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The most common methods for analysis of platelet adenine nucleotides have been either firefly-luciferase or enzyme based spectrophotometric assay. However HPLC provides ah alternative with advantages of direct, rapid and efficient analysis. Although this has usually been achieved by the use of ion exchange columns, this study relates to the development of a rapid 10 minute assay using reverse phase partition. Platelet preparations were extracted with perchloric acid, neutralised and subjected to a sample ‘clean up’step prior to injection. The selected manning conditions were isocratic elution using 0.15M pH5.45 NH4H2PO4 as solvent and a 4 minute linear gradient change in flow rate from 4 to 6 mls/min. at ambient temperature. Analyses were initially performed on yBondapak C18 columns of conventional type but the running conditions described relate to the use of radial compression types of this column. Sample peaks were identified by comparison with known standard retention times. The following normal range was established ATP 5.82 ± 1.14 n moles/108 plats. ADP 4.14 ± 1.45. AMP 0.28 ± 0.14 and ATP/ADP ratio 1.43 ± 0.48. ‘Within man’ precision showed that the coefficient of variation for ATP was 2.55%, ADP 3.26%, AMP 4.64%, whilst ‘between run’ precision for ATP was 9.03% and 11.98% for ADP. The value of nucleotide analysis was demonstrated in a group of five patients with DIC. Four patients had impaired platelet aggregation responses to ADP and collagen. Nucleotide analysis revealed significant reduction of intraplatelet ADP and ATP and increased ATP/ADP ratios. This confirms previous suggestions that acquired storage pool defects occur in DIC. In addition the low ATP levels indicate that in DIC there is also a reduction in metabolically active nucleotides.

Measurement Of Platelet Nucleotides By Reverse-Phase HPLC, And The Demonstration Of Acquired Storage Pool Defect In D.I.C