Upregulated plasma and urinary levels of nucleosides as biological markers in the diagnosis of primary gallbladder cancer

We first detected aberrant nucleoside levels in the plasma, urine, bile, and tissues from cases and controls to explore them as biomarkers in the diagnosis of gallbladder cancer. Reversed‐phase high‐performance liquid chromatography was used to assess the levels of ten nucleosides in these samples from gallbladder cancer patients, gallstone patients, and healthy controls. Plasma and urine samples were collected from patients with gallbladder cancer (n = 202), patients with gallstones (n = 203), and healthy controls (n = 205); bile and tissue samples were collected from 91 gallbladder cancer patients, 93 gallstone patients; and 90 were donated after cardiac death. Of the ten nucleosides analyzed, eight urinary nucleosides, five plasma nucleosides, three bile nucleosides, and one tissue nucleoside were significantly upregulated in the gallbladder cancer patients compared to control groups (p < 0.05). Among these upregulated nucleosides, the sensitivity, specificity, and accuracy of urinary nucleosides in the diagnosis of gallbladder cancer patients were 89.4, 97.1, and 95.7%, respectively, those of plasma nucleosides were 91.2, 95.6, and 94.2%, respectively, those of bile nucleosides were 95.3, 96.4, and 95.1%, respectively, and those of tissue nucleosides were 86.2, 93.8, and 92.6%, respectively. These results suggest that nucleosides may be as useful as biological markers for gallbladder cancer.     

Direct determination of nucleosides in the urine of patients with breast cancer using column-switching liquid chromatography-tandem mass spectrometry

We developed an analytical method for a simple, sensitive and simultaneous determination of oxidized nucleosides in urine using column-switching liquid chromatography-electrospray/tandem mass spectrometry (LC-ESI/MS/MS). We connected two columns through a six-way switching valve and effectively separated nucleosides in the urine from the interference by column-switching liquid chromatography. We monitored separated nucleosides using positive ionization tandem mass spectrometry in selective reaction monitoring (SRM) mode. The calibration ranges of nucleosides were 0.2-100 nmol/mL. The linearity of the method was 0.994-0.999, and the limits-of-detection (LOD) at a signal-to-noise (S/N) ratio of 3 were 0.1-0.2 nmol/mL. The coefficients of variation were in the range 2.28-11.74% for within-day variation and 4.36-11.15% for day-to-day variation, respectively. To explore the relationship between breast cancer and the nucleosides level in human urine, we measured the concentrations of nucleosides in female patients with breast cancer (n = 30) and in normal female subjects (n = 30). The concentration of nucleosides was significantly increased in patients with breast cancer when compared with the normal controls (1-methyladenosine; p < 0.005, N(2),N(2)-dimethylguanosine; p < 0.01, 5-hydroxymethyl-2'-deoxyuridine; p < 0.001, 8-hydroxy-2-deoxyguanosine; p < 0.001). Therefore, the elevated levels of nucleosides could be used as an important biomarker for breast-cancer research.     

Development and validation of a hydrophilic interaction chromatography-tandem mass spectrometry method with on-line polar extraction for the analysis of urinary nucleosides. Potential application in clinical diagnosis

The present paper describes the development, validation and application of a quantitative method for the determination of endogenous nucleosides and nucleobases in urine based on the on-line coupling of a solid-phase extraction step with hydrophilic interaction chromatography-tandem mass spectrometry. The method combines the use of a highly polar restricted-access material (RAM), based on an N-vinylacetamide copolymer, for efficient analyte extraction and matrix removal, with separation by zwitterionic hydrophilic interaction chromatography (ZIC-HILIC), that revealed a satisfactory retention of the polar analytes studied. Detection using a triple quadrupole analyser allowed reliable identification and high-sensitivity quantitation of the target compounds. The on-line configuration developed, RAM-ZIC-HILIC-MS/MS, provides a convenient approach to automate the application to urine analysis, with minimum sample manipulation. The whole method was validated according to European Legislation for bioanalytical methods. The validation steps included the verification of matrix effects, calibration curve, precision, accuracy, selectivity, stability and carry-over in real samples. The results of the validation process revealed that the proposed method is suitable for the reliable determination of nucleosides and nucleobases in human urine, showing limits of detection from 0.1 to 1.3 ng mL(-1). The application to clinical samples was also checked; the results obtained in analyses of urine samples from healthy volunteers and cancer patients using Principal Component Analysis, Hierarchical Cluster Analysis and Soft Independent Modeling of Class Analogy are also shown.     

毛细管电泳-小波神经网络法辅佐诊断乳腺癌的研究

采用毛细管电泳法测定了46个健康人和26个乳腺癌病人尿样中的13种正常核苷和修饰核苷,以小波神经网络作为模式识别工具对健康人和乳腺癌病人的分类作了研究,随机选取的训练集的识别率达到100％,相应的预测集判别率正确性在96％以上,与经典的前向多层神经网络相比,小波神经网络具有更强的信息提取和逼近能力.研究结果还表明,小波神经网络的预测能力强于主成分分析和线性判别分析,毛细管电泳法与小波神经网络的结合有望成为乳腺癌的辅助诊断手段.     

固相萃取-高效阴离子交换色谱-积分脉冲安培法检测人体尿液中的异黄蝶呤

建立了固相萃取-高效阴离子交换色谱-积分脉冲安培法(SPE-HPAEC-IPAD)测定人体尿液中异黄蝶呤的分析方法。尿液经ENVI-18与732型阳离子交换柱串联萃取后,除去了大量干扰物质。采用IonPac AS21分析柱(250 mm×2 mm),以0.025 mol/L NaOH溶液为淋洗液,流速为0.40 mL/min,在优化的安培检测波形条件下,异黄蝶呤的质量浓度在0.005～0.200 mg/L范围内与峰面积呈良好的线性关系,相关系数为0.998 4,检出限为0.003 mg/L。健康人及癌症病人尿液在2 mg/L和5 mg/L两个添加水平的平均回收率在95.4%～96.8%之间,相对标准偏差小于5%。此方法环保、快速、准确,可用于健康人与癌症病人尿液中异黄蝶呤的测定。     

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.     

Reversed-phase high-performance liquid chromatographic investigation of mucosal nucleosides and bases and urinary modified nucleosides of gastrointestinal cancer patients

Reversed-phase high-performance liquid chromatography (HPLC) was used to determine the levels of nucleosides, bases and their metabolites in perchloric acid extracts of gastrointestinal mucosa. By comparing the levels of these compounds in the normal portion with the neoplastic portion of mucosa resected from malignant cancer patients, it was found that there was significant elevation of the uracil level in the neoplastic mucosa of all eight patients with colorectal cancer (2.7-fold in normal mucosa), but only in the neoplastic mucosa of one out of four patients with gastric cancer. The levels of hypoxanthine and uridine in the colorectal cancer mucosa samples and the inosine in gastric cancer samples were also significantly higher than those in normal mucosa. The urinary modified nucleosides were prefractionated with a boronate affinity gel column, and their levels were determined by the same HPLC method. There was no significant difference in the concentrations of pseudouridine, 1-methylguanosine N2-methylguanosine and N2,N2-dimethylguanosine between urine samples taken before and after surgery from eight patients with malignant colorectal cancer. Contrary to other reports, no significant differences in modified nucleoside levels were observed between urine samples from patients with colorectal cancer and those from normal subjects.     

Quantitative high-performance liquid chromatography of nucleosides in biological materials

A rigorous, comprehensive, and reliable reversed-phase high-performance liquid chromatographic (HPLC) method has been developed for the analysis of ribonucleosides in urine (psi, m1A, m1I, m2G, A, m2(2)G). An initial isolation of ribonucleosides with an affinity gel containing an immobilized phenylboronic acid was used to improve selectivity and sensitivity. Response for all nucleosides was linear from 0.1 to 50 nmoles injected and good quantitation was obtained for 25 microliter or less of sample placed on the HPLC column. Excellent precision of analysis for urinary nucleosides was achieved on matrix dependent and independent samples, and the high resolution of the reversed-phase column allowed the complete separation of 9 nucleosides from other unidentified UV absorbing components at the 1-ng level. Supporting experimental data are presented on precision, recovery, chromatographic methods, minimum detection limit, retention time, relative molar response, sample clean-up, stability of nucleosides, boronate gel capacity, and application to analysis of urine from patients with leukemia and breast cancer. This method is now being used routinely for the determination of the concentration and ratios of nucleosides in urine from patients with different types of cancer and in chemotherapy response studies.     

Evaluation of a gas sensor array and pattern recognition for the identification of bladder cancer from urine headspace

Previous studies have indicated that volatile compounds specific to bladder cancer may exist in urine headspace, raising the possibility that headspace analysis could be used for diagnosis of this particular cancer. In this paper, we evaluate the use of a commercially available gas sensor array coupled with a specifically designed pattern recognition algorithm for this purpose. The best diagnostic performance that we were able to obtain with independent test data provided by healthy volunteers and bladder cancer patients was 70% overall accuracy (70% sensitivity and 70% specificity). When the data of patients suffering from other non-cancerous urological diseases were added to those of the healthy controls, the classification accuracy fell to 65% with 60% sensitivity and 67% specificity. While this is not sufficient for a diagnostic test, it is significantly better than random chance, leading us to conclude that there is useful information in the urine headspace but that a more informative analytical technique, such as mass spectrometry, is required if this is to be exploited fully.     

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.     

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)。     

Activation analysis of selenium in cancer research

The method described in a previous work to separate trace amounts of selenium in organic samples without using a carrier, based on the adsorption on active carbon filters of the complex formed with ammonium pyrrolidindithiocarbamate (APDC) at pH 1.5–2, has been applied to urine samples from 15 females patients suffering from cervical uterine cancer. With this type of sample the method reaches a maximum sensitivity (few ppb) with a good statistical variation (±12%). Since the highest concentration of selenium in human tissues is found in the kidney, and the elimination of this element is mainly by the urine, the method seems to be a powerful tool in the research about the human metabolism of selenium. This paper shows a possible relation of selenium concentration in human urine and the evolution time of cervical uterine cancer, in spite of limits imposed by the statistical error plus the inhomogeneity of the sample.     

彩色多普勒超声结合弹性成像技术在鉴别乳腺良恶性肿瘤中的应用价值

本文对彩色多普勒超声(CDUs)结合弹性成像技术(UE)在鉴别乳腺良恶性肿瘤中的应用价值进行了分析。选取2018年1月~2019年2月本院乳腺肿瘤患者103例为研究对象,所有患者均给予CDUs、UE检查,以病理检查为对照,分析CDUs、UE及二者结合对乳腺良恶性肿瘤的鉴别价值。病理检查显示,103例乳腺肿瘤患者中,恶性48例(46.60%)、良性55例(53.40%);乳腺肿瘤恶性者CDUs、UE得分明显高于良性者,差异有统计学意义(P<0.05);在鉴别乳腺良恶性肿瘤的敏感度、特异度、准确度中,CDUs为79.17%、72.73%、75.73%,UE为75.00%、65.45%、69.90%,CDUs与UE结合为95.83%、90.91%、93.20%,CDUs结合UE明显高于CDUs、UE,差异有统计学意义(P<0.05)。本文证实CDUs、UE对乳腺良恶性肿瘤具有良好的鉴别价值,且CDUs结合UE的鉴别价值更高。     

A rapid and sensitive method for quantitation of nucleosides in human urine using liquid chromatography/mass spectrometry with direct urine injection

Oxidized nucleosides are biochemical markers for tumors, aging, and neurodegenerative diseases. However, during the last decade, the analytical methods for nucleosides by gas chromatography/mass spectrometry (GC/MS) and high-performance liquid chromatography (HPLC) with single-parameter detectors like electron-capture detection (ECD) have not been sufficiently rapid or reliable to detect nucleosides in urine and to analyze clinical samples. It has been reported (Dudley et al., Rapid Commun Mass Spectrom. 2000; 14: 1200) that liquid chromatography with electrospray mass spectrometry (LC/ESI-MS) is more specific and sensitive for analysis of nucleosides than HPLC with conventional detectors; however, this method required complex extraction steps. In the present work a direct LC/ESI-MS method for nucleosides without extraction of urine samples has been developed. Analysis of nucleosides using positive-ion mode with selected reaction monitoring effectively eliminated potential interferences from endogenous constituents of the urine. This highly selective and sensitive method made it possible to analyze urinary nucleosides with a lower limit of quantitation of 0.2 nmol/mL. The method has been validated, with both excellent linearity and reproducibility, in the calibration range from 0.2-400 nmol/mL. The correlation coefficients of the calibration curves were higher than 0.987. The coefficients of variation were in the range 0.03-14.92% (inter-day) and 0.54-14.39% (intra-day), respectively.     

Analysis of urinary nucleosides. IV. Identification of urinary purine nucleosides by liquid chromatography/electrospray mass spectrometry

Modified urinary nucleosides are potentially invaluable in cancer diagnosis. High-performance liquid chromatography (HPLC) was combined with full scan mass spectrometry (MS), tandem mass spectrometry and MSn analysis in order to identify purine nucleosides purified from urine. UV peaks evident in the chromatogram were examined by the various mass spectrometric techniques and adenosine, 1-methyladenosine, xanthosine, N1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, N2,N2,N7-trimethylguanosine, inosine, and 1-methylinosine were each identified in the urine samples from cancer patients. The benefits of the use of LC/MS compared with HPLC alone are discussed.     

Modified nucleosides in human serum.

Methylated purines and pyrimidines derived from the degradation of transfer ribonucleic acid have been shown to be excreted in abnormal amounts in the urine of patients with cancer. Recent technology developed by Gehrke and Kuo has allowed the separation and quantification of modified nucleosides in serum using reversed-phase high-performance liquid chromatography with diode-array measurement. Serum levels of ten modified nucleosides were measured in 37 normal healthy adults to establish normal values and to correlate activity with age and sex. In addition, serum levels of patients with several malignancies were measured to determine activity in these diseases. Levels of modified nucleosides in normal individuals were consistently reproducible and showed no significant variation among males versus females or with age. Patients with malignant diseases showed consistent elevations and these were highest in patients with more advanced disease. The evidence of no significant differences in the mean levels of modified nucleosides in serum with age or sex in normal adults and elevations in patients with malignancies demonstrate the potential value of modified nucleosides as cancer biomarkers.     

乳腺癌代谢物组模式特征发现方法及HPLC/M S/M S分析

提出一种基于单独最优特征组合和BP神经网络的代谢物组模式特征发现方法,并用其寻找到尿样中与乳腺癌最为相关的4种核苷,组成一组特异性检测参数.经HPLC/MS/MS联用法鉴定,它们是乳清酸核苷、1-甲酰化腺苷、S-腺苷-L-蛋氨酸及N²-甲酰化鸟苷.将这4种核苷作为输入变量,用BP神经分类网络建立乳腺癌诊断模型.留一法交叉验证和独立验证结果表明,该模型预测准确率达到90%以上.