Quantifying the cellular NAD+ metabolome using a tandem liquid chromatography mass spectrometry approach

Introduction

Nicotinamide adenine dinucleotide (NAD⁺) is an essential pyridine nucleotide that serves as a key hydride transfer coenzyme for several oxidoreductases. It is also the substrate for intracellular secondary messenger signalling by CD38 glycohydrolases, DNA repair by poly(adenosine diphosphate ribose) polymerase, and epigenetic regulation of gene expression by a class of histone deacetylase enzymes known as sirtuins. The measurement of NAD⁺ and its related metabolites (hereafter, the NAD⁺ metabolome) represents an important indicator of cellular function.

Objectives

A study was performed to develop a sensitive, selective, robust, reproducible, and rapid method for the concurrent quantitative determination of intracellular levels of the NAD⁺ metabolome in glial and oocyte cell extracts using liquid chromatography coupled to mass spectrometry (LC/MS/MS).

Methods

The metabolites were separated on a versatile amino column using a dual HILIC-RP gradient with heated electrospray (HESI) tandem mass spectrometry detection in mixed polarity multiple reaction monitoring mode.

Results

Quantification of 17 metabolites in the NAD⁺ metabolome in U251 human astroglioma cells could be achieved. Changes in NAD⁺ metabolism in U251 cell line, and murine oocytes under different culture conditions were also investigated.

Conclusion

This method can be used as a sensitive profiling tool, tailoring chromatography for metabolites that express significant pathophysiological changes in several disease conditions and is indispensable for targeted analysis.

     

Upregulation of Glycolytic Enzymes,Mitochondrial Dysfunction and Increased Cytotoxicity in Glial Cells Treated with Alzheimer’s Disease Plasma

Alzheimer’s disease (AD) is a neurodegenerative disorder associated with increased oxidative stress and neuroinflammation. Markers of increased protein, lipid and nucleic acid oxidation and reduced activities of antioxidant enzymes have been reported in AD plasma. Amyloid plaques in the AD brain elicit a range of reactive inflammatory responses including complement activation and acute phase reactions, which may also be reflected in plasma. Previous studies have shown that human AD plasma may be cytotoxic to cultured cells. We investigated the effect of pooled plasma (n = 20 each) from healthy controls, individuals with amnestic mild cognitive impairment (aMCI) and Alzheimer’s disease (AD) on cultured microglial cells. AD plasma and was found to significantly decrease cell viability and increase glycolytic flux in microglia compared to plasma from healthy controls. This effect was prevented by the heat inactivation of complement. Proteomic methods and isobaric tags (iTRAQ) found the expression level of complement and other acute phase proteins to be altered in MCI and AD plasma and an upregulation of key enzymes involved in the glycolysis pathway in cells exposed to AD plasma. Altered expression levels of acute phase reactants in AD plasma may alter the energy metabolism of glia.     

Effects of release method on the survival,somatic growth,and body condition of headstarted turtles

Headstarting involves ex situ rearing of vulnerable life stages, then releasing individuals into the wild once they are larger and less vulnerable to predation. Sometimes, headstarted animals display underdeveloped behaviors that may lead to an acclimation period of reduced survival and growth after release. Using data from a 6-year headstarting program, we tested whether the early release condition affected survival, body condition, and somatic growth rate in 2 groups of headstarted Blanding's turtles (Emydoidea blandingii) released into Rouge National Urban Park (RNUP) in Toronto, Ontario, Canada. The first group included turtles released directly into the wild (i.e., hard release). The second group included turtles released into an in situ enclosure in which individuals remained for a week without food supplementation before being fully released into the wild (i.e., delayed release). Release condition did not affect survival or growth rate. In the delayed-release group, body condition initially declined rapidly and remained low for up to 1 year after release. Given the lack of wild juveniles in RNUP, we compared body condition of headstarted turtles at various time points since release to similar-sized wild juveniles from 2 other Ontario populations, one from Algonquin Provincial Park (APP) and one near Lake Erie (LE). Body condition of headstarted turtles was similar to those of wild APP turtles regardless of release method, and higher than those of wild LE turtles. Our results indicate that delayed release did not improve post-release outcomes for headstarted turtles in an urban landscape and headstarted turtles sustain similar health metrics as wild turtles.