Abstract
Following an outline of the chemistry and biochemistry of the purine and pyrimidine bases and their nucleotides the analytical approaches to their quantiation have been reviewed. High performance liquid chromatographic methods for nucleotides, nucleosides and bases have been developed and anion-exchange and ion-paired reversed-phase separations have been evaluated for use with biological extracts. The importance of pH to the selectivity of the IEC has been defined. The ion-exchange system gave the simplest chromatograms since nucleosides and bases were also retained on the optimum ion-paired system which used triethylamine as the counter ion. Detection principles relying on uv, electrochemical (ECD) and fluoresence (FL) derivatisation have been optimised and their relative suitabilities discussed. UV detection was the most generally useful but EC and FL are more selective and sensitive. ECD was best suited to nucleosides and bases with a high degree of oxygenation. Fluorescent N6-etheno derivatives which can be resolved by ion-paired HPLC is very sensitive but restricted to adenine derivatives. The extraction of nucleotides from red cells has been investigated in detailed. The final extraction conditions using trichloroacetic acid gave highest recoveries with minimal hydrolysis of nucleotides. The final assays were reliable and reproducible. Combinations of the above systems were used to determine normal values for nucleotides in red cells, platelets and lymphocytes from control subjects. Nucleotide data from red cells from 84 controls was obtained but no significant correlations were observed. The changes in red cell nucleotide pattern in various diseases including those associated with adenosine deaminase deficiency and purine nucleoside phosphorylase deficiency, when deoxynucleotides accumulate, were determined. Abnormalities in the nucleotide content in rheumatoid arthritis (RA) were found. ATP concentration were significantly below normal. These and other findings in RA have been discussed in relation to a disease mechanism involving reperfusion injury.