Adenine nucleotides (ANs)—adenosine 5′-triphosphate (ATP), adenosine 5′-diphosphate (ADP), and adenosine 5′-monophosphate (AMP)—are essential for energy transfer and the supply of countless processes within cellular metabolism. Their concentrations can be expressed as adenylate energy charge (AEC), a measure of cellular metabolic energy that directly correlates with the homeostasis of the organism. AEC index has broad diagnostic potential, as reduced ATP levels are associated to various conditions, such as inflammatory diseases, metabolic disorders, and cancer. We introduce a novel methodology for rapid isolation, purification, and quantification of ANs from a single drop of capillary blood. Of all the stationary phases tested, activated carbon proved to be the most efficient for the purification of adenine nucleotides, using an automated micro-solid phase extraction (µ-SPE) platform. An optimized µ-SPE method, coupled with RP-HPLC and a run time of 30 min, provides a reliable analytical framework for adenine nucleotide analysis of diverse biological samples. AN concentrations measured in capillary blood samples were 1393.1 µM, 254.8 µM, and 76.9 µM for ATP, ADP, and AMP molecules aligning with values reported in the literature. Overall, this study presents a streamlined and precise approach for analyzing ANs from microliters of blood, offering promising applications in clinical diagnostics.

Adenine nucleotides (AN): adenosine 5’-triphosphate (ATP), adenosine 5’-diphosphate (ADP), and adenosine 5’-monophosphate (AMP) are essential for energy transfer and supply of countless processes within the cellular metabolism. Their concentrations can be expressed as adenylate energy charge (AEC), a measure of cellular metabolic energy that directly correlates with the homeostasis of the organism. AEC index can be used as a clinical health indicator with broad diagnostic potential, as a decrease in ATP levels is associated with various conditions, such as inflammatory diseases, metabolic disorders, and cancer. We introduce a novel methodology for rapid isolation, purification, and quantification of AN from just a drop of capillary blood obtained from a healthy volunteer. Of all the stationary phases tested, activated carbon proved to be the most efficient for the purification of adenine nucleotides, using an automated micro-solid phase extraction (µ-SPE) platform. By removing interfering substances, we achieved high chromatographic selectivity and resolution, facilitating the quantification of AN. The analyzed AN concentrations from capillary blood correspond to the values found in the literature, which corroborates reliability of the developed extraction method. Overall, this study presents a streamlined and precise approach for analyzing AN from microliters of blood that offers promising applications in clinical diagnostics.

Autophagy is a fundamental intracellular pathway responsible for maintaining cellular homeostasis under both physiological and stress conditions. Through the degradation of damaged proteins and organelles and the recycling of intracellular material, it contributes to cellular adaptation and preservation of cellular function. In addition, autophagy interacts with multiple forms of regulated cell death (RCD) and influences processes associated with cell survival and cell death. These interactions involve signaling networks that respond to metabolic changes, oxidative damage, and environmental challenges. This chapter describes the molecular basis of autophagy and discusses its contribution to cellular function under physiological and stress conditions. Interactions between autophagy and RCD pathways, including apoptosis, ferroptosis, necroptosis, and pyroptosis, are also addressed. Cancer is discussed as an example that illustrates how the consequences of autophagy can vary according to biological context. While autophagy may restrict early tumor development, it can also support the survival and progression of established tumors. This chapter further considers the use of autophagy modulation in therapeutic strategies. Together, these topics provide an overview of the involvement of autophagy in cellular physiology, disease, and RCD.