Abstract
The physiological response of the human body to several diseases can be reflected by the metabolite pattern in biological fluids. Cancer, like other diseases accompanied by metabolic disorders, causes characteristic effects on cell turnover rate, activity of modifying enzymes, and RNA/DNA modifications. This results in an altered excretion of modified nucleosides and biochemically related compounds. In the course of our metabolic profiling project, we screened 24-h urine of patients suffering from lung, rectal, or head and neck cancer for previously unknown ribosylated metabolites. Therefore, we developed a sample preparation procedure based on boronate affinity chromatography followed by additional prepurification with preparative TLC. The isolated metabolites were analyzed by ion trap mass spectrometry (IT MS) and Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). IT MS was applied for LC-auto MS3 screening runs and MSn(n=4–6) syringe pump infusion experiments, yielding characteristic fragmentation patterns. FTICR MS measurements enabled the calculation of corresponding molecular formulae based on accurate mass determination (mass accuracy: 1–5 ppm for external and sub-ppm values for internal calibration). We were able to identify 22 metabolites deriving from cellular RNA metabolism and related metabolic pathways like histidine metabolism, purine biosynthesis, methionine/polyamine cycle, and nicotinate/nicotinamide metabolism. The compounds 1-ribosyl-3-hydroxypyridinium, 1-ribosyl-pyridinium, and 3-ribosyl-1-methyl-l-histidinium as well as a series of ribosylated histamines, conjugated to carboxylic acids at the Nω-position were found as novel urinary constituents. The occurrence of the modified nucleosides 2-methylthio-N 6-(cis-hydroxyisopentenyl)-adenosine, 5-methoxycarbonylmethyl-2-thiouridine, N 6-methyl-N 6-threonylcarbamoyladenosine, and 2-methylthio-N 6-threonylcarbamoyladenosine in human urine is verified for the first time.
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Paik, S. Incorporating Genomics into the Cancer Clinical Trial Process. Semin. Oncol. 2001, 28, 305–309.
Kim, S. Y.; Hahn, W. C. Cancer Genomics: Integrating Form and Function. Carcinogenesis 2007, 28, 1387–1392.
Rajapakse, J. C.; Duan, K.; Yeo, W. K. Proteomic Cancer Classification with Mass Spectrometry Data. Am. J. Pharmacogenomics 2005, 5, 281–292.
Maurya, P.; Meleady, P.; Dowling, P.; Clynes, M. Proteomic Approaches for Serum Biomarker Discovery in Cancer. Anticancer Res. 2007, 27, 1247–1255.
Griffin, J. L.; Shockcor, J. P. Metabolic Profiles of Cancer Cells. Nat. Rev. Cancer 2004, 4, 551–561.
Kind, T.; Tolstikov, V.; Fiehn, O.; Weiss, R. H. A Comprehensive Urinary Metabolomic Approach for Identifying Kidney Cancer. Anal. Biochem. 2007, 363, 185–195.
Gamache, P. H.; Meyer, D. F.; Granger, M. C.; Acworth, I. N. Metabolomic Applications of Electrochemistry/Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2004, 15, 1717–1726.
Ryan, D.; Robards, K. Metabolomics: The greatest Omics of Them All? Anal. Chem. 2006, 78, 7954–7958.
Tormey, D. C.; Waalkes, T. P.; Gehrke, C. W. Biological Markers in Breast Carcinoma—Clinical Correlations with Pseudouridine, N2,N2-Dimethylguanosine, and 1-Methylinosine. J. Surg. Oncol. 1980, 14, 267–273.
Itoh, K.; Konno, T.; Sasaki, T.; Ishiwata, S.; Ishida, N.; Misugaki, M. Relationship of Urinary Pseudouridine and 1-Methyladenosine to Activity of Leukemia and Lymphoma. Clin. Chim. Acta 1992, 206, 181–189.
Nakano, K.; Shindo, K.; Yasaka, T.; Yamamoto, H. Reversed-Phase High-Performance Liquid Cchromatographic Investigation of Mucosal Nucleosides and Bases and Urinary Modified Nucleosides of Gastrointestinal Cancer Patients. J. Chromatogr. 1985, 343, 21–33.
Waalkes, T. P.; Abeloff, M. D.; Ettinger, D. S.; Woo, K. B.; Gehrke, C. W.; Kuo, K. C.; Borek, E. Modified Ribonucleosides as Biological Markers for Patients with Small Cell Carcinoma of the Lung. Eur. J. Cancer Clin. Oncol. 1982, 18, 1267–1274.
Borek, E.; Baliga, B. S.; Gehrke, C. W.; Kuo, C. W.; Belman, S.; Troll, W.; Waalkes, T. P. High Turnover Rate of Transfer RNA in Tumor Tissue. Cancer Res. 1977, 37, 3362–3366.
Tsutsui, E.; Srinivasan, P. R.; Borek, E. TRNA Methylases in Tumors of Animal and Human Origin. Proc. Natl. Acad. Sci. U. S. A. 1966, 56, 1003–1009.
Dudley, E.; Lemiere, F.; Van Dongen, W.; Langridge, J. I.; El-Sharkawi, S.; Games, D. E.; Esmans, E. L.; Newton, R. P. Analysis of Urinary Nucleosides. III. Identification. of 5′-Deoxycytidine in Urine of a patient with head and neck cancer. Rapid Commun. Mass Spectrom. 2003, 17, 1132–1136.
Gehrke, C. W.; Kuo, K. C. Ribonucleoside Analysis by Reversed-Phase High-Performance Liquid Chromatography. J. Chromatogr. 1989, 471, 3–36.
Xu, G.; Schmid, H. R.; Lu, X.; Liebich, H. M.; Lu, P. Excretion Pattern Investigation of Urinary Normal and Modified Nucleosides of Breast Cancer Patients by RP-HPLC and Factor Analysis Method. Biomed. Chromatogr. 2000, 14, 459–463.
Xu, G.; Lu, X.; Zhang, Y.; Lu, P.; Di, S. C.; Lehmann, R.; Liebich, H. Two Approaches for Determining the Urinary Excretion Patterns of Nucleosides—HPLC and CE. Sepu 1999, 17, 97–101.
Kammerer, B.; Frickenschmidt, A.; Muller, C. E.; Laufer, S.; Gleiter, C. H.; Liebich, H. Mass Spectrometric Identification of Modified Urinary Nucleosides Used as Potential Biomedical Markers by LC-ITMS Coupling. Anal. Bioanal. Chem. 2005, 382, 1017–1026.
Qian, M.; Yang, S.; Wu, H.; Majumdar, P.; Leigh, N.; Glaser, R. Ammonia Elimination from Protonated Nucleobases and Related Synthetic Substrates. J. Am. Soc. Mass. Spectrom. 2007, 18, 2040–2057.
Zhang, Q.; Wang, Y. Differentiation of 2′-O- and 3′-O-Methylated Ribonucleosides by Tandem Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2006, 17, 1096–1099.
Takeda, N.; Nakamura, M.; Yoshizumi, H.; Tatematsu, A. Structural Characterization of Modified Nucleosides in tRNA Hydrolysates by Frit-Fast Atom Bombardment Liquid Chromatography/Mass Spectrometry. Biol. Mass Spectrom. 1994, 23, 465–474.
Crow, F. W.; Tomer, K. B.; Gross, M. L.; McCloskey, J. A.; Bergstrom, D. E. Fast Atom Bombardment Combined with Tandem Mass Spectrometry for the Determination of Nucleosides. Anal. Biochem. 1984, 139, 243–262.
Bullinger, D.; Frickenschmidt, A.; Pelzing, M.; Zey, T.; Zurek, G.; Laufer, S.; Kammerer, B. Identification of Urinary Nucleosides by ESI-TOF-MS. LC-GC Eur. 2005, 16–17.
Kammerer, B.; Frickenschmidt, A.; Gleiter, C. H.; Laufer, S.; Liebich, H. MALDI-TOF MS Analysis of Urinary Nucleosides. J. Am. Soc. Mass Spectrom. 2005, 16, 940–947.
McCloskey, J. A.; Crain, P. F. The RNA Modification Database—1998. Nucleic Acids Res. 1998, 26, 196–197.
Bullinger, D.; Neubauer, H.; Fehm, T.; Laufer, S.; Gleiter, C. H.; Kammerer, B. Metabolic Signature of Breast Cancer Cell Line MCF-7: Profiling of Modified Nucleosides via LC-IT MS Coupling. BMC Biochem. 2007, 8, 25.
Liebich, H. M.; Di, S. C.; Wixforth, A.; Schmid, H. R. Quantitation of Urinary Nucleosides by High-Performance Liquid Chromatography. J. Chromatogr. A 1997, 763, 193–197.
Barcelo-Barrachina, E.; Moyano, E.; Galceran, M. T. Determination of Heterocyclic Amines by Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry. J. Chromatogr. A 2004, 1054, 409–418.
Tuytten, R.; Lemiere, F.; Van Dongen, W.; Esmans, E. L.; Witters, E.; Herrebout, W.; Van der Veken, B.; Dudley, E.; Newton, R. P. Intriguing Mass Spectrometric Behavior of Guanosine Under Low Energy Collision-Induced Dissociation: H2O Adduct Formation and Gas-Phase Reactions in the Collision Cell. J. Am. Soc. Mass Spectrom. 2005, 16, 1291–1304.
Frycak, P.; Huskova, R.; Adam, T.; Lemr, K. Atmospheric Pressure Ionization Mass Spectrometry of Purine and Pyrimidine Markers of Inherited Metabolic Disorders. J. Mass Spectrom. 2002, 37, 1242–1248.
Chheda, G. B.; Patrzyc, H. B.; Tworek, H. A.; Dutta, S. P. Isolation and Characterization of 5-Carbamoylmethyluridine and 5-Carbamoylmethyl-2-Thiouridine from Human Urine. Nucleosides Nucleotides 1999, 18, 2155–2173.
Chheda, G. B.; Tworek, H. A.; Bhargava, A. K.; Rachlin, E.; Dutta, S. P.; Patrzyc, H. B. Isolation and Characterization of 3-(3-Amino-3-Carboxypropyl)Uridine from Human Urine. Nucleosides Nucleotides 1988, 7, 417–429.
Imamura, I.; Watanabe, T.; Sakamoto, Y.; Wakamiya, T.; Shiba, T.; Hase, Y.; Tsuruhara, T.; Wada, H. N. τ-Ribosylhistidine, a Novel Histidine Derivative in Urine of Histidinemic Patients: Isolation, Structure, and Tissue Level. J. Biol. Chem. 1985, 260, 10528–10530.
Piraud, M.; Vianey-Saban, C.; Petritis, K.; Elfakir, C.; Steghens, J.; Morla, A.; Bouchu, D. ESI-MS/MS Analysis of Underivatized Amino Acids: A New Tool for the Diagnosis of Inherited Disorders of Amino Acid Metabolism: Fragmentation Study of 79 Molecules of Biological Interest in Positive and Negative Ionization mode. Rapid Commun. Mass Spectrom. 2003, 17, 1297–1311.
van der Heiden, C.; Wadman, S. K.; de Bree, P. K.; Wauters, E. A. Increased Urinary Imidazolepropionic acid, N-Acetylhistamine, and Other Imidazole Compounds in Patients with Intestinal Disorders. Clin. Chim. Acta 1972, 39, 201–214.
van der Heiden, C.; Wauters, E. A.; Duran, M.; Wadman, S. K.; Ketting, D. Gas Chromatographic Analysis of Urinary Tyrosine and Phenylalanine Metabolites in Patients with Gastrointestinal Disorders. Clin. Chim. Acta 1971, 34, 289–296.
Morris, G. S.; Simmonds, H. A.; Davies, P. M. Use of Biological Fluids for the Rapid Diagnosis of Potentially Lethal Inherited Disorders of Human Purine and Pyrimidine Metabolism. Biomed. Chromatogr. 1986, 1, 109–118.
Schram, K. H. Urinary Nucleosides. Mass Spectrom. Rev. 1998, 17, 131–251.
Chheda, G. B. Isolation and Characterization of N6-Succinyladenosine from Human Urine. Nucleic Acids Res. 1977, 4, 739–746.
Mills, J. S.; Mills, G. C.; McAdoo, D. J. Isolation and Identification of 5′-Methylthioadenosine Sulfoxide from Human Urine. Nucleosides Nucleotides 1983, 2, 465–478.
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Data of ethics proposal: request number of ethics commission: 280/2005V, date of agreement: 31. 10. 2005.
Published online June 28, 2008
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Bullinger, D., Fux, R., Nicholson, G. et al. Identification of urinary modified nucleosides and ribosylated metabolites in humans via combined ESI-FTICR MS and ESI-IT MS analysis. J Am Soc Mass Spectrom 19, 1500–1513 (2008). https://doi.org/10.1016/j.jasms.2008.06.015
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DOI: https://doi.org/10.1016/j.jasms.2008.06.015