title: Measurement of free and bound fractions of extracellular ATP in biological solutions using bioluminescence.
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 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.
authors.affiliation: Department of Surgery, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7228, USA.
authors.forename: Christelle D
authors.initials: CD
authors.lastname: Douillet
authors.forename: Sihong
authors.initials: S
authors.lastname: Suy
authors.forename: Ben L
authors.initials: BL
authors.lastname: Zarzaur
authors.forename: William P
authors.initials: WP
authors.lastname: Robinson
authors.forename: Peter M
authors.initials: PM
authors.lastname: Milano
authors.forename: Richard C
authors.initials: RC
authors.lastname: Boucher
authors.forename: Preston B
authors.initials: PB
authors.lastname: Rich
mesh_terms: 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
chemical_list: D000418:Albumins; D012996:Solutions; D000255:Adenosine Triphosphate