The NAD+/NADH Ratio and Aerobic Respiration

by CLRLY LLC August 19, 2024

Welcome Back!

I hope you guys have been able to enjoy brat summer for me, I’ve been too busy gatekeeping to find the time. But here we are, approaching the end of August, and school is back in session.

Over the next few blog posts, CLRLY enjoyers can expect the biggest doc drop to hit the health space since the ’23 liver detox archives were declassified. We will continue to distill the complexities of the metabolism into an understanding of the essentials, and in the process, you will come to know the simplicity of its Nature and master its function like never before.

Hop on the CLRLY magic school bus as we work our way backwards from the Radical Biology 101 blog post in which we covered oxidative phosphorylation, and let’s move on to examining the bigger picture. First up, the NAD+/NADH ratio and aerobic respiration.

Aerobic Glycolysis

Glucose (6-carbon chain) is taken into the cytoplasm of the cell and is split into two pyruvate molecules (two 3-carbon chains). This generates a net +2 ATP, +2 NADH.

Pyruvate is then transported into the mitochondria and converted into two acetyl-CoA molecules by the enzyme pyruvate dehydrogenase (PDH). This generates an additional +2 NADH.

Tricarboxylic Acid Cycle (TCA cycle, Kreb’s cycle, citric acid cycle)

The two acetyl-CoAs enter the TCA cycle and generate +6 NADH (as well as +2 FADH2 which are transported to Complex II and oxidized.

The +10 total NADH carry their electrons to Complex I of the ETC where they are oxidized and recycled back to NAD+ as they give up their electrons.

The electrons are transported through the ETC protein complexes to power the formation of a hydrogen ion gradient across the inner mitochondrial membrane used to generate ATP at Complex V.

How do you determine a cell's redox state?

NAD+ helps catalyze the above reactions by accepting electrons. When it gains electrons, it is reduced to NADH which then transports these electrons to Complex I, thus driving ATP production. The ratio of NAD+ to NADH is representative of the redox balance of the cell. Read that again. More NAD+ pushes the metabolism towards an oxidative state ie. faster metabolic rate. More NADH pushes the metabolism towards a reductive state ie. slower metabolic rate. There is always a large pool of NAD+ relative to NADH in the mitochondria-- usually about 10:1-- but even a moderate increase in NADH can impact the redox state.

How much ATP is produced in aerobic respiration?

Ideally, the cell is able to generate approximately 36 ATP total from the breakdown of one glucose molecule through aerobic respiration. However, we need efficient oxidation, or efficient use of oxygen, in order to produce the maximum number of ATP with the proper amount of oxidative stress. Achieving this requires optimally functioning oxidative phosphorylation (OXPHOS)— always the number one goal.

How does mitochondrial dysfunction lead to a slower metabolism?

As covered in the Radical Biology 101 blog post, oxidation of the mitochondrial protein complexes due to things like heavy metals and free iron/copper can impair protein function and damage the mitochondrial membrane. This slows down electron transport and causes electrons to “leak” from the ETC. Consequently, these leaked electrons can prematurely bind with oxygen and form the superoxide radical which contributes to oxidative stress, especially when undergoing the Fenton reaction with iron or copper in which the problematic hydroxyl radical is produced.

Up until now, we’ve primarily focused on the downstream effects of this happening such as diminished ATP production and mtDNA mutations, but now let’s look at what’s happening upstream of the ETC, specifically NADH accumulation.

When electron transport slows as a result of damaged ETC proteins (Complex I-IV), electrons “back up” and oxidation of NADH at Complex I decreases. When NADH can’t get rid of its electrons, it begins to accumulate, along with the upstream products acetyl-CoA and pyruvate. At the same time, NAD+ levels drop as it isn’t recycled back into the system, pyruvate dehydrogenase activity decreases and aerobic glycolysis begins to falter.

How does anaerobic activity impact NADH levels?

In addition to impaired ETC function, a hypoxic environment, such as that induced during intense exercise, slows down electron transport. When there isn’t adequate oxygen available to accept electrons at Complex IV, the final stage of the ETC, electrons again begin to back up and NADH accumulates. As aerobic respiration slows, the cell shifts over to anaerobic glycolysis to maintain ATP production.

Is obesity caused by too much NADH?

Lastly, NADH can accumulate as a result of overfeeding, or phrased another way, under activity. Diabetic/overweight individuals are an all-too-common example of this. Too much energy in, not enough energy out. The cell has plenty of electrons (NADH surplus) and nothing to do with them, so it begins storing this energy as fat via a process called de novo lipogenesis; ideal for a squirrel bulking before a harsh winter, not ideal for Aunt Karen living indoors at a year-round 70 degrees.

What is "reductive stress"?

I’m sure that there will soon be a scourge of influencers discussing the NAD+/NADH ratio who will make an effort to replace the term oxidative stress with “reductive stress”, a concept popularized by Brad Marshall who has done a lot of commendable research in this field. He examines things mostly from a diet/weight loss perspective, specifically excess PUFA intake, and attributes the cause of metabolic dysfunction to an excess of reduced products such as acetyl-CoA and NADH, while largely ignoring mineral imbalances and oxidation of the ETC proteins within the mitochondria. It’s too much to unpack in this post, but I think that though an individual’s metabolic state should be taken into consideration when assessing the proper treatment strategy, elevated NADH is not the root cause of oxidative stress but rather a symptom of it.

Conclusion

The takeaways here: a high NAD+ to NADH ratio is essential for a fast metabolic rate— what many here are seeking. When we have a high NAD+/NADH ratio and OXPHOS is optimized, we efficiently use oxygen to derive the maximum amount of ATP from glucose through aerobic respiration. In situations where the ETC is damaged, where there isn’t enough oxygen, or when the ATP pool is full, NADH accumulates and OXPHOS begins to slow down. This leads to an accumulation of reduced products, referred to as reductive stress. As this progresses, the metabolism shifts towards glycolysis in an effort to maintain ATP production... and an unsung hero takes center stage.

To be continued...