A Comparison of Normal Red Cell ATP Levels as Measured by the Firefly System and the Hexokinase System

Widely divergent normal red cell ATP levels have been reported by investigators using different methods. In order to clarify the cause of thesediscrepancies and to establish correct normal values for red cell ATP, the firefly technic for measuring ATP levels was compared with other methods. TheATP content of TCA filtrates of red cells was the same when determined bythe firefly method as by the hexokinase-linked technic. Relatively low concentrations of protein were found to stimulate light output when lyophilyzedfirefly extract, but not freshly prepared firefly extract, was used. Thus, falsely,high values were obtained when red cell extracts were examined, unless protein was also added to the standard. Storage of heparinized blood for as littleas 1 hour resulted in a substantial decrease in red cell ATP levels. The losswith ACD blood was less, and could be obviated entirely by using an ACDsolution with a pH adjusted to between 3.5 and 4.0. Removing the buffy coator washing cells in saline resulted in no further loss of red cell ATP. Extraction of washed red cells with TCA resulted in an average loss of 4.6 per cent ofATP, while extraction of whole blood with TCA resulted in a 14 per cent lossof ATP. In contrast, perchloric acid extraction resulted in no ATP loss. If ATPdeterminations are carried out using the firefly method, protein should beadded to the standard. If red cells must be stored for any period of time priorto extraction of ATP, an ACD solution with a pH of 3.5 to 4.0 should be used.If extracts of red cells are made, perchloric acid appears significantly superiorto trichloroacetic acid.

Submitted on January 10, 1967 Accepted on March 1, 1967