Measurement of free and bound fractions of extracellular ATP in biological solutions using bioluminescence.
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 micromol/L to 33.2% ATP bound at 1000 micromol/L) but independent of ATP concentration (29.3%, 10-1000 nmol/L ATP in 602 micromol/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.
Douillet
Christelle D
CD
Department of Surgery, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7228, USA.
Suy
Sihong
S
Zarzaur
Ben L
BL
Robinson
William P
WP
Milano
Peter M
PM
Boucher
Richard C
RC
Rich
Preston B
PB
D000255:Adenosine Triphosphate / Q000032:analysis*; D000418:Albumins / Q000737:chemistry; D000818:Animals; D008163:Luminescent Measurements / Q000379:methods*; D008297:Male; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D012680:Sensitivity and Specificity; D012996:Solutions / Q000737:chemistry
D000418:Albumins; D012996:Solutions; D000255:Adenosine Triphosphate