A robust redox extraction protocol for quantitative and reproducible metabolite isolation and recovery has been developed for simultaneous measurement of nicotinamide adenine dinucleotide (NAD) and its reduced form, NADH, from Saccharomyces cerevisiae. Following culture in liquid media, yeast cells were harvested by centrifugation and then lysed under nonoxidizing conditions by bead blasting in ice-cold, nitrogen-saturated 50 mM ammonium acetate. To enable protein denaturation, ice cold nitrogen-saturated CH(3)CN/50 mM ammonium acetate (3:1 v/v) was added to the cell lysates. Chloroform extractions were performed on supernatants to remove organic solvent. Samples were lyophilized and resuspended in 50 mM ammonium acetate. NAD and NADH were separated by HPLC and quantified using UV-Vis absorbance detection. NAD and NADH levels were evaluated in yeast grown under normal (2% glucose) and calorie restricted (0.5% glucose) conditions. Results demonstrate that it is possible to perform a single preparation to reliably and robustly quantitate both NAD and NADH contents in the same sample. Robustness of the protocol suggests it will be (i) applicable to quantification of these metabolites in other cell cultures; and (ii) amenable to isotope labeling strategies to determine the relative contribution of specific metabolic pathways to total NAD and NADH levels in cell cultures.

/pdf/single-sample-extraction-protocol-for-the-quantification-of-1youdu3v0w.pdf

Single sample extraction protocol for the quantification of NAD and NADH redox states in Saccharomyces cerevisiae

Glassy carbon (GC) electrode is modified by stepwise manual casting of successive layers of ionic liquid crystals (ILC)/carbon nanotubes (CNTs) and magnetite nanoparticles. The composite [GC/(ILC-CNTs)/Fe 3 O 4 )] is used as a successful NADH biosensor. Molecular interaction of NADH is improved through the conductive and dense structure of ILC/CNTs. Magnetite nanoparticles act as electron-exchange “antennas” resulting in enhanced charge transfer at the interface, selective and sensitive oxidation of NADH. The type of ionic liquid used is key factor for sensor performance. The figures of merit for the proposed NADH sensor are: linear dynamic range of 5–700 μmol L −1 , a sensitivity of 0.0102 μA μmol −1  L, a detection limit of 34.6 nmol L −1 and a limit of quantification of 0.115 μmol L −1 . The sensor showed a stable amperometric response and anti-interfering ability in presence of ascorbic acid, tryptophan, ibuprofen and morphine.

A new strategy for NADH sensing using ionic liquid crystals-carbon nanotubes/nano-magnetite composite platform

Nicotinamide adenine dinucleotide (NAD{sup +}) is synthesized via two major pathways in prokaryotic and eukaryotic systems: the de novo biosynthesis pathway from tryptophan precursors, or by the salvage biosynthesis pathway from either extracellular nicotinic acid or various intracellular NAD{sup +} decomposition products. NAD{sup +} biosynthesis via the salvage pathway has been linked to an increase in yeast replicative lifespan under calorie restriction (CR). However, the relative contribution of each pathway to NAD{sup +} biosynthesis under both normal and CR conditions is not known. Here, we have performed lifespan, NAD{sup +} and NADH (the reduced form of NAD{sup +}) analyses on BY4742 wild type, NAD+ salvage pathway knockout (npt1{Delta}), and NAD+ de novo pathway knockout (qpt1{Delta}) yeast strains cultured in media containing either 2% glucose (normal growth) or 0.5% glucose (CR). We have utilized {sup 14}C labeled nicotinic acid in the culture media combined with HPLC speciation and both UV and {sup 14}C detection to quantitate the total amounts of NAD{sup +} and NADH and the amounts derived from the salvage pathway. We observe that wild type and qpt1{Delta} yeast exclusively utilize extracellular nicotinic acid for NAD{sup +} and NADH biosynthesis under both the 2% and 0.5% glucose growth conditions suggestingmore » that the de novo pathway plays little role if a functional salvage pathway is present. We also observe that NAD{sup +} concentrations decrease in all three strains under CR. However, unlike the wild type strain, NADH concentrations do not decrease and NAD{sup +}:NADH ratios do not increase under CR for either knockout strain. Lifespan analyses reveal that CR results in a lifespan increase of approximately 25% for the wild type and qpt1{Delta} strains, while no increase in lifespan is observed for the npt1{Delta} strain. In combination these data suggest that having a functional salvage pathway is more important than the absolute levels of NAD{sup +} or NADH for lifespan extension under CR.« less

/pdf/quantitation-of-nad-biosynthesis-from-the-salvage-pathway-in-19ehavf4pm.pdf

Quantitation of NAD+ biosynthesis from the salvage pathway in Saccharomyces cerevisiae.

A cavity ring-down spectroscopy (CRDS) instrument was developed using mature, robust hardware for the measurement of carbon-14 in biological studies. The system was characterized using carbon-14 elevated glucose samples and returned a linear response up to 387 times contemporary carbon-14 concentrations. Carbon-14 free and contemporary carbon-14 samples with varying carbon-13 concentrations were used to assess the method detection limit of approximately one-third contemporary carbon-14 levels. Sources of inaccuracies are presented and discussed, and the capability to measure carbon-14 in biological samples is demonstrated by comparing pharmacokinetics from carbon-14 dosed guinea pigs analyzed by both CRDS and accelerator mass spectrometry. The CRDS approach presented affords easy access to powerful carbon-14 tracer techniques that can characterize complex biochemical systems.

/pdf/quantifying-carbon-14-for-biology-using-cavity-ring-down-11uyckrool.pdf

Quantifying Carbon-14 for Biology Using Cavity Ring-Down Spectroscopy.

Accelerator mass spectrometry (AMS) is sometimes called ‘the art of counting atoms one by one’. In addition to counting individual atoms, AMS is also capable to determine both mass number (A) and a...

https://www.tandfonline.com/doi/pdf/10.1080/23746149.2016.1224603

Accelerator mass spectrometry: state of the art and perspectives

The publication rate of 3 H and 14 C use in biomedical research decreased by a factor of three since 1990 when the first applications of AMS in biomedicine were published. Against this decrease, the high sensitivity of AMS for these isotopes in small isolated samples has made significant contributions. New smaller spectrometers and increased commercial availability of AMS have solved some of the issues surrounding availability and cost, but improved quantitation in non-isotopic methods now compete with some early uses of AMS. We review the strength of AMS for quantifying rare biochemical events and chemical passages through individual people or cells and consider these as the frontiers of quantitation leading to profitable science unavailable to other techniques.

Biochemical paths in humans and cells: Frontiers of AMS bioanalysis

A robust redox extraction protocol for quantitative and reproducible metabolite isolation and recovery has been developed for simultaneous measurement of nicotinamide adenine dinucleotide (NAD) and its reduced form, NADH, from Saccharomyces cerevisiae. Following culture in liquid media, approximately 10{sup 8} yeast cells were harvested by centrifugation and then lysed under non-oxidizing conditions by bead blasting in ice-cold, nitrogen-saturated 50-mM ammonium acetate. To enable protein denaturation, ice cold nitrogen-saturated CH{sub 3}CN + 50-mM ammonium acetate (3:1; v:v) was added to the cell lysates. After sample centrifugation to pellet precipitated proteins, organic solvent removal was performed on supernatants by chloroform extraction. The remaining aqueous phase was dried and resuspended in 50-mM ammonium acetate. NAD and NADH were separated by HPLC and quantified using UV-VIS absorbance detection. Applicability of this procedure for quantifying NAD and NADH levels was evaluated by culturing yeast under normal (2% glucose) and calorie restricted (0.5% glucose) conditions. NAD and NADH contents are similar to previously reported levels in yeast obtained using enzymatic assays performed separately on acid (for NAD) and alkali (for NADH) extracts. Results demonstrate that it is possible to perform a single preparation to reliably and robustly quantitate both NAD and NADH contents in the same sample. Robustness of the protocol suggests it will be (1) applicable to quantification of these metabolites in mammalian and bacterial cell cultures; and (2) amenable to isotope labeling strategies to determine the relative contribution of specific metabolic pathways to total NAD and NADH levels in cell cultures.

/pdf/single-sample-extraction-and-hplc-processing-for-128x10i8r3.pdf

M.M. Kabir

Papers

Biochemical paths in humans and cells: Frontiers of AMS bioanalysis

Single sample extraction and HPLC processing for quantification of NAD and NADH levels in Saccharomyces cerevisiae