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Luminescent Assays to detect NADH/NADPH

NAD+: It's Not Just Metabolism Anymore

IMG_8764 Michele Arduengo
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When I was an undergraduate, I don’t remember just how many courses included several days spent poring over the Krebs Cycle, counting carbons and following NAD (P)+/NAD(P)H as we winded our way, converting  the energy from sugar into redox potential for the cell. Then we did it again—a couple of times—in graduate school. When I was teaching undergraduates, my students and I acted like sleuths once again, following those carbons through the Krebs cycle then following the protons into the electron transport system. NAD(P)+/NAD(P)H were the molecules of redox potential, the story of energy in oxidative phosphorylation, the way you got to ATP.

In other classes, completely separate from the study of metabolism, was the study of cell signaling. We looked at MAP kinases, JAK/STAT pathways, G proteins, and steroid receptors–studying how signals from the environment, other cells and even from within cells were translated into the cellular activities of protein synthesis, protein degradation, cell division and even cell death. Cell signaling and metabolism were separate subjects. They did not collide.

Many things have happened in the biological sciences since I completed my graduate studies: Dolly the sheep, the sequencing of the human genome, the isolation and culture of human stem cells in the lab, and a fundamental change in our understanding of the role that nicotinamide adenine dinucleotides  (NAD(P)/NAD(P)H) play in the cell. The first items made for sensational headlines, but the final one is having a lasting impact on cardiology, cancer research and even our understanding of aging. It turns out nicotinamide adenine dinucleotides are key in linking metabolism to other activities in the cell such as transcriptional control of many genes, DNA damage and repair and interaction with and regulation of cell signaling networks (for a review of some of these, see the introduction to this PubHub article).

Because of the diverse roles that the nicotinamide adenine dicnucleotides play in cellular regulation, gene expression and disease, biologists are now looking at these molecules as indicators of drug mechanism of action or treatment toxicities. For instance, like ATP, NAD(P) and NAD(P)H are key metabolites and good indicators of the metabolic health of cells,Measurement of total NAD +NADH or NADP + NADPH dinucleotides can provide researchers with an idea of the metabolic health of a cellular population.

Additionally because of their roles in cell signaling and in tying metabolism to signaling networks within the cell, these molecules are becoming more important in drug discovery research efforts. Researchers are increasingly performing inhibitor screens with purified enzymes that consume or produce these dinucleotides.

Finally, dysregulation of metabolism during disease states can shift the oxidative state of cells as a result of the change in the ratio of oxidized and reduced forms of nicotinamide adenine dinucleotides, being able to detect such a shift by measuring the individual oxidized and reduced dinucleotide forms is useful.

We have developed three assays that allow the investigation of the role of the nicatinamide dinucleotides in cell health and metabolism: the NAD(P)H-Glo™ Detection System, the NAD/NADH-Glo™ Assay, and the NADP/NADPH-Glo™ Assay. We have two articles about these assays, one describing the technology and assay chemistry and a second discussing the application of the assays to research questions. You can read each of the articles by following the links below.

Part I: Technology and Features Part II: Choosing the Right Product for Your Application

This family of assays provides robust, simple and flexible tools for cellular and biochemical analysis of nicotinamide adenine dinucleotides for many applications. Measure purified enzymes, perform rapid screens for inhibitors or activators of NAD-dependent enzymes, assess the metabolic health of your cells, and study the dysregulation of metabolic pathways.