High-Performance Liquid Chromatographic Assay of the Diadenosine Polyphosphates in Human Platelets

✍ Scribed by Joachim Jankowski; Wilhelm Potthoff; Markus van der Giet; Martin Tepel; Walter Zidek; Hartmut Schlüter

• Publisher: Elsevier Science
• Year: 1999
• Tongue: English
• Weight: 86 KB
• Volume: 269
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1999.3097

✦ Synopsis

Diadenosine pentaphosphate (Ap

5 A) and diadenosine hexaphosphate (Ap 6 A) were recently identified in human platelets and were shown to be important modulators of cardiovascular function. Here we describe an HPLC assay for quantitating Ap 3 A, Ap 4 A, Ap 5 A, and Ap 6 A contents in human platelets simultaneously. Di(1,N 6 -ethenoadenosine) hexaphosphate was used as internal standard. The extraction procedure consists of (a) deproteinization, (b) selective concentration of the diadenosine polyphosphates with a boronate affinity chromatography, and (c) desalting prior to the HPLC analysis. The assay was validated by PSD-MALDI-mass spectrometry and by addition of authentic diadenosine polyphosphate to platelet samples. The assay was carried out by an ion-pair reversed-phase perfusion chromatography. In platelets from human blood the following amounts of diadenosine polyphosphates were determined: Ap 3 A, 192.5 ؎ 151.0 nM; Ap 4 A, 223.8 ؎ 172.3 nM; Ap 5 A, 100.2 ؎ 81.1 nM; Ap 6 A, 32.0 ؎ 19.6 nM (mean ؎ SD, n ‫؍‬ 105). The described assay can be used with less than 20 ml blood and allows quantitation of the diadenosine polyphosphates in the picomole range.