Bioluminescent assay of adenine nucleotides: rapid analysis of ATP and ADP in red cells and platelets using the LKB luminometer

Abstract

The assessment of both red cell and platelet function requires assay of adenine nucleotides. We describe the use of the new LKB luminometer to measure adenosine triphosphate (ATP) and adenosine diphosphate (ADP) by bioluminescence in both normal and abnormal red cells and platelets. ATP was measured a in a lysate of red cells suspended in buffer, pH 7.4, b in ethanol extracts of platelets from platelet-rich plasma (PRP) and c in ethanol extracts of supernatant platelet-poor plasma (PPP) following platelet aggregation by collagen. ADP was measured as ATP after phosphorylation by pyruvate kinase (PK) with phosphoenolpyruvate (PEP). Duplicate assays showed a variance of less than 3%. Red cell lysates and ethanol extracts of PRP and PPP stored at -40 degrees C were stable for 4 weeks. Duplicate assays of ATP and ADP in eight samples plus standards could be performed in 2 h. Normal values were: red cells (microM/ml red cells) (n = 20), ATP 1.15 +/- 0.17 (1 SD), ADP 0.22 +/- 0.07, ATP/ADP (mean ratio) 5.76:1, platelets (nm/10(9) platelets) (n = 20), ATP 53.3 +/- 7.6, ADP 27.8 +/- 5.8, ATP/ADP (mean ratio: 1.96:1). Abnormalities of red cell ATP and/or ADP could be demonstrated in chronic renal failure, hereditary glycolytic enzyme deficiencies and other haemolytic states. In myeloproliferative disorders defective platelet aggregation associated with storage pool deficiency and/or impaired release of ADP and ATP could be shown. We conclude that this is a reliable, rapid and economical technique for measuring red cell and platelet adenine nucleotides.