HPLC-UV measurements of metabolites in the supernatant of endothelial cells exposed to oxidative stress

Abstract

2,8-Dihydroxyadenine (2,8-DHA) was identified by high-performance liquid chromatography with ultraviolet detection as a major metabolite in the supernatant of endothelial cells of the pulmonary artery (PAECs) and aorta (AECs), in addition to hypoxanthine, xanthine, uric acid, and uracil. Under normoxic, hypoxic, and hyperoxic conditions, the concentrations of all the identified metabolites change with time, marking the response of endothelial cells to stress, as a result of changes in cellular metabolism. Thus, the metabolites can serve as stress markers, and their concentrations can indicate the type and the level of cell stress. The results verify that PAECs adapt to survive oxidative stress of hyperoxia. However, AECs can adapt to hypoxia only for a short time and do not survive prolonged hypoxia. The role of the polyamine synthesis pathway in the formation of the unsalvaged adenine, as a possible source of 2,8-DHA, is discussed.

The assessment of the quality of the graft in an animal model for lung transplantation using the metabolomics 1H high‐resolution magic angle spinning NMR spectroscopy

“…Cell stress generated by reactive oxygen species lead to the release of adenosine triphosphate and uridine triphosphate in the extracellular fluid. After 2 h, the adenosine triphosphate levels decrease resulting in its degradation to adenosine (24). Finally, the adenosine triphosphate production decreases under cell stress; this generates adenosine monophosphate (AMP) and adenosine diphosphate (ADP) which degraded into final metabolites.…”

“…On the other hand, we observe an opposite situation with the uracil signal. Indeed, the uracil signal was absent in the control samples and remained absent over time during cold ischemia with Perfadex V R solution, whereas it markedly increased over time in the absence of perfusion whether at 24 or 4 C. Recent studies report a change in the levels of cellular purine or pyrimidine, such as uracil, following oxidative stress (24,25), indicative of cell damage. Cell stress generated by reactive oxygen species lead to the release of adenosine triphosphate and uridine triphosphate in the extracellular fluid.…”

Rapid measurements of 2,8-dihydroxyadenine (2,8-DHA) with a nanostructured electrochemical sensor in 5-fold diluted supernatants of endothelial cells exposed to oxidative stress

“…Measurements of 2,8-DHA as a stress marker in cell supernatants are attractive because this metabolite has been found to be present in cells at much higher concentrations than other metabolites. 2 During normal cellular work in endothelial cells, 5 0 -methylthioadenosine (MTA), which is a byproduct of the polyamine synthesis pathway (Fig. 2), is degraded to adenine by 5 0 -methylthioadenosine phosphorylase (MTAP).…”

“…This compound is formed by pathways which control cell stress and has been identified recently in endothelial cells. 2 Although electrochemical sensors have not been tested previously in the direct screening of metabolites in cellular samples, 1 FSV with the N-CFS sensor has been applied to the screening of urine samples. 3 FSV measurements require only several seconds to complete [3][4][5] while, for example, separation-based approaches are significantly more time-consuming.…”

“…This noise reduction accounts for the increased S/N. 2 The resulting high sensitivity of FSV with the N-CFS permits dilution of biological samples to limit the effects of sample composition on sensor response. 3 Advantages of FSV measurements with a N-CFS in rapid screening of low molecular weight metabolites in biological fluids have been demonstrated.…”