 to 5.30 μmol/L (r = 0.9997), with a relative standard deviation of 1.1–6.8%, and the recovery for spiked urine samples was 84 ± 3%. The newly developed method was successfully applied to determine urinary samples of healthy volunteers, coking plant workers, and cancer patients. The 8-OHdG level in cancer patients was significantly higher than that in healthy people.     

Detection and quantification of 8‐hydroxy‐2′‐deoxyguanosine in Alzheimer's transgenic mouse urine using capillary electrophoresis

8‐Hydroxy‐2′‐deoxyguanosine (8‐OHdG) is one of the major forms of oxidative DNA damage, and is commonly analyzed as an excellent marker of DNA lesions. The purpose of this study was to develop a sensitive method to accurately and rapidly quantify the 8‐OHdG by using CE‐LIF detection. The method involved the use of specific antibody to detect the DNA lesion (8‐OHdG) and consecutive fluorescence labeling. Next, urinary 8‐OHdG fluorescently labeled along with other constituents were resolved by capillary electrophoretic system and the lesion of interest was detected using a fluorescence detector. The limit of detection was 0.18 fmol, which proved sufficient sensitivity for detection and quantification of 8‐OHdG in untreated urine samples. The relative standard deviation was found to be 11.32% for migration time and 5.52% for peak area. To demonstrate the utility of this method, the urinary concentration of 8‐OHdG in an Alzheimer's transgenic mouse model was determined. Collectively, our results indicate that this methodology offers great advantages, such as high separation efficiency, good selectivity, low limit of detection, simplicity and low cost of analysis.     

A method for routine quantitation of urinary 8-hydroxy-2'-deoxyguanosine based on solid-phase extraction and micro-high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

8-Hydroxy-2'-deoxyguanosine (8OHdG), one of the major oxidative DNA lesions induced by radical agents, is commonly used as a biomarker for oxidative stress, nowadays preferably in urine. In the absence of a commercially available internal standard a micro-high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (micro-HPLC/ESI-MS/MS) method, suitable for routine analysis of 8OHdG in human urine using external calibration, was developed. Evaluation of the matrix effect showed that the method allows highly sensitive and accurate quantitation despite the absence of an internal standard. HPLC analysis was performed using gradient elution at a flow rate of 10 microL min(-1) using a capillary reversed-phase column and an injection volume of 0.5 microL, with detection of 8OHdG in positive multiple reaction monitoring (MRM) mode. The absolute limit of detection was 0.35 fmol using m/z 168 as a quantifier (fragment) ion. A linear (R2> 0.999) calibration curve in urine was obtained over a range 0.2-10 ng mL(-1). This method is about 20 times more sensitive than previously described procedures, and is characterized by high accuracy (mean 90%) and good reproducibility (RSD <10%). The optimized method was applied to determination of 8OHdG in 18 urinary samples derived from three healthy volunteers. 8OHdG urinary excretion ranged from 3.0-7.9 microg/day, and a large intra-individual variation was found. This method, which effectively circumvents the need for isotopically labeled 8OHdG (internal standard), is suitable for routine monitoring of exposure to DNA-damaging factors in a large number of subjects.     

Determination of urinary oxidative DNA damage marker 8-hydroxy-2'-deoxyguanosine and the association with cigarette smoking

8-hydroxy-2′-deoxyguanosine (8OHdG) has been widely used as a biomarker of oxidative DNA damage in both animal models and human studies. To evaluate the effect of cigarette smoking on oxidative stress, we studied the levels of urinary 8OHdG from smokers and non-smokers and investigated the association with cigarette smoking. The urinary 8OHdG concentrations were determinated by capillary electrophoresis with end-column amprometric detection (CE-AD) after a single-step solid phase extraction (SPE), and then quantitatively expressed as a function of creatinine excretion. To increase the concentration sensitivity, a dynamic pH junction was used and the focusing effect was obvious when using 30 mM phosphate (pH 6.50) as sample matrix. The limit of detection is 4.3 nM (signal-to-noise ratio S/N=3). The relative standard deviation (R.S.D.) was 1.1% for peak current, and 2.3% for migration time. Based on the selected CE-AD method, it was found that the mean value of urinary 8OHdG levels in the smokers significantly higher than that in non-smokers ( versus , P=0.0004; creatinine versus creatinine, P=0.028).     

Novel approach for developing urinary nucleosides profile by capillary electrophoresis-mass spectrometry

A simple, rapid and efficient capillary electrophoresis-mass spectrometry (CE-MS) method was developed to analyze urinary nucleosides for the first time. The composition of CE buffer and MS parameters were systematically optimized. The optimum buffer was 150 mM acetic acid containing 15% methanol and 15% ethanol. The optimum MS parameters were: methanol containing 0.5% acetic acid was selected as the sheath liquid and the flow rate was 5 microL/min; the flow rate and temperature of drying gas were 6L/min and 150 degrees C, respectively; the pressure of nebulizing gas was 2 psig; and the fragmentor and ESI voltage were 100 V and 4000 V, respectively. Under the optimum CE-MS conditions, the urinary nucleosides were separated within 18 min. The linearity between the relative peak areas and the corresponding concentration of nine nucleosides markers were excellent. The limits of detection (S/N=3) of markers were 0.00862-3.82 nmol/mL. The optimum CE-MS method was applied to analyze urine from 20 bladder cancer patients and 20 healthy volunteers. Considering the standards of many nucleosides cannot be obtained, it is not the ratios of the concentrations of nucleosides to that of creatinine in the literatures, but the ratios of the relative peak area of nucleosides to the concentration of creatinine that used for pattern recognition. And, the statistical analysis result indicated this method was feasible.     

高效液相色谱-程序波长紫外检测法同时测定血浆中的色氨酸及其代谢物

建立了一种高效液相色谱-程序波长紫外检测法同时测定血浆中色氨酸(Trp)及其主要代谢产物犬尿氨酸(Kyn)和5-羟色胺(5-HT)。以茶碱为内标(IS),采用BDS-Hypersil-C8柱(150 mm×4.6 mm, 5 μm)分离。流动相为10 mmol/L醋酸钠缓冲液(pH 4.5)-乙腈(94:6, v/v),流速为0.6 mL/min;柱温为25 ℃;紫外检测波长设定: Kyn和IS为360 nm, 5-HT为220 nm, Trp为302 nm。3种物质的平均回收率为87%～113%;线性范围分别为3.97～400 μmol/L(Trp), 0.421～20.2 μmol/L(Kyn), 4.36～980 nmol/L(5-HT);检出限分别为0.134 μmol/L(Trp), 0.0160 μmol/L(Kyn), 2.03 nmol/L(5-HT)。利用该方法对15例抑郁症患者和15例健康志愿者的血浆进行测定,结果表明两组间Trp的代谢存在显著的差异。     

高效液相色谱－电化学检测法测定尿液中的多胺

在已报道的高效液相色谱－电化学检测多胺方法研究的基础上，又进一步探讨了该方法用于尿样本测定的各种条件，并测定了健康志愿者及肿瘤患者尿液中的多胺。结果显示肿瘤患者未水解及水解尿液中的腐胺、尸胺的平均值高于健康志愿者。