HPLC assay with UV detection for determination of RBC purine nucleotide concentrations and application for biomarker study in vivo.
ATP and other purine nucleotides are important biomarkers for ischemia and may have considerable potential as targets for management of ischemic heart disease and stroke. The main objective of the study is to develop a rapid HPLC assay, which has adequate sensitivity and specificity for measuring concentrations of ATP, ADP, AMP, GTP, GDP and GMP in erythrocytes (RBC). The assays used ion-pair chromatography coupled with ultraviolet detection at 254 nm to separate and detect the purine nucleotides. Using 50-100 microL of RBC lysate as blank biologic matrix, the assay was linear from 100 to 2000 microg/mL for ATP and ADP, and 20-400 microg/mL for AMP, GTP, and GDP with coefficients of determination (r(2)) >0.99. GDP and GMP were not measurable in the study because of low concentrations and interference from endogenous materials, respectively. The intra-assay and inter-assay variations over a period of 1 year were less than 10% and 20%, respectively for most of the nucleotides. The assay was successfully applied to two pilot biomarker studies to measure RBC concentrations of the purine nucleotides in rats under restraining and exercise conditions. Preliminary results showed that the RBC concentrations of ATP and GTP were higher in the spontaneously hypertensive rats (SHR) compared to the Sprague-Dawley (SD) rats, and that exercise increased RBC concentrations of ATP in rats treated with the calcium channel blocker diltiazem.
Yeung
Pollen
P
Pharmacokinetics & Metabolism Laboratory, College of Pharmacy, Dalhousie University, Halifax, NS, Canada. Pollen.Yeung@Dal.Ca
Ding
Lixia
L
Casley
William L
WL
D000244:Adenosine Diphosphate / Q000097:blood; D000249:Adenosine Monophosphate / Q000097:blood; D000255:Adenosine Triphosphate / Q000097:blood; D000818:Animals; D001681:Biological Assay; D015415:Biomarkers / Q000097:blood; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004110:Diltiazem / Q000494:pharmacology; D004912:Erythrocytes / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000097:blood; D008297:Male; D010805:Physical Conditioning, Animal; D010865:Pilot Projects; D011685:Purine Nucleotides / Q000097:blood*; D051381:Rats; D011918:Rats, Inbred SHR; D017207:Rats, Sprague-Dawley; D012015:Reference Standards; D012680:Sensitivity and Specificity; D052616:Solid Phase Extraction; D013045:Species Specificity; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*
D015415:Biomarkers; D002121:Calcium Channel Blockers; D011685:Purine Nucleotides; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D004110:Diltiazem