Determination of Adenylate Nucleotides in Amphipod Gammarus fossarum by Ion-Pair Reverse Phase Liquid Chromatography: Possibilities of Positive Pressure Micro-Solid Phase Extraction

Abstract

Adenine nucleotides—adenosine monophosphate, diphosphate, and triphosphate—are of utmost importance to all living organisms, where they play a critical role in the energy metabolism and are tied to allosteric regulation in various regulatory enzymes. Adenylate energy charge represents the precise relationship between the concentrations of adenosine monophosphate, diphosphate, and triphosphate and indicates the amount of metabolic energy available to an organism. The experimental conditions of adenylate extraction in freshwater amphipod Gammarus fossarum are reported here for the first time and are crucial for the qualitative and quantitative determination of adenylate nucleotides using efficient and sensitive ion-pair reverse phase LC. It was shown that amphipod calcified exoskeleton impeded the neutralization of homogenate. The highest adenylate yield was obtained by homogenization in perchloric acid and subsequent addition of potassium hydroxide and phosphate buffer to achieve a pH around 11. This method enables separation and accurate detection of adenylates. Our study provides new insight into the complexity of adenylate extraction and quantification that is crucial for the application of adenylate energy charge as a confident physiological measure of environmental stress and as a health index of G. fossarum.

Energy Status of Stygophilous Amphipod Synurella ambulans as a Promising Biomarker of Environmental Stress in the Hyporheic Zone

“…Composite samples of S. ambulans for quantification of adenylates were prepared as described by Redžović et al [37], who applied a modified method used by Díaz Enrich et al [38]. Amphipods were pooled due to their small size and mass (10-50 specimens per sample, mass of a sample: 0.012-0.034 g; Table S4).…”

“…The AMP, ADP and ATP concentrations determined in the S. ambulans samples were calculated from the peak area of the respective nucleotide and expressed as µmol g −1 wet weight (w.w.). The final result was obtained by multiplying the adenylate concentration by the extraction recovery of internal standard ADN [37].…”