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A New Sensitive Method for Determination of Intracellular 1-ß-D-arabinofuranosylcytosine 5'-triphosphate Content in Human Materials in Vivo¹

First Department of Internal Medicine, Fukui Medical School, 23-3 Shimoaizuki, Matsuoka, Fukui 910-11, Japan

A new sensitive method for the measurement of 1-ß-D-arabinofuranosyl-CTP (ara-CTP), an intracellular active metabolite of 1-ß-D-arabinofuranosylcytosine (ara-C), in human materials in vivo has been established. An acid-soluble fraction containing ara-CTP was extracted from blastic cells by ara-C treatment with trichloroacetic acid (final concentration, 0.3 M) neutralized with an equal volume of cold freon containing 0.5 M tri-n-octylamine. The ara-CTP fraction was separated from the acid-soluble fraction by high-performance liquid chromatography (TSK gel diethylaminoethyl-2 SW column) eluted with 0.05 M phosphate buffer (pH 6.9) and 20% acetonitrile. ara-CTP was lyophilized, dephosphorylated to ara-C by incubation with 10 units alkaline phosphatase for 12 h at 55°C, and measured by RIA using anti-ara-C serum. Recovery through the whole procedure was 92%. In the human chronic myelogenous leukemia cell line K562, the intracellular ara-CTP levels produced when the cells were incubated with ara-C were assayed as above, and they showed a linear increase depending on ara-C concentrations from 0.01 to 10 µM, demonstrating a very close correlation with the labeled ara-CTP levels yielded by cells on incubation with radiolabeled ara-C (r² = 0.99). The detection limit was 0.1 pmol/5 x 10⁶ cells, and a sample amount of only 5 x 10⁶ cells was enough for each assay. In the clinical applications, our method proved capable of detecting a wide concentration range of ara-CTP produced when patients were treated with ara-C or its derivatives from very low to intermediate doses. No radiolabeled drug was necessary. The method was very useful for in vivo pharmacodynamic studies of ara-C therapy.

¹ This work was supported in part by a Grant-in-Aid from the Ministry of Health and Welfare, Japan.

² To whom requests for reprints should be addressed.

Received 9/26/95. Accepted 2/16/96.

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