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  • Late Papers from the 17th International Symposium on Chromatography, Vienna, Austria, September 25–30, 1988
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Ion-pair reversed phase HPLC determination of nucleotides, nucleosides and nucleobases — Application to nucleotide metabolism in hepatocytes

Chromatographia volume 27, pages 639–643 (1989)Cite this article

Summary

Three groups of metabolites were analyzed in extracts of rat hepatocytes by an HPLC method: (i) nucleotides (ATP, ADP, AMP, GTP, GDP, UTP, UDP, IMP, UMP), (ii) nucleosides and nucleobases (adenosine, adenine, guanosine, inosine, xanthine, hypoxanthine, uridine) and (iii) inhibitors of xanthine oxidoreductase (oxypurinol, allopurinol). Perchloric acid extracts were neutralized with K2CO3/triethanolamine and analyzed at 254 nm by reversed-phase ion-pair high-performance liquid chromatography. The nucleotides and their derivatives were separated with a gradient elution using 10 mM NH4H2PO4/2 mM t-butylammonium-phosphate and acetonitrile. Recovery values were estimated for the extraction procedure used. The method was applied to the investigation of nucleotide metabolism of hepatocytes of starved rats at anoxia and reoxygenation.

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References

  1. R. A. Hartwick, P. R. Brown, J. Chromatogr.112, 651 (1975).

    Google Scholar 

  2. D. Perrett, in: C. K. Lim (Ed.), HPLC of small molecules — a practical approach, IRL Press, Oxford, 1986, p. 221.

    Google Scholar 

  3. S. C. Cho, J. Chromatogr.377, 356 (1986).

    Google Scholar 

  4. P. R. Brown, HPLC in nucleic acid research, methods and applications, Marcel Dekker Inc., New York, 1984, p. 77.

    Google Scholar 

  5. G. S. Morris, H. A. Simmonds, J. Chromatogr., Biomed. Appl.344, 101 (1985).

    Google Scholar 

  6. K. Nakano, K. Shindo, T. Yasaka, H. Yamamoto, J. Chromatogr.332, 127 (1985).

    Google Scholar 

  7. R. A. Hartwick, A. M. Krstulovič, P. R. Brown, J. Chromatogr.186, 659 (1979).

    Google Scholar 

  8. W. Stocchi, L. Cucchiarini, M. Magnani, L. Chiarantini, P. Palma, G. Crescentini, Anal. Biochem.146, 118 (1985).

    Google Scholar 

  9. A. Werner, W. Siems, H. Schmidt, I. Rapoport, G. Gerber, R. T. Toguzov, Y. V. Tikhonov, A. M. Pimenov, J. Chromatogr., Biomed. Appl.421, 257 (1987).

    Google Scholar 

  10. D. R. Webster, G. D. Boston, D. M. Paton, J. Liquid Chromatogr.8, 603 (1985).

    Google Scholar 

  11. A. Werner, W. Siems, G. Gerber, H. Schmidt, S. Gruner, H. Becker, Chromatographia25, 237 (1988).

    Google Scholar 

  12. P. A. Perrone, P. R. Brown, J. Chromatogr.307, 53 (1984).

    Google Scholar 

  13. P. A. Perrone, P. R. Brown, J. Chromatogr.317, 301 (1984).

    Google Scholar 

  14. D. P. Jones, T. Y. Aw, F. G. Kennedy, in: N. W. Cornell and R. A. Harris (Eds.), Isolation, characterization and use of hepatocytes, Elsevier biomedical, New York, 1983, p. 323.

    Google Scholar 

  15. H. Hummerich, H. de Groot, T. Noll, S. Soboll Biochem. J.,250, 641 (1988).

    Google Scholar 

  16. M. N. Berry, D. S. Friend, J. Cell Biology43, 506 (1969).

    Google Scholar 

  17. G. van den Berghe, F. Bontemps, H. G. Hers, Biochem. J.188, 913 (1980).

    Google Scholar 

  18. K. K. Tekkanat, I. H. Fox, Clin. Chem.34, 925 (1988).

    Google Scholar 

  19. V. Stocchi, L. Cucchiarini, F. Canestrari, M. P. Piacentini, G. Fornaini, Anal. Biochem.167, 181 (1978).

    Google Scholar 

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Authors and Affiliations

  1. Institute of Biochemistry, Humboldt University, Hessische Straße 3-4, 1040, Berlin, GDR

    A. Werner, W. Schneider, W. Siems, T. Grune & C. Schreiter

Authors
  1. A. Werner
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  2. W. Schneider
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  3. W. Siems
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  4. T. Grune
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  5. C. Schreiter
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Werner, A., Schneider, W., Siems, W. et al. Ion-pair reversed phase HPLC determination of nucleotides, nucleosides and nucleobases — Application to nucleotide metabolism in hepatocytes. Chromatographia 27, 639–643 (1989). https://doi.org/10.1007/BF02258995

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  • DOI: https://doi.org/10.1007/BF02258995

Key Words

  • Column liquid chromatography
  • Reversed-phase
  • Ion-pair
  • Recoveries
  • Hepatocytes
  • Purine compounds