A new sensitive method for determination of intracellular 1-beta-D-arabinofuranosylcytosine 5'-triphosphate content in human materials in vivo.
A new sensitive method for the measurement of 1-beta-D-arabinofuranosyl-CTP (ara-CTP), an intracellular active metabolite of 1-beta-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 degrees 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 microns, 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 10(6) cells, and a sample amount of only 5 x 10(6) 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.
Yamauchi
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T
First Department of Internal Medicine, Fukui Medical School, Japan.
Ueda
T
T
Nakamura
T
T
D000469:Alkaline Phosphatase; D001085:Arabinofuranosylcytosine Triphosphate / Q000032:analysis* / Q000097:blood; D001752:Blast Crisis; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002855:Chromatography, Thin Layer / Q000379:methods; D003561:Cytarabine / Q000378:metabolism / Q000627:therapeutic use; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive; D015470:Leukemia, Myeloid, Acute / Q000097:blood / Q000188:drug therapy; D009190:Myelodysplastic Syndromes / Q000097:blood / Q000188:drug therapy; D011863:Radioimmunoassay / Q000379:methods; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D003561:Cytarabine; D001085:Arabinofuranosylcytosine Triphosphate; D000469:Alkaline Phosphatase