The nucleotides in normal human blood.

Approximately 15 ml. of venous blood were withdrawn and expressed into a chilled tube containing 0.30 ml. (300 U.S.P. units) of sodium heparin solution. After thorough but gentle stirring, 10.0 ml. were transferred by Folin pipette to 10 ml. of ice cold 10 per cent trichloroacetic acid. The mixture was thoroughly homogenized with a heavy stirring rod and centrifuged in the cold. The supernatant was poured into a glassstoppered graduate, and the residue was re-extracted twice with lo-ml. portions of cold 5 per cent trichloroacetic acid. The combined supernatant solutions were extracted with equal volumes of cold ether until neutral, aerated for 5 minutes to remove as much as possible of the remaining ether, and stored frozen. The extra blood was centrifuged for hematocrit determination and the plasma saved for other determinations. The separation of blood nucleotides followed the method of Hurlbert et al. (5) with the use of 20 x l-cm. columns of Dowex 1-formate resin, except that gradient elution was not employed. The trichloroacetic acid extract of blood was poured onto a column, and distilled water was allowed to flow through the column until no more ultraviolet-absorbing material came through into the effluent (approximately 1 hour). The eluting solutions ranged from 0.13 N formic acid to 4 N formic acid-O.8 M ammonium formate. Fractions of 5 ml. were collected. An ultraviolet monitor from Gilson Medical Electronics, Middelton, Wisconsin, was used to delineate the elution pattern. The absorbance of each fraction was read in the Beckman DU spectrophotometer at 260 rnp.l The material from each peak was evaporated to dryness on a steam bath and the free purines or pyrimidines liberated by hydrolysis for 1 hour at 100” in 72 per cent HCIOI. The neutralized solutions were spotted on Whatman No. 1 paper for descending chromatography in the isopropanol-HCl system of Wyatt (6) followed by ascending chromatography in the butanol-