Removal of Polysorbate 80 by complexation prior to LC-MS analysis.

The presence of Polysorbate 80 in samples can challenge liquid chromatography-electrospray ionisation mass spectrometry (LC-ESI-MS) analysis as it is easily ionised and detected. In this study, we demonstrate that interference from Polysorbate 80 can be reduced by complexation with a metal ion followed by precipitation by thiocyanate. The precipitation procedure was tested on a mixture of low molecular weight compounds (e.g. amino acids and non-amino organic acids) and it was shown that none of the tested compounds were precipitated.

NAD+ regulates nucleotide metabolism and genomic DNA replication.

The intricate orchestration of enzymatic activities involving nicotinamide adenine dinucleotide (NAD+) is essential for maintaining metabolic homeostasis and preserving genomic integrity. As a co-enzyme, NAD+ plays a key role in regulating metabolic pathways, such as glycolysis and Kreb's cycle. ADP-ribosyltransferases (PARPs) and sirtuins rely on NAD+ to mediate post-translational modifications of target proteins. The activation of PARP1 in response to DNA breaks leads to rapid depletion of cellular NAD+ compromising cell viability. Therefore, the levels of NAD+ must be tightly regulated. Here we show that exogenous NAD+, but not its precursors, has a direct effect on mitochondrial activity. Short-term incubation with NAD+ boosts Kreb's cycle and the electron transport chain and enhances pyrimidine biosynthesis. Extended incubation with NAD+ results in depletion of pyrimidines, accumulation of purines, activation of the replication stress response and cell cycle arrest. Moreover, a combination of NAD+ and 5-fluorouridine selectively kills cancer cells that rely on de novo pyrimidine synthesis. We propose an integrated model of how NAD+ regulates nucleotide metabolism, with relevance to healthspan, ageing and cancer therapy.