Measurement of free and bound fractions of extracellular ATP in biological solutions using bioluminescence

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Abstract
Measurement of extracellular ATP in biological solutions is complicated by protein-binding and rapid enzymatic degradation. We hypothesized that the concentration of extracellular ATP could be determined luminometrically by limiting degradation and measuring the free and protein-bound fractions. ATP was added (a) at constant concentration to solutions containing varying albumin concentrations; (b) at varying concentrations to a physiological albumin solution (4 gm/dL); (c) at varying concentrations to plasma. After centrifugation, a fraction of each supernatant was heated. ATP in heated and unheated samples was measured luminometrically. Blood was drawn into saline or an ATP-stabilizing solution and endogenous plasma ATP measured. ATP-albumin binding was a linear function of albumin concentration (3.5% ATP bound at 100 µmol/L to 33.2% ATP bound at 1000 µmol/L) but independent of ATP concentration (29.3%, 10–1000 nmol/L ATP in 602 µmol/L albumin). Heating released the majority of bound ATP from albumin-containing solutions (94.8 ± 1.7%) and plasma (97.6 ± 5.1%). Total endogenous plasma ATP comprised 93 ± 27 nmol/L (free) and 150 ± 40 nmol/L (total fraction). Without stabilizing solution, degradation of free endogenous plasma ATP occurred. Within a physiological range (10–1000 nmol/L), ATP binds albumin independently of ATP concentration. Heating releases bound ATP, enabling accurate luminometric measurement of total extracellular ATP (free and bound) in biological samples. Copyright © 2005 John Wiley & Sons, Ltd.