HPLC analysis of nucleotides in tissues subjected to induced metabolic changes

• Main Author: Williams, S. P.
• Published: Swansea University 1982
• Subjects: 572.85
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.636628

Metabolic changes were induced in rat tissues by the administration of various hormones, nitrogenous bases and other metabolites and pharmacologically active compounds. Free nucleotides were extracted in perchloric acid, rapidly neutralized with an amine/Freon solution and then partially purified by ligand exchange chromatography. The extraction procedure was designed to minimize formation of artefacts; and the recovery of nucleotides was high (ca. 95%). Nucleotide extracts were analysed by HPLC, using a weak anion-exchange, microparticulate column (u Bondapak-NH2) and a linear phosphate gradient. This system separated approximately twenty of the naturally occurring ribonucleotides including acid-breakdown products of the pyridine nucleotides. Of the sixteen induced metabolic changes examined, seven markedly altered the nucleotide profile of rat tissues. Both acute and chronic administration of ethanol, by injection and ingestion respectively, caused a large decrease in the concentration of ATP and other NTPs, with a concomitant increase in the concentrations of the corresponding NMPs, in the liver. No changes were observed in the nucleotide profiles of other tissues after ethanol administration. Similar changes in the hepatic nucleotides were found after repeated daily injection of phenobarbitone (i.p.), and within minutes of the injection of adrenalin (i.p.). Injection of fructose (i.p.) also reduced the hepatic ATP concentration, however, the total adenine nucleotide pool and other nucleotides were also greatly depleted. In contrast, injection of adenine (i.p.) increased the hepatic +ATP concentration. Injection of nicotinamide caused a massive increase in the NAD concentration, plus other associated metabolic changes, in the liver. It was also found that allopurinol increased the IMP concentration in spleen, but not in the liver or kidney. These results were discussed in relation to the known metabolic effects of the compounds examined. Also the application of the present HPLC analytical system to the detection of alterations in metabolism was discussed. A procedure was also developed to facilitate the analysis of cyclic nucleotides from tissues and from urine, by HPLC.