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J. Biol. Chem., Vol. 281, Issue 43, 32057-32064, October 27, 2006

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A Novel Nucleotide Found in Human Erythrocytes, 4-Pyridone-3-carboxamide-1--D-ribonucleoside Triphosphate^*

Ewa M. Slominska, Elizabeth A. Carrey^¶, Henryk Foks^||, Czeslawa Orlewska^||, Ewa Wieczerzak^**, Pawel Sowinski, Magdi H. Yacoub, Anthony M. Marinaki, H. Anne Simmonds, and Ryszard T. Smolenski¹

From the Department of Biochemistry, Medical University of Gdansk, 80-211 Gdansk, Poland, Purine Research Unit, Guy's Hospital, London SE9 RT, United Kingdom, ^¶University College London Institute of Child Health, London WC1N EH, United Kingdom, ^||Department of Organic Chemistry, Medical University of Gdansk, 80-416 Gdansk, Poland, ^**Department of Organic Chemistry, University of Gdansk, 80-952 Gdansk, Poland, Laboratory of NMR Spectroscopy, Chemical Faculty, Gdansk University of Technology, 80-952 Gdansk, Poland, Heart Science Centre, Imperial College London, Harefield UB9 6JH, United Kingdom

We report the identification of a hitherto unknown nucleotide that is present in micromolar concentrations in the erythrocytes of healthy subjects and accumulates at levels comparable with the ATP concentration in erythrocytes of patients with chronic renal failure. The unknown nucleotide was isolated and identified by liquid chromatography with UV and tandem mass detection, ¹H nuclear magnetic resonance and infrared spectroscopy as 4-pyridone-3-carboxamide-1--D-ribonucleoside triphosphate (4PYTP), a structure indicating association with metabolism of the oxidized nicotinamide compounds. Subsequently, we demonstrated formation of 4PYTP in intact human erythrocytes during incubation with the chemically synthesized nucleoside precursor 4-pyridone-3-carboxamide-1--D-ribonucleoside (4PYR). We noted preferential accumulation of monophosphate of 4PYR (4PYMP) over 4PYTP as well as a decrease in erythrocyte ATP concentration during incubation with 4PYR. Both the 4PYR phosphorylation and ATP depletion were blocked by an inhibitor of adenosine kinase. Plasma concentration of 4PYR was detectable but very low (0.013 ± 0.006 µM) in contrast with the high daily urine excretion of this compound (26.7 ± 18.2 µmol/24 h) in healthy subjects, indicating much greater renal clearance than other nicotinamide metabolites, nucleosides, or creatinine. We also noted a 40-fold increase in 4PYR plasma concentration in patients with chronic renal failure (0.563 ± 0.321 µM). We suggest that 4PYTP formation in the erythrocytes is a hitherto unknown process aimed at sequestering potentially toxic 4PYR in a form that could be safely transported and subsequently released and excreted during passage of erythrocytes through the kidney.

Received for publication, August 7, 2006

^* This study has been supported by the Dr. Hadwen Trust for Humane Research, the Ministry of Science and Information Society Technologies of Poland (W-109 and PBZ-KBN-101/T09/2003/17), and the Magdi Yacoub Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental material.

¹ To whom correspondence should be addressed: Heart Science Centre, Imperial College London, Harefield UB9 6JH, UK. Tel.: 44-1895-828829; Fax: 44-1895-828864; E-mail: r.smolenski{at}ic.ac.uk.

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