Urinary Nucleosides Research Articles

Artificial neural networks based on principal component analysis input selection for clinical pattern recognition analysis

Two clinical data sets were applied for pattern recognition in order to discover the correlation between urinary nucleoside profiles and tumours. One data set contains 168 clinical urinary samples, of which 84 specimens are from female thyroid cancer patients (malignant tumour group), and the other samples were collected from healthy women (normal group). However, 168 clinical urinary samples comprised the second data set, too. In all the specimens, each number of the samples for both uterine cervical cancer patients (malignant tumour group) and healthy females (normal group) is 60, and the other 48 samples were collected from uterine myoma patients (benign tumour group). For the two data sets, the separation and quantitative determination of the clinical urinary nucleosides were performed by capillary electrophoresis (CE). The pattern recognition was achieved applying multiple layer perceptron artificial neural networks (MLP ANN) based on conjugate gradient descent training algorithm. Moreover, applying the proposed principal component analysis (PCA) input selection scheme to MLP ANN, the accuracy rate of the pattern recognition was improved to some extent (or without any deterioration) even by much simpler structure of MLP ANN. The study showed that MLP ANN based on PCA input selection was a promising tool for pattern recognition.

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 μL/min; the flow rate and temperature of drying gas were 6 L/min and 150 °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.

Increased urinary level of oxidized nucleosides in patients with mild-to-moderate Alzheimer's disease

Objective: Oxidative stress may play an important role in the pathogenesis of Alzheimer's disease (AD). Design and methods: To investigate the possible role of oxidative DNA damage in the pathogenesis of AD, we measured the metabolite concentrations of oxidized nucleosides (pseudouridine, 1-methyladenosine, 5-methylcytidine, 5-methyl-2′-deoxycytidine, 3-methyluridine, N 2, N 2-dimethylguanosine, 8-hydroxy-2′-deoxyguanosine, 5-deoxyadenosine and 2-deoxyguanosine) in urine between AD ( n = 36) and control subjects ( n = 34) using liquid chromatography-mass spectrometry (LC-MS) without urine preparation. Results: In AD, the 3-methyluridine, 1-methyladenosine, 8-hydroxy-2′-deoxyguanosine ( p < 0.05, respectively), 2-deoxyguanosine ( p < 0.01) and pseudouridine, N 2, N 2-dimethylguanosine ( p < 0.001, respectively) were significantly increased when compared with the control subjects. Conclusion: The results indicate that oxidized urinary nucleosides may be useful as biomarkers for AD in early stages.

Multilayer perceptron tumour diagnosis based on chromatography analysis of urinary nucleosides

Nucleosides in human urine are of interest as a biochemical marker for cancer, acquired immune deficiency syndrome (AIDS) and the whole-body turnover of RNAs. A reversed-phase high-performance liquid chromatography (RP-HPLC) method with photodiode-array detection was used to quantitatively analyze urinary normal and modified nucleosides. 55 persons with malignant tumors of various types, 13 persons with benign tumors and 41 healthy controls were investigated within a clinical intervention study. Artificial neural networks (ANN) have been used as a practical pattern recognition tool to distinguish cancer patients from healthy persons. Using a multilayer perceptron (MPL), a specificity of 85%, and a sensitivity of 97% in differentiation between tumor patients and healthy persons was achieved. The differentiation between benign and malignant tumors had a sensitivity of 60% and a specificity of 84%. These results verify the usefulness of ANN and the RP-HPLC method for tumor recognition in agreement with existing studies.

Urinary profiling investigation of metabolites with cis‐diol structure from cancer patients based on UPLC‐MS and HPLC‐MS as well as multivariate statistical analysis

Nucleosides are an important class of metabolites and have been investigated as potential tumor biomarkers. A method based on ultra peerformance liquid chromatography (UPLC)-TOF MS was developed to analyze urinary nucleosides and other metabolites with cis-diol structure to distinguish between cancer patients and healthy persons and compare the results with those obtained by HPLC. The data showed that the UPLC method used about one third of the time required by HPLC and achieved a much better chromatographic resolution and increased sensitivity, the number of peaks detected by UV being 79 and 94 for HPLC and UPLC, respectively. With UPLC-TOF MS, more information was obtained about metabolites, the separation of cancer patients from healthy persons was significantly improved, and more potential biomarkers were found. The method based on UPLC-TOF MS is a powerful technique for the study of metabolite profiles.

Synthesis and Application of a Nitrobenzeneboronic Acid-substituated Silica for Affinity Chromatography

A boronic acid-substituted silica was synthesized using γ-glycidoxypropyltrimethoxysilane as the spacer arm and 3-amino-4-nitrobenzeneboronic acid as the ligand. The 3-amino-4-nitrobenzeneboronic acid was modified from m-aminobenzeneboronic acid. Then, boronic acid-substituted silica was packed in a stainless-steel column (35 mm × 4.6 mm I.D.) under a pressure of 20.7 MPa as an affinity pre-column. A coupled-column liquid chromatographic method for the direct analysis of 11 urinary nucleosides is described. Efficient on-line clean-up and concentration of 11 nucleosides from urine samples were obtained using the affinity pre-column as the first column (extraction column) and an ODS column (250 mm × 4.6 mm I.D.) as the second column (analytical column). The mobile phases applied consisted of 0.25 M ammonium acetate (pH 7.95) on the extraction column, and 25 mM potassium dihydrogen phosphate (pH 4.5) on the analytical column, respectively. Determination of urinary nucleosides was performed on the analytical column using a linear gradient elution comprising 25 mM potassium dihydrogen phosphate (pH 4.5) and methanol-water (60:40, v/v) with UV detection at 260 nm. Calibration plots of 11 standard nucleosides showed good RSD, good recovery, and excellent linearity (r > 0.995).

Increasing conclusiveness of metabonomic studies by cheminformatic preprocessing of capillary electrophoretic data on urinary nucleoside profiles

Nowadays, bioinformatics offers advanced tools and procedures of data mining aimed at finding consistent patterns or systematic relationships between variables. Numerous metabolites concentrations can readily be determined in a given biological system by high-throughput analytical methods. However, such row analytical data comprise noninformative components due to many disturbances normally occurring in analysis of biological samples. To eliminate those unwanted original analytical data components advanced chemometric data preprocessing methods might be of help. Here, such methods are applied to electrophoretic nucleoside profiles in urine samples of cancer patients and healthy volunteers. The electrophoretic nucleoside profiles were obtained under following conditions: 100 mM borate, 72.5 mM phosphate, 160 mM SDS, pH 6.7; 25 kV voltage, 30 °C temperature; untreated fused silica capillary 70 cm effective length, 50 μm I.D. Different most advanced preprocessing tools were applied for baseline correction, denoising and alignment of electrophoretic data. That approach was compared to standard procedure of electrophoretic peak integration. The best results of preprocessing were obtained after application of the so-called correlation optimized warping (COW) to align the data. The principal component analysis (PCA) of preprocessed data provides a clearly better consistency of the nucleoside electrophoretic profiles with health status of subjects than PCA of peak areas of original data (without preprocessing).

Synthesis of silica-based benzeneboronic acid affinity materials and application as pre-column in coupled-column high-performance liquid chromatography

Three silica-based benzeneboronic acid affinity materials were synthesized by using an m-aminobenzeneboronic acid as the ligand and using three different spacer arms. Under high-pressure, three affinity pre-columns were packed with these materials and the retention of every affinity pre-column with 11 urinary nucleosides was studied. With different spacer arms of boronic acid-substituted silica materials, the absorption to vicinal alcohols ( cis-diols) and stability under acidic elution conditions are of great difference. Coupled-column liquid chromatographic methods for the direct analysis of urinary nucleosides were respectively established. As a result, two of three affinity pre-columns showed good chromatographic property in the on-line analysis of urinary nucleosides. The coupled-column system including pre-column I is the best with excellent linearity ( R 2 > 0.995), good recoveries (85.6–96.9%) and reproducibility (R.S.D.: 1.01–4.02%). The pre-column I could at least endure 150 repetitive injections of a 100 μL urinary sample.

Modified nucleosides: an accurate tumour marker for clinical diagnosis of cancer, early detection and therapy control.

Modified nucleosides, regarded as indicators for the whole-body turnover of RNAs, are excreted in abnormal amounts in the urine of patients with malignancies. To test their usefulness as tumour markers and to compare them with the conventional tumour markers, fractionated urine samples were analysed using chromatography. The excretion patterns of nucleosides of 68 cancer patients with malignant and benign tumours and 41 healthy controls have been studied. Significant elevations in the total sum and the concentrations of at least three (or four) of indicator nucleosides cytidine, pseudouridine, 2-pyridone-5-carboxamide-N1-ribofuranoside, N2,N2-dimethylguanine, 1-methylguanosine, 2-methylguanosine and 1-methyladenosine indicate a tumour with a sensitivity of 54% (77%) and a specificity of 86% (98%). Using an artificial neural network analysis, a sensitivity of 97% and a specificity of 85% were achieved in differentiating between tumour and control volunteers. The comparison with carcinoembryonic antigen, cancer antigen 15-3 und tissue polypeptide antigen indicates that urinary nucleosides may be useful tumour markers. This study suggests that the simultaneous determination of modified nucleosides and creatinine in urine samples of patients with cancer leads to an advantage to current methods and is a useful method to detect cancer early and to control the success of therapy.

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.

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.

Normal and modified urinary nucleosides represent novel biomarkers for colorectal cancer diagnosis and surgery monitoring

Up to now, there is still no ideal tumor marker in early diagnosis and effective monitoring, especially for surgical resection of colorectal cancer (CRC). The aim of the present study was to evaluate the application of urinary normal and modified nucleosides in diagnosis and surgery monitoring of CRC. Between October 2002 and July 2003, 52 consecutive patients with pathological confirmed CRC were enrolled. Spontaneous urine samples were collected 1 day before surgery and on day 8 postoperatively, and 14 urinary nucleosides were determined by reverse-phase high-performance liquid chromatography (RP-HPLC). Another 62 healthy people were also studied as control. The clinical routine tumor markers, serum carcinoembryonic antigen (CEA), carbohydrate antigen (CA)199, CA125, and alpha-fetoprotein (AFP) of CRC patients, were correspondingly evaluated by electrochemiluminescent immunoassay. The levels of 11 out of 14 of the determined urinary nucleosides in the CRC group were much higher than those of normal controls. Through the principal component analysis of these 14 nucleosides, 76.9% of CRC patients were correctly classified. The sensitivity of this analysis was much higher than that of CEA (38.5%), CA199 (40.4%), CA125 (15.4%), and AFP (17.3%; P < 0.01). Receiver operating characteristic (ROC) curve analysis of 1-methylguanosine (m1G) and pseudouridine (Pseu) showed good sensitivity-specificity profiles of the diagnosis of CRC. The elevated levels of the nine nucleosides significantly decreased after curative resection of 40 CRC cases. The data also showed that the preoperative levels of some nucleosides were positively related with tumor size and Dukes staging of CRC. The evaluation of normal and modified urinary nucleosides might become novel tumor markers, which will be facilitated in the clinical setting and helpful in the diagnosis, management and follow up of CRC. Pseu and m1G may be more promising for clinical use and be worthy of further studies in the near future.

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 MS(5) (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.

Application of urinary nucleosides in the diagnosis and surgical monitoring of colorectal cancer

To evaluate the value of urinary normal and modified nucleosides in diagnosis and surgical monitoring of colorectal cancer (CRC). Between October 2002 and July 2003, 52 consecutive patients with pathological confirmed CRC were included in this study. Spontaneous urine samples were collected 1 d before and 8 d after surgery and 14 kinds of urinary nucleosides in the samples were determined by reversed-phase high-performance liquid chromatography (RP-HPLC) method. Another 62 healthy volunteers were also enrolled as controls. The routine clinical tumor markers, including serum CEA, CA199, CA125 and AFP levels of CRC patients were evaluated by electrochemical-luminescence immunoassay simultaneously. The mean levels of pseudouridine (Pseu), adenosine (A), cytidine (C), 1-methyladenosine (m1A), 1-methylinosine (m1I), 3-methyluridine + 5-methyluridine (mU), 2,2-methylguanosine (m22G), inosine (I), 1-methylguanosine (m1G), N4-acetylcytidine (ac4C), N6-methyladenosine (m6A) among 14 kinds of determined urinary nucleosides in CRC group were much higher than those of controls (P < 0.05). Based on principal component analysis, 76.9% of CRC patients were correctly identified, which was much higher than that of CEA (38.5%), CA199 (40.4%), CA125 (15.4%), and AFP (17.3%) (P < 0.01). ROC curve analysis of m1G, and Pseu showed good sensitivity-specificity profiles to CRC. Two classification equations, Y(normal) = -3.009 + 0.0272 x Pseu + 4.918 x m1G and Y(CRC) = -8.057 + 0.0667 x Pseu + 8.258 x m1G, were established by Bayes stepwise discriminate analysis for predicting carcinogenesis of CRC. The elevated levels of Pseu, C, U (uridine), m1A, m1I, m1G, ac4C, A, m22G dramatically decreased after curative resection of 40 cases of CRC. And our data also showed that the preoperative levels of Pseu, m1G, m1A and m22G were positively related with tumor size and the preoperative levels of m1A, m22G and ac4C were positively related with Duke's staging of CRC (P < 0.05). Normal and modified urinary nucleosides may become additional tumor markers which are feasible in the clinical setting and will prove helpful in the diagnosis, management and follow-up of CRC, and Pseu and m1G may be more promising for clinical application.

MALDI-TOF MS Analysis of Urinary Nucleosides

As RNA turnover seems to be impaired in cancer patients, modified nucleosides have been evaluated as potential tumor markers. Modified nucleosides are mainly formed post-transcriptionally in tRNA, set free during RNA metabolism, and excreted in urine. Especially methylated nucleosides play an important role, as their levels are higher in urine from cancer patients. For structural elucidation of known and unknown nucleosides from urine samples of cancer patients, MALDI-TOF MS and MALDI-PSD were used for the first time. This technique generally ensures high sensitivity, mass resolution, and accuracy. In our analytical approach we prepurified nucleosides from urine by affinity chromatography and subsequently separated them by semipreparative high performance liquid chromatography. The different fractions were collected separately and analyzed by MALDI-TOF MS and PSD-MALDI using a mixture of six low molecular weight calibrants for internal or external calibration. The molecular totals formulas based on a mass accuracy of 10 ppm and below were calculated and a systematic data base search was performed. The inherent problem of the MALDI-technique, the reduced sensitivity for low molecular weight substances caused by matrix suppression effects, was reduced by our technique. We identified several nucleosides in urine, which were previously identified via retention times and UV spectra of standards after HPLC analysis. Eight further nucleosides were observed. This work demonstrates for the first time the potential of MALDI-TOF and PSD-MALDI in combination with semipreparative HPLC for assignment of nucleosides in urine. The particularly high mass accuracy of this mass spectrometric method provides opportunities for identifying unknown compounds.

Determination of urinary nucleosides by direct injection and coupled-column high-performance liquid chromatography

A coupled-column liquid chromatographic method for the direct analysis of 14 urinary nucleosides is described. Efficient on-line clean-up and concentration of 14 nucleosides from urine samples were obtained by using a boronic acid-substituted silica column (40 mm × 4.0 mm I.D.) as the first column (Col-1) and a Hypersil ODS 2 column (250 mm × 4.6 mm I.D.) as the second column (Col-2). The mobile phases applied consisted of 0.25 mol/L ammonium acetate (pH 8.5) on Col-1, and of 25 mmol/L potassium dihydrogen phosphate (pH 4.5) on Col-2, respectively. Determination of urinary nucleosides was performed on Col-2 column by using a linear gradient elution comprising 25 mmol/L potassium dihydrogen phosphate (pH 4.5) and methanol–water (60:40, v/v) with UV detection at 260 nm. Urinary nucleosides analysis can be carried out by this procedure in 50 min requiring only pH adjustment and the protein precipitation by centrifugation of urine samples. Calibration plots of 14 standard nucleosides showed excellent linearity ( r > 0.995) and the limits of detection were at micromolar levels. Both of intra- and inter-day precisions of the method were better than 6.6% for direct determination of 14 nucleosides. The validated method was applied to quantify 14 nucleosides in 20 normal urines to establish reference ranges.

Urinary nucleosides as biological markers for patients with colorectal cancer

Fourteen urinary nucleosides, primary degradation products of tRNA, were evaluated to know the potential as biological markers for patients with colorectal cancer. The concentrations of 14 kinds of urinary nucleosides from 52 patients with colorectal cancer, 10 patients with intestinal villous adenoma and 60 healthy adults were determined by column switching high performance liquid chromatography method. The mean levels of 12 kinds of urinary nucleosides (except uridine and guanosine) in the patients with colorectal cancer were significantly higher than those in patients with intestinal villous adenoma or the healthy adults. Using the levels of 14 kinds of urinary nucleosides as the data vectors for principal component analysis, 71% (37/52) patients with colorectal cancer were correctly classified from healthy adults, in which the identification rate was much higher than that of CEA method (29%). Only 10% (1/10) of patients with intestinal villous adenoma were indistinguishable from patients with colorectal cancer. The levels of m1G, Pseu and m1A were positively related with tumor size and Duke's stages of colorectal cancer. When monitoring the changes in urinary nucleoside concentrations of patients with colorectal cancer associated with surgery, it was found that the overall correlations with clinical assessment were 84% (27/32) and 91% (10/11) in response group and progressive group, respectively. These findings indicate that urinary nucleosides determined by column switching high performance liquid chromatography method may be useful as biological markers for colorectal cancer.

Age-dependence of urinary normal and modified nucleosides in childhood as determined by reversed-phase high-performance liquid chromatography

Modified nucleosides have been characterized as tumor markers for a number of malignant diseases. In order to use these markers in children, the age-dependence of the nucleoside levels in healthy children has to be established and taken into account in diagnostic decisions. In this study, the levels of 12 normal and modified nucleosides in urine of 166 healthy children and adolescents with an age between 1 day and 19 years are determined by reversed-phase HPLC, and age-dependent reference ranges are defined. The urinary nucleoside concentrations are related to the creatinine concentrations, which allows the use of randomly collected urine samples. All nucleoside levels in urine of children decrease with age, most pronounced during the first 4 years of life, and the age-dependence of the reference values of the individual nucleosides can be approximated by a mathematical function y = b 0 + b 1 (1/ x) with the regression coefficients b 0 and b 1, the nucleoside levels y and the age x between 1 year and 19 years. In the very young children, the shifts in the nucleoside concentrations are more differentiated. Starting with low levels on the first day of life, the concentrations of all studied nucleosides rise up to an age of 1–2 months, when they reach their absolute maximum for all age periods, and then decrease.

Clinical significance and prognostic value of urinary nucleosides in breast cancer patients

Objective: Thirteen urinary nucleosides, primarily degradation products of tRNA, were evaluated as potential tumor markers for breast cancer patients. Design and methods: The micellar electrokinetic chromatography (MEKC) method has been used to analyze the urinary nucleosides in 41 healthy controls, 20 patients with benign breast tumors, and 26 breast cancer patients. Results: Urinary nucleoside concentrations of breast cancer patients were found to increase significantly compared to those of patients with benign breast tumors and healthy controls. By using 13 nucleoside concentrations as data vectors for principal component analysis (PCA), 73% (19/26) of breast cancer patients were correctly identified from healthy controls, while only 20% (4/20) of patients with benign breast tumors were indistinguishable from breast cancer patients. The mean level of all forms of urinary nucleosides in patients with metastatic breast cancer was higher than that in patients with primary breast cancer. The levels of modified nucleosides tended to decrease and return to normal after surgery. Conclusion: The results indicate that urinary nucleosides may be useful as tumor markers for breast cancer.

Diagnosis of liver cancer using HPLC-based metabonomics avoiding false-positive result from hepatitis and hepatocirrhosis diseases

Metabonomics, the study of metabolites and their roles in various disease states, is a novel methodology arising from the post-genomics era. This methodology has been applied in many fields. Current metabonomics practice has relied on mass spectrometry (MS), gas chromatography–mass spectrometry (GC–MS), liquid chromatography–mass spectrometry (LC–MS) and nuclear magnetic resonance (NMR) to analyze metabolites. In this study, a novel approach of using high-performance liquid chromatography (HPLC) in conjunction with developed software was employed. Using the principal components analysis method (PCA), all (113) peaks of urinary metabolites with a cis-diol structure from patients with hepatitis and hepatocirrhosis were compared to those from liver cancer patients. The results showed that the metabonomics-PCA method might be useful to differentiate between patients with hepatocirrhosis and hepatitis from patients with liver cancer while lowering false-positive rate. These findings also suggest that a subset of the urinary nucleosides identified with metabonomics correlate better with cancer diagnosis than the traditional single tumor marker alpha-fetoprotein (AFP).