Determination of acetyl coenzyme A in human whole blood by ultra-performance liquid chromatography-mass spectrometry

Acetyl coenzyme A is involved in several key metabolic pathways. Its concentration can vary considerably in response to physiological or pathological conditions making it a potentially valuable biomarker. However, little information about the measurement and concentration of acetyl CoA in human whole blood is found in the literature. The aim of this study was the development of an accurate method for the determination of acetyl CoA in human whole blood by LC-MS/MS. The method, involving extraction from whole blood by a rapid protein precipitation procedure was thoroughly validated: limit of quantitation was 3.91 ng mL-1. Accuracy and precision were calculated at five concentrations and were within ±15%. The average endogenous level of acetyl CoA in human whole blood was determined in 17 healthy individuals to be 220.9 ng mL-1 (ranging from 124.0 to 308.0 ng mL-1). This represents, to our knowledge, the first report of acetyl CoA levels in human whole blood, and the first practical and reliable method for its determination

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field