Identification and Quantification of Diadenosine Polyphosphate Concentrations in Human Plasma

Abstract

Objective-Diadenosine polyphosphates have been demonstrated to be involved in the control of vascular tone as well as the growth of vascular smooth muscle cells and hence, possibly, in atherogenesis. In this study we investigated the question of whether diadenosine polyphosphates are present in human plasma and whether a potential source can be identified that may release diadenosine polyphosphates into the circulation. Methods and Results-Plasma diadenosine polyphosphates (Ap n A with nϭ3 to 6) were purified to homogeneity by affinity-, anion exchange-, and reversed phase-chromatography from deproteinized human plasma. Analysis of the homogeneous fractions with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) revealed molecular masses ([MϩH] ϩ ) of 757, 837, 917, and 997 d. Comparison of the postsource decay matrix-assisted laser desorption/ionization mass spectrometry mass spectra of these fractions with those of authentic diadenosine polyphosphates revealed that these isolated substances were identical to Ap 3 A, Ap 4 A, Ap 5 A, and Ap 6 A. Enzymatic analysis showed an interconnection of the phosphate groups with the adenosines in the 5Ј-positions of the ribose moieties. The mean total plasma diadenosine polyphosphate concentrations (mol/L; mean Ϯ SEM) in cubital veins of normotensive subjects amounted to 0.89Ϯ0.59 for Ap 3 A, 0.72Ϯ0.72 for Ap 4 A, 0.33Ϯ0.24 for Ap 5 A, and 0.18Ϯ0.18 for Ap 6 A. Cubital venous plasma diadenosine polyphosphate concentrations from normotensives did not differ significantly from those in the hypertensive patients studied. There was no significant difference between arterial and venous diadenosine polyphosphate plasma concentrations in 5 hemodialysis patients, making a significant degradation by capillary endothelial cells unlikely. Free plasma diadenosine polyphosphate concentrations are considerably lower than total plasma concentrations because approximately 95% of the plasma diadenosine polyphosphates were bound to proteins. There were no significant differences in the diadenosine polyphosphate plasma concentrations depending on the method of blood sampling and anticoagulation, suggesting that platelet aggregation does not artificially contribute to plasma diadenosine polyphosphate levels in significant amounts.The Ap n A (with nϭ3 to 6) total plasma concentrations in adrenal veins were significantly higher than the plasma concentrations in both infrarenal and suprarenal vena cava

Uridine triphosphate (UTP) is released during cardiac ischemia

“…Furthermore, ADP and AMP had a similar accumulation pattern although not as marked as ATP and UTP. The baseline ATP levels were in the micromolar range, which is similar to previously reported levels of circulating ATP and diadenosine polyphosphates in man [15,20]. Baseline levels of adenosine in the pig plasma were markedly lower than those of its nucleotides, most likely due to both rapid uptake into the red blood cells and degradation.…”

“…Platelet contamination was excluded by Burker chamber examination. A similar method used to determine dinucleotides was validated against best practice conditions for limiting platelet activation, with no significant differences [15]. The plasma was aspirated and mixed with an equal amount of 10% trichloroacetic acid (TCA) to precipitate all proteins.…”

A Bench to Bedside View of Uremic Toxins

“…Furthermore, diadenosine polyphosphates are strong vasoconstrictors with direct effects on vascular tone mediated by P2X receptors. 48,49 Thus, diadenosine polyphosphates may be one as yet unidentified cause of hypertension in renal failure.…”

Endogenous Diadenosine Tetraphosphate, Diadenosine Pentaphosphate, and Diadenosine Hexaphosphate in Human Myocardial Tissue

“…17 The lyophilized eluate of analytical reversed-phase chromatography was analyzed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and postsource decay MALDI mass spectrometry using the conditions as described in Jankowski et al, 22 UV spectroscopy, 17 and enzymatic cleavage experiments. 23 …”