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 these discrepancies and to establish correct normal values for red cell ATP, the firefly technic for measuring ATP levels was compared with other methods. The ATP content of TCA filtrates of red cells was the same when determined by the firefly method as by the hexokinase-linked technic. Relatively low concentrations of protein were found to stimulate light output when lyophilyzed firefly 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 little as 1 hour resulted in a substantial decrease in red cell ATP levels. The loss with ACD blood was less, and could be obviated entirely by using an ACD solution with a pH adjusted to between 3.5 and 4.0. Removing the buffy coat or 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 of ATP, while extraction of whole blood with TCA resulted in a 14 per cent loss of ATP. In contrast, perchloric acid extraction resulted in no ATP loss. If ATP determinations are carried out using the firefly method, protein should be added to the standard. If red cells must be stored for any period of time prior to 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 superior to trichloroacetic acid.