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An ion-pair reversed-phase HPLC method for determination of fresh tissue adenine nucleotides avoiding freeze-thaw degradation of ATP
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Zur Nedden, Stephanie, Eason, Robert, Doney, Alexander S. and Frenguelli, Bruno G.. (2009) An ion-pair reversed-phase HPLC method for determination of fresh tissue adenine nucleotides avoiding freeze-thaw degradation of ATP. Analytical Biochemistry, Vol.388 (No.1). pp. 108-114. ISSN 0003-2697
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Official URL: http://dx.doi.org/10.1016/j.ab.2009.02.017
Abstract
Knowledge of the energetic state of tissue is required in a wide range of experimental studies, particularly those investigating the decline and recovery of cellular metabolism after metabolic stress. Such information can be obtained from high-performance liquid chromatography (HPLC) determination of tissue levels of adenine nucleotides (ATP, ADP, and AMP) and their interrelationship in the tissue energy charge (EC). Accordingly, a large range of techniques with which to measure these molecules and their downstream metabolites have been reported. However, the accurate determination of the tissue EC also depends on the nucleotide extraction procedure given that changes in adenine nucleotide levels take place very quickly when ATPases are not inactivated immediately. In this article, we describe all ion-pair reversed-phase HPLC method by which separation of adenine nucleotides can be performed rapidly, allowing multiple analyses in 1 day, with both high sensitivity and extraction efficiency and using fresh samples, thereby avoiding freeze-thaw degradation of nucleotides. We applied this method to hippocampal brain slice extracts and show that same-day extraction and analysis results ill a more accurate determination of the in situ energetic state than does the commonly used snap-freezing in liquid nitrogen. (C) 2009 Elsevier Inc. All rights reserved.
| Item Type: | Journal Article |
|---|---|
| Subjects: | Q Science > QD Chemistry |
| Divisions: | Faculty of Science > Life Sciences (2010- ) > Biological Sciences ( -2010) |
| Journal or Publication Title: | Analytical Biochemistry |
| Publisher: | Academic Press |
| ISSN: | 0003-2697 |
| Date: | 1 May 2009 |
| Volume: | Vol.388 |
| Number: | No.1 |
| Number of Pages: | 7 |
| Page Range: | pp. 108-114 |
| Identification Number: | 10.1016/j.ab.2009.02.017 |
| Status: | Peer Reviewed |
| Publication Status: | Published |
| Access rights to Published version: | Restricted or Subscription Access |
| Funder: | Research into Ageing |
| URI: | http://wrap.warwick.ac.uk/id/eprint/28136 |
Data sourced from Thomson Reuters' Web of Knowledge
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