The acid-soluble nucleotides of milk. II. Isolation and identification of two novel uridine nucleotide oligosaccharide conjugates from human milk and colostrum.

Materials and Methods Preparation of UDP-X1 and UDP-X3-Twenty litres of fresh human milk was deproteinized by adding 1/4 volume of cold 2 N perchloric acid. The supernatant was neutralized with 2 N KOH and the resulting potassium perchloric acid and solidified lipid fraction were removed by filtration. The filtrate was again acidified with 1/10 volume of N HC1 and passed through a charcoal column. After washing with water, the absorbed nucleotides were eluted with 2 litres of ammoniacal ethanol. The eluate was concentrated to approximately 200 ml. under reduced pressure, mounted on a column of Dowex-l (formate form) and subjected to chromatography using a slight modi fication of H u r l b e r is gradient elution system (2). The chromatographic pattern is shown in Figure 1. Both UDP-X, fraction (tube No. 144-152) and UDPX3 fraction (tube No. 127-137) were freed from formic acid and ammonium formate by treatment with charcoal, and subjected to the second ion exchange column chromatography, which was carried out by gradient elution with sodium formate (Fig. 2). The main peaks were desalted by the charcoal treat ment and purified further by paper chromatography using solvent A (vide infra). Almost all ultraviolet absorbing material migrated as a single band in each