Extraction of intracellular nucleosides and nucleotides with acetonitrile.
Extraction of intracellular nucleosides and nucleotides with acetonitrile (ACN) and water was compared with extraction with 60 g/L perchloric acid (PCA), followed by neutralization with KOH, 1 mol/L. Freshly isolated rat bone-marrow and intestinal cells were incubated with radiolabeled 5-fluorouracil (FUra) and 5-fluorodeoxyuridine. The ribose and deoxyribose nucleosides and nucleotides of FUra, and ADP and ATP in the soluble extracts were separated by HPLC and measured by scintillation counting or ultraviolet absorbance. The insoluble precipitates were digested in 1 mol/L NaOH and analyzed for the radioactive macromolecule-bound nucleotides. Both extraction methods yielded the same total (i.e., soluble and insoluble) amount of radioactivity. However, the ACN method yielded significantly more FUra nucleosides and triphosphate nucleotides and ATP in the soluble fraction, and more proteins and macromolecule-bound nucleotides in the insoluble fraction. In the PCA method, the soluble fraction contained more monophosphate nucleotides and ADP than in the ACN method. The PCA extraction procedure promoted decomposition of ATP to ADP and interfered with the ion-pairing reversed-phase HPLC assay. The ACN extraction is faster (less than 5 min) than the PCA extraction (greater than 10 min). Moreover, the ACN in the soluble extract fraction can be removed by evaporation and thus does not interfere with the HPLC analysis. Thus the ACN method evidently is suitable for extraction of nucleosides and nucleotides.
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J L
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College of Pharmacy, Ohio State University, Columbus 43210.
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Wientjes
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D000097:Acetonitriles; D000818:Animals; D001853:Bone Marrow / Q000032:analysis / Q000378:metabolism; D011232:Chemical Precipitation; D002851:Chromatography, High Pressure Liquid; D005260:Female; D005467:Floxuridine / Q000378:metabolism; D005472:Fluorouracil / Q000378:metabolism; D006878:Hydroxides; D007202:Indicators and Reagents; D007413:Intestinal Mucosa / Q000378:metabolism; D007422:Intestines / Q000032:analysis; D009705:Nucleosides / Q000302:isolation & purification* / Q000378:metabolism; D009711:Nucleotides / Q000302:isolation & purification* / Q000378:metabolism; D010472:Perchlorates; D011188:Potassium; D017680:Potassium Compounds; D051381:Rats; D014316:Tritium
D000097:Acetonitriles; D006878:Hydroxides; D007202:Indicators and Reagents; D009705:Nucleosides; D009711:Nucleotides; D010472:Perchlorates; D017680:Potassium Compounds; D005467:Floxuridine; D014316:Tritium; D011188:Potassium; D005472:Fluorouracil; C029943:potassium hydroxide; C032159:acetonitrile