In their groundbreaking work, Arthur Harden and William John Young sought to learn more about how yeast perform fermentation. They tried to reproduce the process outside of the yeast cells. Using laboratory techniques, they were able to break open yeast cells and separate their contents into two fractions. One fraction was sensitive to heat, meaning that heat destroyed its ability to perform the fermentation reaction. The other fraction was not heat-sensitive.
By separating and then recombining the fractions, Harden and Young were able to show that the fermenting ability of the heat-sensitive fraction depended on the heat stable fraction. They surmised that the heat-sensitive fraction contained proteins responsible for fermentation, and the heat-stable fraction contained co-factors (like NAD molecules) and other stable molecules that helped the proteins perform the reactions.
Originally a student of art, Hans von Euler-Chelpin continued Harden and Young’s work by studying the details of the reactions that occur in the fermentation process. In this work, von Euler-Chelpin was able to further separate components of the heat-stable fraction of the yeast cells. In doing so, he purified the NAD molecule. von Euler-Chelpin is credited with uncovering the first information about the chemical shape and properties of the co-factor that allowed fermentation reactions to proceed.
Otto Heinrich Warburg studied chemistry fermentation reactions and discovered that NAD is needed for a certain type of chemical reaction, called a hydride transfer. Hydride transfer reactions involve the exchange of a hydrogen atom and its accompanying electrons. These types of reactions are essential to cellular metabolism and many other chemical processes that are required to sustain life. Warburg’s work showed that, in fermentation, the nicotinamide part of NAD+ accepts the hydride to become NADH and allows the reaction to proceed.
In the early 1900s, Pellagra was a common disease that caused symptoms such as diarrhea and dementia. Joseph Goldberger performed the initial experiments that identified Pellagra as a nutritional deficiency, but his experiments performed in humans were controversial. His later experiments crossed ethical lines by inducing pellagra in prison inmates by withholding certain nutritious foods from the diet.
Conrad Elvehjem furthered this work by performing controlled experiments in dogs. Elvehem noted that when dogs get pellagra, due to a poor diet, their tongues turn black. This model animal system allowed Elvehjem to give dogs different food extracts and see which ones helped dogs recover from the “black tongue” disease. Through careful purification of the food extracts, Elvehjem discovered that nicotinic acid cured pellagra, or “black tongue” disease, in dogs.
After Hans von Euler-Chelpin’s early purification of NAD and Conrad Elvehjem’s discovery of nicotinic acid as the nutrient that prevented pellagra, Arthur Kornberg studied the way that NAD is made in the body. By this time, methods for the purification of proteins and co-enzymes had advanced to the point that scientists could purify all the components they thought would be needed for a reaction. They could then test their theories by combining the purified components and looking for evidence that the reaction happened.
Kornberg purified the components needed for the NAD-generating reaction from yeast cells and combined them in an experimental set-up to demonstrate that they were responsible for creating NAD. His experiments were the first to demonstrate the chemical reaction cells use to create NAD from the precursor molecule nicotinamide mononucleotide (NMN).
Conrad Elvehjem showed that nicotinic acid was the agent that prevented pellagra, but Arthur Kornberg’s work only showed how nicotinamide mononucleotide, not nicotinic acid, was used by cells to create NAD. Jack Preiss and Philip Handler did work to uncover how nicotinic acid was converted to NAD. In their work, they showed that nicotinic acid is converted to NAD in three steps and identified the enzymes responsible for each. Today we refer to the “pathway” by which nicotinic acid is converted to NAD as the Preiss-Handler pathway.
Researchers up to this point had shown that NAD was important for fermentation in yeast and health in humans and animals. They had uncovered various ways in which NAD was generated in the cell. Although quite a bit was known about the ways that the NAD molecule was “built” in the cell, no research up until this point had shown a chemical reaction in which the NAD molecule was taken apart, or broken down into its component parts. Mandel’s work identified a reaction that broke NAD into two parts, nicotinamide and ADP-ribose.
Sirtuin enzymes were discovered in yeast for their exciting ability to extend their lifespan. Biochemical work dissecting how yeast sirtuins affect longevity led to the discovery that they use NAD to help keep certain genes in the cell “silent” so they cannot function. To do this, the sirtuin enzymes break NAD and use its components to “deacetylate” other proteins in the cell. Deacetylating histone proteins associated with DNA, for example, can change how the cell accesses nearby genes in the DNA.
Like the discoveries of nicotinic acid by Conrad Elvehjem and Preiss and Handler, Brenner and colleagues first identified a new precursor or building block of NAD and discovered the enzymes that eukaryotic cells use to convert that precursor to NAD. This work uncovered a two-step pathway by which nicotinamide riboside (NR) is converted to NAD. Follow-up work showed that feeding nicotinamide riboside to cells resulted in increased NAD levels and extended lifespan in yeast.
As researchers continue studying NAD, they also continue to broaden our understanding of this molecule and the many ways cells use and create it. Inspired by their experimental findings, scientists all over the world are actively investigating strategies for increasing NAD and its promising potential to support our health. Even in the consumer landscape, NAD supplementation is starting to trend, from IV therapy to utilization of NAD-boosting supplements. New studies examining the effects of NAD precursors such as NADH is publishing practically every week. Registered human trials at varying stages of completion also promise to help us more fully understand the potential for NADH and other precursors to impact human health.
