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.     

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.     

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.     

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.     

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.     

Analysis of urinary nucleosides. III. Identification of 5'-deoxycytidine in urine of a patient with head and neck cancer

Modified nucleosides in the urine have been postulated to be diagnostic indices of disease, particularly of cancer. The urine of a patient with terminal head and neck cancer has been found to contain a modified nucleoside with a protonated molecule of m/z 228. By means of high-performance liquid chromatography/ion trap mass spectrometry (HPLC/ITMS) and capillary liquid chromatography/triple quadruple mass spectrometry (CapLC/TQMS) we have identified the compound as 5'-deoxycytidine. This is the first report of 5'-deoxycytidine in man: in addition to the elucidation of its structure, its possible origins and the potential significance of its occurrence are discussed.     

Separation and identification of purine nucleosides in the urine of patients with malignant cancer by reverse phase liquid chromatography/electrospray tandem mass spectrometry

Urinary‐modified nucleosides have a potential role as cancer biomarkers for a number of malignant diseases. High performance liquid chromatography (HPLC) was combined with full‐scan mass spectrometry, MS/MS analysis and accurate mass measurements in order to identify purine nucleosides purified from urine. Potential purine nucleosides were assessed by their evident UV absorbance in the HPLC chromatogram and then further examined by the mass spectrometric techniques. In this manner, numerous modified purine nucleosides were identified in the urine samples from cancer patients including xanthine, adenosine, N1‐methyladenosine, 5′‐deoxy‐5′‐methylthioadenosine, 2‐methyladenosine, N6‐threonylcarbamoyladenosine, inosine, N1‐methylinosine, guanosine, N1‐methylguanosine, N7‐methylguanine, N2‐methylguanosine, N2,N2‐dimethyguanosine, N2,N2,N7‐trimethylguanosine. Furthermore, a number of novel purine nucleosides were tentatively identified via critical interpretation of the combined mass spectrometric data including N3‐methyladenosine, N7‐methyladenine, 5′‐dehydro‐2′‐deoxyinosine, N3‐methylguanine, O6‐methylguanosine, N1,N2,N7‐trimethylguanosine, N1‐methyl‐N2‐ethylguanosine and N7‐methyl‐N1‐ethylguanosine. Copyright © 2009 John Wiley & Sons, Ltd.     

Detection of new radiation-induced DNA lesions by liquid chromatography coupled to tandem mass spectrometry

High-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) has been used to search for the formation of as yet unidentified radiation-induced DNA lesions. For that purpose, the characteristic fragmentation of most of 2'-deoxyribonucleosides that corresponds to the loss of the 2-deoxyribose moiety (loss of 116 mass units) has been utilized to specifically detect modified nucleosides. Aerated aqueous solutions of DNA were exposed to ionizing radiation, and subsequently DNA was digested to nucleosides with a cocktail of endo- and exonucleases. HPLC/ESI-MS/MS analysis of the resulting 2'-deoxyribonucleoside mixture allowed us to detect four novel DNA modifications. In a subsequent step, the sensitivity of the tandem mass spectrometer was used to search for the formation of the newly detected lesions in the DNA of gamma-irradiated cells. Thus, one of the four newly detected lesions was found to be significantly generated in cellular DNA upon exposure to ionizing radiation. In addition, the latter lesion was also shown to be present in untreated cells, indicating that the modified nucleoside could be formed endogenously.     

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.     

Bioinformatical evaluation of modified nucleosides as biomedical markers in diagnosis of breast cancer

It is known that patients suffering from cancer diseases excrete increased amounts of modified nucleosides with their urine. Especially methylated nucleosides have been proposed to be potential tumor markers for early diagnosis of cancer. For determination of nucleosides in randomly collected urine samples, the nucleosides were extracted using affinity chromatography and then analyzed via reversed phase high-performance liquid chromatography (HPLC) with UV-detection. Eleven nucleosides were quantified in urine samples from 51 breast cancer patients and 65 healthy women.The measured concentrations were used to train a Support Vector Machine (SVM) and a k-nearest-neighbor classifier (k-NN) to discriminate between healthy control subjects and patients suffering from breast cancer. Evaluations of the learned models by computing the leave-one-out error and the prediction error on an independent test set of 29 subjects (15 healthy, 14 breast cancer patients) showed that by using the eleven nucleosides, the occurrence of breast cancer could be forecasted with 86% specificity and 94% sensitivity when using an SVM and 86% for both specificity and sensitivity with the k-NN model.     

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。     

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。     

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.     

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.     

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.     

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.     

Study of normal and modified nucleosides in serum by RP-HPLC

Summary Modified nucleosides derived predominantly from transfer ribonucleic acid (tRNA) have been studied as possible tumor markers. In this paper a reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been applied to study 15 normal and modified nucleosides in serum. The nucleoside levels in normal human serum were established, and the concentrations of 15 nucleosides in serum from 19 cancer patients were determined. It was found that the concentrations of modified nucleosides were significantly higher in patient sera. Based on 15 nucleoside concentrations in serum, factor analysis could classify correctly 90% of cancer patients from the normal humans. Further work showed that urine was slightly better than serum when determining nucleosides as biological marker candidates. More work is ongoing to determine the clinical usefulness of modified nucleosides as tumor markers.     

Synthesis of 2'-O-[2-[(N,N-dimethylamino)oxy]ethyl] modified nucleosides and oligonucleotides.

A versatile synthetic route has been developed for the synthesis of 2'-O-[2-[(N,N-dimethylamino)oxy]ethyl] (abbreviated as 2'-O-DMAOE) modified purine and pyrimidine nucleosides and their corresponding nucleoside phosphoramidites and solid supports. To synthesize 2'-O-DMAOE purine nucleosides, the key intermediate B (Scheme 1) was obtained from the 2'-O-allyl purine nucleosides (13a and 15) via oxidative cleavage of the carbon-carbon bond to the corresponding aldehydes followed by reduction. To synthesize pyrimidine nucleosides, opening the 2,2'-anhydro-5-methyluridine 5 with the borate ester of ethylene glycol gave the key intermediate B. The 2'-O-(2-hydroxyethyl) nucleosides were converted, in excellent yield, by a regioselective Mitsunobu reaction, to the corresponding 2'-O-[2-[(1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl)oxy]ethyl] nucleosides (18, 19, and 20). These compounds were subsequently deprotected and converted into the 2'-O-[2-[(methyleneamino)oxy]ethyl] derivatives (22, 23, and 24). Reduction and a second reductive amination with formaldehyde yielded the corresponding 2'-O-[2-[(N,N-dimethylamino)oxy]ethyl] nucleosides (25, 26, and 27). These nucleosides were converted to their 3'-O-phosphoramidites and controlled-pore glass solid supports in excellent overall yield. Using these monomers, modified oligonucleotides containing pyrimidine and purine bases were synthesized with phosphodiester, phosphorothioate, and both linkages (phosphorothioate and phosphodiester) present in the same oligonucleotide as a chimera in high yields. The oligonucleotides were characterized by HPLC, capillary gel electrophoresis, and ESMS. The effect of this modification on the affinity of the oligonucleotides for complementary RNA and on nuclease stability was evaluated. The 2'-O-DMAOE modification enhanced the binding affinity of the oligonucleotides for the complementary RNA (and not for DNA). The modified oligonucleotides that possessed the phosphodiester backbone demonstrated excellent resistance to nuclease with t(1/2) > 24 h.