Introduction: Measurement of ATP, ADP, and AMP (adenylate pools) is essential for establishing mitochondrial function and metabolic response to stressors or pharmacological interventions. HPLC analysis is useful due to its compatibility with multiple samples and UV/mass spectroscopy for confirmation. However, HPLC is limited by quality of sampling, storage, and technique. Following sampling, ATP levels in a tissue decline rapidly due to enzymatic degradation leading to results unrepresentative of in situ. Attempted correction is made by flash freezing with liquid nitrogen and storing at -80°C to halt enzymatic degradation. Effectiveness of this method is in question due to reactivation of enzymes with thawing of samples. Analyzing tissues immediately after sampling is the most reliable practice but is inconvenient in most lab settings due to being increasingly time consuming with more samples. Objective & Hypothesis: The goal of this analysis is to provide a methodology for measuring adenylate pools in mice tissues and determining the best practice for storage of samples: preparing samples with perchloric acid and centrifugation (PCA) or flash frozen (FF) samples. We hypothesize that PCA will yield closer results to in situ than FF due to precipitating protein from the sample thereby eliminating enzymatic activity. Methods: Utilizing mice liver tissue from a total of 5 mice (N=5), HPLC was run for FF samples and PCA samples immediately following sampling and at 1,2,3, and 4 weeks. Both FF and PCA samples were stored at -80°C. Additionally, adenylate energy change (AEC) was calculated as follows, AEC=(ATP+1/2 ADP)/(ATP+ADP+AMP) for each measurement. AEC is a more stable metric than individual adenylate pool concentrations in healthy tissues and thus serves as a more informative marker of metabolic status. The methodology was verified using heart, liver, lung, and kidney tissues from both female and male mice to provide reference concentrations. Statistical analysis was performed in SPSS version 30 with P values < 0.05 considered as significant. A nonparametric, Related-Samples Wilcoxon Signed Rank test was utilized to evaluate the difference from baseline to last time point. Results: For PCA there were no significant differences between baseline and last time point for ATP (1.53 ± 0.93μmol/g vs. 1.16 ± 0.50μmol/g, p=0.345), ADP (0.99 ± 0.58μmol/g vs. 0.98 ± 0.37μmol/g, p=0.893), AMP (0.91 ± 0.69μmol/g vs. 0.62 ± 0.27μmol/g, p=0.345), and AEC (0.58 ± 0.12 vs. 0.59 ± 0.13, p= 0.225). For FF there were significant differences between baseline and last time point for ATP (1.62 ± 0.77μmol/g vs. 0.08 ± 0.06μmol/g, p=0.043), AMP (0.95 ± 0.63μmol/g vs. 2.47 ± 1.28μmol/g, p=0.043), and AEC (0.60 ± 0.11 vs. 0.14 ± 0.09, p=0.043) and no significant differences for ADP (1.05 ± 0.46μmol/g vs. 0.56 ± 0.18μmol/g, p=0.080). There were also significant differences in the change from baseline to last measurement for ATP (PCA 0.37 ± 0.77μmol/g vs. FF 1.54 ± 0.81μmol/g, p=0.043), ADP (PCA 0.01 ± 0.45μmol/g vs. 0.50 ± 0.44μmol/g, p=0.043), and AEC (PCA -0.02 ± 0.03 vs. FF 0.46 ± 0.13, p=0.043) with a non-significant difference for AMP (PCA 0.28 ± 0.63μmol/g vs. FF -1.52 ± 0.98μmol/g, p=0.080). Conclusion: This analysis supports PCA as the best option for preserving in situ adenylate pool concentrations while providing a viable HPLC methodology for analyzing adenylate pool concentrations in mice tissues. Thus, utilization of this methodology and storage technique should bolster the reliability of metabolic analyses. Further improvements to this methodology should involve verification with different species and tissue types. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.
Bedard et al. (Fri,) studied this question.