Simplified Determination of Blood Adenosine Triphosphate Using the Firefly System

Abstract

A simplified method is described for the determination of red cell ATP using the firefly lantern extract method. Variables investigated include the effect of the time of reading, dilution of firefly extract and the effective range of the method. Excellent recoveries were obtained. Optimal extraction of ATP from red cells was achieved with a hypotonic buffer at pH 9.2. The method could be used with acid-citrate-dextrose, heparin or EDTA as an anticoagulant. The method was found to be highly specific when the nucleotides found in normal human blood were investigated; only adenosine diphosphate and guanosine triphosphate gave slight readings, neither of which would significantly affect ATP determinations of human blood. Normal human values were found to be 5.45 µmoles of ATP/Gm. of hemoglobin or 1.83 µmoles/ml. red cells in heparinized blood samples. This method is believed to be more rapid, more reproducible and more accurate than any previously described method of ATP determination.

Some Effects of pH and Temperature upon Normal Human Red Cell Adenosine Triphosphate

“…Values obtained using a hexokinase assay (Brewer & Powell 1966) were, after recalculation, 73-117 pmoles of ATP per 100 ml. of packed cells, whilst the luciferase assay (Beutler & Baluda 1964) yielded 183 i -6 pmoles of ATP per 100 ml. of packed cells.…”

A technique for the processing of blood samples for subsequent assay of ATP, and an investigation of the method of standardization of the firefly-luciferase ATP assay

“…Even when correcting the data for a 25% overestimation error, the firefly luminescence method still gives a significantly higher (about 25%) mean level of ATP in human erythrocytes than the enzymatic method. As Beutler et al 13 have suggested, this may be due to some loss of ATP during the rather long extraction procedure involved in the enzymatic method. It would appear that when the "luminescence" method is used, either the standards should be assayed in the presence of hemoglobin, or an arbitrary 20% correction, based on the present data, should be employed.…”

Experimental Studies on the Transfusion of Blood Obtained Postmortem

“…Survival of Cr51-labeled donor cells in recipienft animals; 11. Chemical and morphological properties of erythrocytes obtained postmortem as determined by (a) morphological appearance, (b) osmotic fragility (1 l), (c) pH values, (d) adenosine triphosphate (ATP) values (1). 111.…”