SIMULTANEOUS DETERMINATION OF ADENOSINE AND RELATED PURINES IN TISSUES AND HEMOLYMPH OF MUSSEL BY HPLC

Abstract

It has been shown that adenosine protects against the lack of oxygen in hypoxia/anoxia intolerant organisms such as mammals, and in some tolerant vertebrates such as freshwater turtles of the genera Chrysemys and Trachemys, and crucian carp. By contrast, little is known about the possible protective role of adenosine in invertebrates, although invertebrates comprise the major number of hypoxia/anoxia tolerant organisms, for example, mussels. With the aim to explore the adenosynergic system in these organisms, we have developed an HPLC method for assessing adenosine, its putative precursors (ATP, ADP, AMP, and cAMP), some of its degradation metabolites (inosine, hypoxanthine, and xanthine), and IMP. The method consists of an ion-pair reversed-phase chromatography with gradient elution and UV detection with a diode array detector that allows us to evaluate all the compounds of interest in a single chromatogram within twenty-five min. The method is suitable for both hemolymph and extracts from different tissues.

Keywords:

• adenosine
• hypoxia
• invertebrates
• metabolic arrest
• mussel

ACKNOWLEDGMENT

This study was supported by The Autonomous Government of Galicia, Spain (grant PGIDT02 RMA26101PR).

Notes

^a The accuracy and the intra-assay precision were determined by repeated analysis of six aliquots of a gill sample without spike, six aliquots spiked with low amounts of standards and six aliquots spiked with high amounts of standards. The accuracy was calculated as the percent deviation from the nominal concentration. To determine the precision was calculated the coefficient of variation.

^b The inter-assay precision and the sample stability at −20°C were determined by analyzing, in ten different days throughout two months, a mantle sample spiked with 500 pmol of Hyp, Xan, Ino, Ado, IMP, and cAMP, 1000 pmol of AMP, and 2000 pmol of ADP and ATP, and stored in ten aliquots at −20°C.