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

Redžović, Zuzana; Erk, Marijana; Svetličić, Ema; Dončević, Lucija; Gottstein, Sanja; Hozić, Amela; Cindrić, Mario (2021) Determination of Adenylate Nucleotides in Amphipod Gammarus fossarum by Ion-Pair Reverse Phase Liquid Chromatography: Possibilities of Positive Pressure Micro-Solid Phase Extraction. Separations, 8 . ISSN 2297-8739

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Official URL: https://www.mdpi.com/2297-8739/8/2/20/htm

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.

Item Type:

Article

Uncontrolled Keywords:

adenylate energy charge ; freshwater amphipods ; ecosystem monitoring ; adenylate extraction ; ecological friendly solutions

Subjects:

NATURAL SCIENCES > Biology

Divisions:

Division of Molecular Medicine

Projects:

Project title	Project leader	Project code	Project type
Qua/Qua Protein: Kvantitativna i kvalitativna analiza proteina za potrebe biomedicine i biotehnološke industrije	Cindrić, Mario	KK.01.1.1.07.0023	EK
Akumulacija, unutarstanično mapiranje i učinci metala u tragovima u akvatičkih organizama	Erk, Marijana	IP-2014-09-4255	HRZZ

Depositing User:

Mario Cindrić

Date Deposited:

23 Mar 2021 13:56

URI:

http://fulir.irb.hr/id/eprint/6356

DOI:

10.3390/separations8020020