尿中核苷排放模式测定的两种方法─-HPLC和CE法(英文)

癌症病人在尿中排放比常人高得多的叶啉和嘧啶,这些改性核耷是RNA的主要组成。由于其增加的排放与癌变的RNA周转有关,已被建议和研究作肿瘤标记物。文章给出测定尿中核苷的反相高效液相色谱法和毛细管电泳方法。两种方法所得数据一致。用此方法建立了正常人尿中核苷的排放水平,并测定了34个癌症病人尿中核苷浓度,观察到改性核着浓度明显的增高现象。结果袭明,两个方法均适于大量尿样的核苷与癌症关系的临床研究。     

胶束电动力学毛细管色谱法测定尿中核苷

用胶束电动力学毛细管色谱法测定尿中核苷.通过苯基硼酸亲和色谱柱对尿中核苷进行纯化,以十二烷基磺酸钠(SDS)-十水合四硼酸钠-磷酸二氢钠为缓冲液(pH=6.96),在未涂层石英毛细管上,于7kV恒压,29℃下进行电泳分离,检测波长为254nm.此方法用于28个正常人尿中14种核苷的测定,确定了正常成年人(汉族)尿中核苷的排放范围.     

肠癌患者尿中核苷排放的高效液相法研究

 用反相高效液相法测定尿中核苷。通过苯基硼酸亲和法提取尿中核苷,在柱(4 6mmi d ×250mm,5μm)上以25mmol/L磷酸二氢钾溶液(pH4 55)和60%的甲醇水溶液作为流动相进行二元梯度淋洗,于22℃下进行反相分离,260nm处紫外检测。用该法测定了41例肠癌患者和52例正常人尿中15种核苷的含量(用核苷与肌酐的摩尔比表示,下同),结果表明肠癌患者中有12种核苷的含量比正常人显著性增高(P<0 001)。以15种核苷的含量作为参量,结合主成分分析区分正常人和肠癌患者,对癌症病人的识别率达76%(31/41)。     

高效毛细管区带电泳测定人尿液中尿酸、肌酐和伪尿核苷的含量

报道了采用高效毛细管区带电泳技术直接将人尿液注入毛细管进行尿液中肌酐、尿酸及伪尿核苷含量测定的新方法。试验表明,以磷酸盐（ｐＨ６．１）作缓冲液,对人体尿液中肌酐、尿酸及伪尿核苷进行直接分析具有较高的灵敏度和较好的重复性。     

毛细管电泳径向基神经网络校正法定量分析核苷

采用径向基神经网络算法对一组已知样品的核苷及内标物浓度与毛细管电泳峰面积数据进行回归计算,建立峰面积与核苷浓度之间的关系模型,对未知样品中待测核苷浓度作出预测,形成了毛细管电泳定量分析新方法．将其用于鸟嘌呤核苷含量测定,所建模型预测结果平均相对误差为0．86％,明显低于线性回归及BP神经网络模型的2．60％和1．07％．研究结果表明,本方法简便易用,能有效提高毛细管电泳定量分析的准确度,优于线性回归及BP神经网络法．     

高效液相色谱-电化学检测多胺的研究

多胺(polyamine)是指生物体内广泛存在的带有两个或两个以上氨基的脂肪胺的总称。主要有精脒(spermidine,SPD)腐胺(putrescine,PUT),精胺(spermine,SPM),和尸胺(cadaverine,CAD)。多胺在核酸,蛋白质代谢及细胞繁殖分化中起调节和控制作用,为人体生长,细胞代谢所必需。1971年Russell首先报道了癌症病人尿中多胺浓度增加,引起了人们广泛的     

高效液相色谱法同时测定假尿核苷和肌酐的研究及应用

本文用高效液相色谱法同时测定人尿中假尿核苷和肌酐,研究了流动相的pH值变化对色谱行为的影响,方法简便、快速,已成功地用于对正常人、肺部感染及肺癌患者的尿样分析,其结果有显著性差异。     

HPLC/ESI-Q-TOF MS鉴定淋巴癌患者尿液中的修饰核苷

尿液修饰核苷反映了机体RNA的代谢速率及细胞增殖状况, 可以作为非常有发展潜力的肿瘤标志物进行研究. 尿液中的修饰核苷采用Oasis®HLB固相萃取柱进行纯化, 利用高效液相色谱与电喷雾质谱联用(HPLC-ESI-MS)、高分辨质谱(HRMS)及串联质谱(MS/MS)技术进行分离鉴定. 对淋巴癌患者尿中修饰核苷研究发现, 9种尿液核苷与标样的信息完全一致, 17种无标样的尿液修饰核苷也被鉴定, 其中包括3-甲基腺苷、7-甲基腺嘌呤、5′-脱氢-2′-脱氧次黄苷、3-甲基鸟嘌呤、O6-甲基鸟苷和7-甲基-1-乙基鸟苷6种未见报道的新尿液修饰核苷. 此方法能在无对照品的情况下快速、准确地提纯、分离和鉴定复杂的生物样品.     

5'-脱氧-5'-氟-5-氟尿核苷的合成和性质

叙述了5'-脱氧-5'-氟尿核苷和5'-脱氧-5'-氟-5-氟尿核苷的合成, 并比较了它们以及尿核苷和5-氟尿核苷的一些性质。     

电化学预处理玻碳电极微分计时电位溶出法测定DNA中的嘌呤碱基及DNA浓度

研究了鸟嘌呤(G)、腺嘌呤(A)、鸟嘌呤核苷、腺嘌呤核苷和变性DNA在电化学预处理玻碳电极上的恒电流微分计时电位溶出行为. 实验结果表明, 用电化学方法预处理玻碳电极操作简单, 效果明显, 预处理玻碳电极对嘌呤及其核苷和DNA的吸附能力大大增强, 用微分计时电位溶出法可以得到灵敏的溶出峰, 溶出峰高(dt/dE)与其浓度在一定范围内呈良好的线性关系, 可用于嘌呤碱基及其核苷的定量检测和DNA浓度的测定. 将该方法应用于酸变性DNA样品中鸟嘌呤与腺嘌呤的同时测定, 选择性好、灵敏度高; 还可获得有关DNA损伤的一些信息.     

Analysis of urinary nucleosides. V. Identification of urinary pyrimidine nucleosides by liquid chromatography/electrospray mass spectrometry

Modified urinary nucleosides are potentially invaluable in cancer diagnosis, as they reflect altered RNA turnovers. High-performance liquid chromatography (HPLC) was combined with full-scan mass spectrometry, tandem mass spectrometry, MS(n) analysis and accurate mass measurements in order to identify pyrimidine nucleosides purified from urine. Potential nucleosides were assessed by their evident UV absorbance in the HPLC chromatogram and then further examined by the various mass spectrometric techniques. In this manner numerous pyrimidine nucleosides were identified in the urine samples from cancer patients including pseudouridine, cytidine, two methylcytidines and an acetylcytidine. Furthermore, a number of novel modified pyrimidine nucleosides were tentatively identified via critical interpretation of the combined mass spectrometric data.     

Study of urinary nucleosides as biological marker in cancer patients analyzed by micellar electrokinetic capillary chromatography

Thirteen normal and modified nucleosides, primarily degradation products of transfer ribonucleic acid (tRNA), were evaluated as potential tumor markers for cancer patients. Their urinary concentrations were determined by means of micellar electrokinetic capillary chromatography (MEKC) in the urine from 54 healthy adults and 70 cancer patients, then quantitatively expressed as a function of creatinine excretion. It was found that urinary nucleosides for cancer patients were on the average significantly higher than those for healthy controls, however, no significant differences were found between male and female or between different ages. Based on 13 urinary nucleoside concentrations, principal component analysis (PCA) could be used to classify 72% of cancer patients from the healthy controls. The present study shows that the precise measurement of urinary nucleosides by MEKC in combining with PCA technique may provide a clinically useful approach for diagnosis of cancer.     

Mass spectrometric identification of modified urinary nucleosides used as potential biomedical markers by LC–ITMS coupling

In diseases accompanied by strong metabolic disorders, like cancer and AIDS, modifying enzymes are up- or down-regulated. As a result, many different types of metabolic end-products, including abnormal amounts of modified nucleosides, are found in urine. These nucleosides are degradation products of an impaired ribonucleic acid (RNA) metabolism, which affects the nucleoside pattern in urine. In several basic experiments we elucidated the fragmentation pathways of 16 characteristic nucleosides and six corresponding nucleic bases that occur in urine using electrospray ionization ion trap MS⁵ (ESI-ITMS) experiments operated in positive ionization mode. For urinary nucleoside analysis, we developed an auto-LC–MS³ method based on prepurification via boronate gel affinity chromatography followed by reversed phase chromatography. For this purpose, an endcapped LiChroCART Superspher RP 18 column with a gradient of ammonium formate and a methanol–water mixture was used. This method gives a limit of detection of between 0.1 and 9.6 pmol for 15 standard nucleosides, depending on the basicity of the nucleoside. Overall, the detection of 36 nucleosides from urine was feasible. It was shown that this auto-LC–MS³ method is a valuable tool for assigning nucleosides from complex biological matrices, and it may be utilized in the diagnosis of diseases associated with disorders in RNA metabolism.     

Analysis of urinary nucleosides as helper tumor markers in hepatocellular carcinoma diagnosis

Hepatocellular carcinoma (HCC) is a common neoplasm in Taiwan, for which early diagnosis is difficult and the prognosis is usually poor. HCC is usually diagnosed by abdominal sonography and serum alpha‐fetoprotein (AFP) detection. Modified nucleosides, regarded as indicators for the whole‐body turnover of RNAs, are excreted in abnormal amounts in the urine of patients with malignancies and can serve as tumor markers. We analyzed the excretion patterns of urinary nucleosides from 25 HCC patients and 20 healthy volunteers by high‐performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI‐MS/MS) under optimized conditions. The HPLC/ESI‐MS/MS approach with selective reaction monitoring (SRM) allowed for the sensitive determination of nucleosides in human urine samples. The mean levels of the urinary nucleosides adenosine, cytidine, and inosine were significantly higher in HCC patients than healthy volunteers (average of 1.78‐, 2.26‐, and 1.47‐fold, respectively). However, the mean levels of urinary 1‐methyladenosine, 3‐methylcytidine, uridine, and 2′‐deoxyguanosine were not significantly different. Combined with the determination of serum AFP levels, the higher levels of urinary adenosine, cytidine, and inosine may be additional diagnosis markers for HCC in Taiwanese patients. Copyright © 2009 John Wiley & Sons, Ltd.     

Analysis of normal and modified nucleosides in urine by capillary electrophoresisa)

Summary Modified nucleosides excreted in urine have been studied as potential diagnostic markers for cancer and AIDS, and as indicators for the whole-body turnover of RNA. Until now, reversed-phase (RP) HPLC and, to some extent, immunoassays are the preferred analytical methods for urinary nucleosides. A new capillary electrophoretic method for the analysis of normal and modified nucleosides in urine has been developed and optimized in our laboratory. The separation of nucleosides extracted from normal human urine on phenyl boronic acid affinity chromatography columns was performed in uncoated 565 mm (500 mm to detection window) × 50 μm i.d. capillary tubing using a 300 mM SDS—25 mM borate—50 mM phosphate buffer (pH 6.7), a 45-s load, a voltage of 7.5 kV (41 μA) and UV detection at 260 and 210 nm. The average recovery of the nucleosides was 91 %. The calibration curves were linear over all physiological and pathophysiological concentration ranges and the limits of detection were at micromolar levels. Reproducibility of migration times were better than 1 % (coefficient of variation,CV), and the reproducibilities of the determined concentrations were better than 5 % for standards and 6–15 % for extracted urine. The developed method was used to quantify 15 normal and modified nucleosides in 25 normal urines to establish reference ranges. The analysis time was less than 45 min. Dedicated to Professor E. Bayer on the occasion of his 70th birthday. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996.     

Analysis of modified nucleosides in the urine of patients with malignant cancer by liquid chromatography/electrospray ionization mass spectrometry

As modified nucleosides reflect altered tRNA turnover which seems to be impaired in the body of cancer patients, they have been evaluated as potential tumor markers. High-performance liquid chromatography/electrosprary ionization quadrupole time-of-flight mass spectrometry (HPLC/ESI-Q-TOFMS) was used to identify nucleosides purified from urine in positive ionization mode. Potential nucleosides were assessed by their evident UV absorbance in HPLC and then further examined by mass spectrometric techniques. In this manner, 21 nucleosides were detected in the urine of a patient with lymphoid cancer including three modified nucleosides 5'-dehydro-2-deoxyinosine, N1,N2,N7-trimethylguanosine and N1-methyl-N2-ethylguanosine, which had never been reported previously.