NAD/NADH Ratio - The One Metabolic Cause To Rule Them All

[maillol]

Inhibition of mitochondrial pyruvate dehydrogenase kinase: a proposed mechanism by which melatonin causes cancer cells to overcome cytosolic glycolysis, reduce tumor biomass and reverse insensitivity to chemotherapy | Request PDF

Not suggesting taking melatonin I just read this and thought it was a good confirmation of what haidut has said.

 

[Highserotonin90]

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[Highserotonin90]

@haidut To date, the only things that can (partially) inhibit glycolysis are turmeric and olive leaves?

 

[Highserotonin90]

@haidut Can the enzyme deficiency of the fumarase gene in the Krebs cycle be bypassed?

 

[Recoen]

Nice post. It's nice to see the Warburg Effect given context. And you brought-up pyruvate decarboxylase.
And this makes me want to rant about the Breslow Mechanism. This is just a minor detail in the grand scheme of things, but it's important in that it highlights how false paradigms can have inertia; there's serious reluctance to change it despite obvious errors. Authoritarianism plays an important role; especially when considering things that cannot even be seen directly – like molecules. Explanations printed in the shiniest books are usually considered to be the truth.

Doctor Alan J. Knell's 1970 doctroral dissertation† provides a 144-page β-slap of Breslow's mechanism for how exactly this enzyme works – for how thiamine removes carbon dioxide from pyruvate.


And he goes into great detail on all of his points.

The Breslow Mechanism in rarely drawn with the entire thiamine molecule. This is done partly to save space and time; but also, the rest of the thiamine molecule actually gets in the way if you try to do this. Knell mentioned in point (a) that it was impossible to build models ― Honestly, it's impossible to even draw.
View attachment 6798
And you would think that Breslow intermediate #3 would be practically irreversible, since this is a carbon-carbon double bond. Knell mentions that "attempts to prepare the pyruvate adduct itself were unsuccessful." So intermediate #2 might not have ever been made, anywhere. Not even inside the enzyme. And even if it had, #3 wouldn't easily be converted back to thiamine like it needs to be. Instead, Knell does one better:
View attachment 6799
The active coenzyme is actually though to be thiamine's isomer, xantho-thiamine.
View attachment 6800
Pyruvate is drawn here with partial charges; the ketone is polarized as usual. The sulfur nucleophilicly-attacks the carbonyl carbon of pyruvate. There is absolutely nothing unusual about such an interaction; this happens all of the time.
View attachment 6813
The σ-bond is free to rotate. [S-linked-pyruvate shown migrating towards pyrimidine rings]
View attachment 6804
It forms a transient bond as electrons are drawn through the heterocycle by the positively-charged histidine* side-chains of the enzyme. There are two of them in close proximity to the pyrimidine rings. (Electrons need to be removed from pyruvate to decarboxlate it to acetyl-.) The ring structure is electronically-conjugated with an extensive π-bond system, so electrons are free to move through it. Conjugated double-bond systems are much more conductive than saturated carbon chains with sigma bonds. Graphite is a semiconductor (~10⁻⁵Ωm) while diamond is not (~10¹¹Ωm). They are both pure carbon.

Another nice thing about this is that you end with an S-linked acetyl group, which is how they're usually shuttled around in the body by coenzyme A and others. It could be thought of as S-acetylthiamine, which transfers to CoA (forming acetyl-CoA) through the second complex (E2) of pyruvate decarboxylase by an enzyme-linked lipoic acid prosthetic group(s). This process also directly involves sulfur atoms; lipoic acid's ring opens to create primary thiols, and these are what bond with the acetyl group in transit to CoA.


Since Knell's paper other interesting things have appeared, and completely overlooked, which support him. Take this 1998 X-ray crystallographic study done on pyruvate decarboxylase and thiamine:
View attachment 6805‡
It shows an open-ring configuration; this is similar to the Knell configuration. The C2 carbon, Breslow's magic carbon that attacks pyruvate, between the nitrogen and sulfur of the thiazole ring is conspicuously absent. Thiamine cannot possible function in the way that Breslow imagined without the C2 carbon. It is so essential to Breslow's mechanism that it may-as-well be called the Breslow Carbon. The authors seem a bit puzzled by this:
Since X-rays were used to image the enzyme – and the thiamine molecule – the researchers just brushed-this-off as radiation damage.

And as late as 2009, the same thing was imaged by and entirely different research group of chemists. They were imaging a different enzyme, but this too was a thiamine-dependent decarboxylase.§
View attachment 6806(click to embiggen)
Again: No electron density where the elusive Breslow carbon should be, but they don't seem to be bothered by this.


Ronald Breslow was president of the American Chemical Society, so most people are careful not to offend him by telling him he's wrong. But he probably is wrong. Alan Knell's explanation is much more theoretically plausible, and is more in accord with experimental observations. This reminds me of Gilbert Ling.

The Breslow Mechanism should should be treated accordingly by being placed next to the membrane pump, the George Wald theory of retinal phototransduction, and the Huxley Crossbridge Theory folders in the filing cabinet; under the section marked "Unicorns."

Unicorn Folder

Dean, Robert B. "Theories of electrolyte equilibrium in muscle." Biol. Symp. Vol. 3. 1941.
Wald, George, Paul K. Brown, and Ian R. Gibbons. "The problem of visual excitation." JOSA 53.1 (1963): 20-35.
Huxley, Andrew F., and Ro M. Simmons. "Proposed mechanism of force generation in striated muscle." Nature 233.5321 (1971): 533-538.
Breslow, Ronald. "On the mechanism of thiamine action. IV. 1 Evidence from studies on model systems." Journal of the American Chemical Society 80.14 (1958): 3719-3726. ​

It's a minor detail, but is highlights a major problem in science. All of the textbooks beat to the same drum, they all have this mechanism; and they all basically just plagiarize each-other. It seems as thought once a wrong idea actually becomes firmly established it will not be corrected by the same people who had created it, or had accepted it, for fear of embarrassment.


*Histidine414 and Histidine114 (see Dobritzsch‡)
*To save space, the squiggly line depicts the pyrophosphate side-chain of thiamine pyrophosphate.
†Knell, Alan John. Thiamine: a study of its chemistry, biochemistry and mechanism of action. Diss. University of Warwick, 1970.
‡Dobritzsch, Doreen, et al. "High Resolution Crystal Structure of Pyruvate Decarboxylase from Zymomonas mobilis." Journal of Biological Chemistry 273.32 (1998): 20196-20204.
§Chakraborty, Sumit, et al. "Detection and time course of formation of major thiamin diphosphate-bound covalent intermediates..." Biochemistry 48.5 (2009): 981-994.

I know this is an old post, but isn’t pyruvate decarboxylase biotin and Mn dependent? Pyruvate dehydrogenase is B1 and Mg dependent.

 

[Highserotonin90]

If, on the other hand, the pyruvate was low, is a low NAD / NADH ratio always the cause?

 

[Highserotonin90]

No, 4 would be the worse, you want a high pyruvate/lactate ratio. So, 1 would be best and potentially 3, while 2 would potentially mean ketosis or some sort of glycolysis disorder while 4 would be highly reduced state and metabolic acidosis.

What does it mean if it's number two?

 

[Peater Pan]

Since B2 riboflavin at higher doses has totally transformed my body's functioning in the last two months, I would like to throw out there the idea that if the B1, B3 etc program discussed in this thread has not yielded the benefits expected, one might add 100 to 400mg B2 to the mix.

It's only logical to think that B2 is also integral to the processes discussed herein.

I remain shocked and astonished at what I can do today compared to what I have never ever been able to conceive of doing only about 2 months ago.

What was your dosing protocol?

 

[GAF]

400mg daily for a long long time. No negative side effects.

 

[dukesbobby777]

400mg daily for a long long time. No negative side effects.

Not even insomnia? You mentioned it energizing you to ultra human levels. Usually such things come with being unable to sleep at night as a side effect.

 

[GAF]

I don't think I ever said ultra human. You should realize my normal operating capacity is probably 50% of average so something that helps me a ton might not be noticed by a semi normal person.

I have taken it at night just as an experiment and I slept normal, for me. Sometimes I took it just before dancing or other exertion in the early evening. Mostly, I take before noon. It's not a stimulant.

In my case it's stamina and breathing and quickness and pain reducing due to better overall body functioning.

 

[Kray]

I don't want to derail this great thread with B2 but here is an interesting discussion of B2 and what happens to deficient pigs. If you take all those deficiency issues and turn them around, that can answer your question. I have been taking plain B2 daily anywhere from 250 to 800mg per day. I take an afternoon dose when I need to keep my body really powered up like a few days ago when I was involved in a move. It was incredible what I was able to do compared to what I could do just a two or so months ago. I theorize that most connective tissue disorders are nothing but riboflavin deficiencies, and that is just one thing it does. I also theorize that B2 serves as sunshine inside your body and once you saturate your blood and extracellular fluid compartment/tissues with it, outside UV rays interact with riboflavin inside and good stuff happens, like cleaning out of pathogens. I can't prove anything. I am just a CPA that goes dancing at night for fun.
https://www.dsm.com/markets/anh/en_US/Compendium/swine/riboflavin.html

B2 has been the only thing added to supplements I was taking long before like thiamine, niacinamide, B6, cordyceps, so I am 100% sure the massive increase in functioning is due to the B2 plus whatever it does to make other supplements and foods work better. Amazon product ASIN B012YIVRM0

@GAF
I am interested in your opinion on the best form of B2? I have the r5P but have not taken it apart from what I get in Energin. Just figuring out the importance of B2 in general, connecting the dots for some of my problems and why I might be struggling in some areas. If you can explain the pros and cons of both form- B2 and r5P- and a reasonable dose for both each (since r5P is the activated form, not sure if the 10mg recommended on the forum refers to plain B2 or r5P), I would really appreciate your help. Thank you!

 

[GAF]

Kray meister.

I have a swallowed a huge amount of both and the effects on my body were the same.

Granted, the I ate a ton of riboflavin for a long time before I ate a ton of R5P, so whatever that might mean.

It's all harmless, if your body needs it. My body just needs boosters now and then nowadays.

I let my instincts determine my B2 needs these days.

 

[PeatBull]

Would be good idea to supplement with NAD?

 

[ecstatichamster]

niacinamide 50mg - 100mg twice a day raises NAD to NADH ratio.

 

[Motorneuron]

I am awaiting a diagnosis of ALS, I tried NADH at the suggestion of the doctor and just one mg aggravated the symptoms. What does this mean?

 

[Jam]

I am awaiting a diagnosis of ALS, I tried NADH at the suggestion of the doctor and just one mg aggravated the symptoms. What does this mean?

You want to increase NAD+, not NADH.

Evaluation of the NAD+ biosynthetic pathway in ALS patients and effect of modulating NAD+ levels in hSOD1-linked ALS mouse models

Amyotrophic lateral sclerosis (ALS) is characterized by progressive degeneration of motor neurons. Astrocytes from diverse ALS models induce motor neu…

www.sciencedirect.com

 

[Motorneuron]

You want to increase NAD+, not NADH.

Evaluation of the NAD+ biosynthetic pathway in ALS patients and effect of modulating NAD+ levels in hSOD1-linked ALS mouse models

Amyotrophic lateral sclerosis (ALS) is characterized by progressive degeneration of motor neurons. Astrocytes from diverse ALS models induce motor neu…

www.sciencedirect.com

Thanks, it's weird because niacinamide also makes it worse.

 

[Jam]

Thanks, it's weird because niacinamide also makes it worse.

Niacinamide makes what exactly worse?

If your NAD+ salvage pathway is impaired , niacinamide (NAM) won't do much. Worst case, none of the B3's will do much at all for increasing NAD+.

However, there are other ways to increase NAD+. For example, quinones such as beta-lapachone (from Pau D'Arco) are known to increase the NAD+/NADH ratio even in the presence of a defective NAD+ salvage pathway. Have you tried Lapodin or Pau D'Arco?

 

[SaltiestSardine]

If using a source of Pau D'Arco is an alcohol extraction superior to a glycerite? I see lots of companies claiming the latter but the studies say otherwise.i have a feeling a lot of the glycerite tinctures claims of superiority are mostly about people who have reservations of taking alchohol, no matter how little quantity