LONGECITY

Great to see results with actual numbers. Interesting that you improved further on the new version of the protocol, even after presumably being on the old one for a while.

Was there any particular reason for choosing AKG over AAKG, and GMS over Sulforaphane?

I still think it could be enlightening to look at before-and-after organic acids tests. That should be a quantitative way of seeing changes in mitochondrial health.

For example, here are the Krebs Cycle metabolites from a typical organic acids test (happens to be my results, but I regularly see similar patterns for others):

 Capture.JPG   37.7KB   0 downloads

Here's the order of the cycle, along with the test results:

High: Citric

Borderline Low: Acotonic

Not measured: Isocitric 

Borderline High: 2-Oxoglutaric (also known as Alpha-Ketoglutaric)

Not measured: Succinyl-CoA

High: Succinic

High: Fumaric

Normal: Malic

Not measured: Oxaloacetic

High: Citric

The questions someone with a biochemistry background should be asking are:

1. If Succinic and Fumaric are High, why is Malic Normal, since it's next in the sequence? The conversion only requires Fumarase and Water.

2. If Citric is High, why is Acotinic Borderline Low, since it's next in the sequence? The conversion only requires Acotinase (and maybe iron, IIRC).

The usual Functional Medicine answer to those questions is to look at intermediates such as minerals and the inputs needed to make the associated enzymes, and ignore both epigenetics and mutated / non-functional mtDNA strands. It seems much more reasonable to me that those latter areas are where the real problems are. If so, then the fission/fusion protocol here should clean up the test results considerably (meaning no more highs or lows).

An updated Mito protocol

 

The previous protocol can be found at post #1366

 

 

Background:

 

Previously I posted methods of cycling mitochondrial morphology to clean up defective mtDNA, which eliminated mutations via the PINK1/Parkin QC process. The normal QC process can detect mutated mtDNA genes during fission as all mito genes are critical and thus the mito membrane potential goes to zero if just one is defective. Greatly magnifying fission and fusion with supplements will aid that process. But there is another source of mitochondrial damage that isn’t so easily eliminated — epigenetic damage. Like nDNA, mtDNA also picks up aberrant methylation with age. This methylation degrades ATP production, but the QC process doesn’t catch it unless the problem is addressed at a critical time, like during biogenesis. If a mitochondrion with one loop of methylated mtDNA runs out of enzymes while involved with replication, then membrane potential may dip to zero and it will get labeled for recycling. Thanks to methylation, it won’t have as much enzyme reserves as other mitochondria, so it will be preferentially targeted. Also, biogenesis is the best time to demethylate mtDNA as methyltransferase can’t operate while there is only one strand.

 

Until recently, mtDNA wasn’t even known to have methylation, and researchers are still confused as to why it is there. Some speak of mtDNA hypermethylation like it is bad while normal methylation has some purpose.

 

See, for instance: Hypermethylation of mitochondrial DNA in vascular smooth muscle cells impairs cell contractility

 

I don’t agree. I say all mtDNA methylation is bad. Methylated mtDNA mooches enzymes off other mtDNA, and because they don’t produce as much ATP they don’t produce as much ROS, and thus have a survival advantage as they are less prone to mutation. Eventually the cell will become full of moochers and result in fatigue and many other problems of aging.

 

So I say get rid of them all, mutations and methylation alike.

 

The new protocol:

 

This new procedure is much simplified. It requires only two doses, Mito1 and Mito2, which are alternated on a daily basis.

 

Mito1 (fission)

● NAM+R, 1 g of each

● AKG, 1 g

● PQQ, 20 mg

 

Mito2 (fusion)

● GMS, 1 g

● AKG, 1 g

● PQQ, 20 mg

 

NAM+R (nicotinamide plus ribose) is a fission promoter, GMS (glycerol monostearate) is a fusion promoter, AKG (alpha-ketoglutarate) is a demethylase promoter, and PQQ is a biogenesis promoter. All of these are fast acting.

 

A two week experiment using reps to failure:

 

Warm water was sufficient to dissolve everything, but the PQQ was taken in a capsule to insure that the other ingredients got a slight head start (probably unnecessary).

 

Mito1 and Mito2 were taken on alternating days. Each dose was taken in the evening and reps of dumbbell curls to failure counted first thing in the morning — five or six hours after dosing — using the same arm.

 

My hypothesis was that the number of reps would reflect mito damage. With mito fusion, enzymes are shared, thus ATP production and reps would be maximum. With fission, methylated (or otherwise damaged) mtDNA produce less ATP and reps would be minimum. The difference would reflect average damage, and if the treatment worked, the difference should decline. If all damage was removed, then the difference should go to zero.

 

Which in fact it did. See the plot below. The y-axis shows the reps and % difference, while the x-axis shows days. The curve labeled baseline is without any treatment, and likely reflects the normal intermediate situation with mito morphology in a dynamic state. It is stable at 16 reps. The upper fusion curve is relatively flat and higher than baseline as expected, while the lower fission curve is lower than baseline, but rises to meet the fusion curve after about two weeks, and stays there. Thus the percent difference goes to zero.

 

Results:

 

Improvement in running endurance, reduced hunger, and reduced need for hypertension medication.

Turnbuckle do you have any idea on how long the 2 week protocol will last before repeating?  Thanks

 

 

The new protocol:

 

This new procedure is much simplified. It requires only two doses, Mito1 and Mito2, which are alternated on a daily basis.

 

Mito1 (fission)

● NAM+R, 1 g of each

● AKG, 1 g

● PQQ, 20 mg

 

Mito2 (fusion)

● GMS, 1 g

● AKG, 1 g

● PQQ, 20 mg

 

NAM+R (nicotinamide plus ribose) is a fission promoter, GMS (glycerol monostearate) is a fusion promoter, AKG (alpha-ketoglutarate) is a demethylase promoter, and PQQ is a biogenesis promoter. All of these are fast acting.

 

 

Very interesting. Are you now using an AKG salt rather than arginine-AKG? 

Since we know AKG can reduce epigenetic age on its own, are you going to drop your stem cell stimulus protocol for a while and see what epigenetic age you get from just using the above?

Great to see results with actual numbers. Interesting that you improved further on the new version of the protocol, even after presumably being on the old one for a while.

 

Was there any particular reason for choosing AKG over AAKG, and GMS over Sulforaphane?

 

I still think it could be enlightening to look at before-and-after organic acids tests. That should be a quantitative way of seeing changes in mitochondrial health.

I had same questions in my mind. I'd like to add more for Turnbuckle, although I know he has previously ignored my questions around taking higher doses than he prescribed, perhaps that was a silly question. But after having read the entire thread, let me see if this makes sense:

Mito1 (fission)

● NAM+R, 1 g of each  - this can be replaced with Tru Niagen (300mg x 8) capsules

● AKG, 1 g -  this can be substituted with AAKG. Perhaps Arginine part of AAKG is not necessary but having it won't interfere, body already has Arginine

● PQQ, 20 mg - not sure if this has a substitute but this is easy enough to find

Mito2 (fusion)

● GMS, 1 g - Stearic Acid, Mango Butter are suitable substitutes, it seems. Sulphrophane may be added because it crosses blood-brain-barrier but is not necessary. I'd add Sulphrophrane though.

● AKG, 1 g - AAKG is fine, see above.

● PQQ, 20 mg - fine.

@Turnbuckle - thank you so much for your work. As I've said before, I've been suffering from Post Finasteride Syndrome and recent age test showed my biological (DNA) age to be 42 (I'm 37). I truly believe following both your protocols to reverse epigenetic is going to cure me of many of the symptoms, if not completely reverse. I haven't even started recovery yet but just having a plan of action is what is keeping me going and optimistic that eventually I may regain my health. Thanks to you and your work for this glimmer of hope.

Very interesting. Are you now using an AKG salt rather than arginine-AKG? 

 

Since we know AKG can reduce epigenetic age on its own, are you going to drop your stem cell stimulus protocol for a while and see what epigenetic age you get from just using the above?

No, I didn't use a salt. Not that salts or other AKG derivatives wouldn't work, but I wanted the fastest acting form for this trial, as I had only one shot at it. And while AKG might be short acting, I saw that as perfect.

And no, I don't believe AKG or its derivatives will prove that useful when used alone.

Adding AKG or AAKG to my SC protocol almost doubled my epigenetic age result, which is now two decades below chronological. This didn't require constant dosing, only during the protocol. And increasing the pools of stem cells is far more useful than what AKG alone can do, which is likely reducing the epigenetic age of TACs -- the rapidly dividing intermediates between SCs (in at least some organs) and somatic cells. So while no treatment is permanent -- as aging marches on when you stop -- intervening at that intermediate level cannot be expected to be the most long lasting. When AKG is used during SC proliferation, however, the result will be much longer lasting, as SCs are a step above TACs in the cellular hierarchy. And as SC pools are magnified, so will the epigenetic effects of demethylase.

 

Mito1 (fission)

● NAM+R, 1 g of each  - this can be replaced with Tru Niagen (300mg x 8) capsules

● AKG, 1 g -  this can be substituted with AAKG. Perhaps Arginine part of AAKG is not necessary but having it won't interfere, body already has Arginine

● PQQ, 20 mg - not sure if this has a substitute but this is easy enough to find

 

Mito2 (fusion)

● GMS, 1 g - Stearic Acid, Mango Butter are suitable substitutes, it seems. Sulphrophane may be added because it crosses blood-brain-barrier but is not necessary. I'd add Sulphrophrane though.

● AKG, 1 g - AAKG is fine, see above.

● PQQ, 20 mg - fine.

Tru Niagen is not an option as nicotinamide ribose has to be digested before it becomes effective. This produces an unacceptable time delay of hours, while mitochondria biogenesis is very fast. If you want an option, try niacin, which will raise NAD+ even faster than nicotinamide, though the average person will not find the flush acceptable, and it can cause acid stomach.

See Fig. 5-b of this paper, and you will understand the problem with Tru Niagen (nicotinamide riboside).

As for food grade stearic acid and mango butter, they have the same problem with digestion. They won't act fast enough for a unitary dose.

As for AAKG, that may also have a problem with speed. It certainly won't be as fast as AKG. You will also have to use 4 times as much due to its higher MW.

Tru Niagen is not an option as nicotinamide ribose has to be digested before it becomes effective. This produces an unacceptable time delay of hours, while mitochondria biogenesis is very fast. If you want an option, try niacin, which will raise NAD+ even faster than nicotinamide, though the average person will not find the flush acceptable, and it can cause acid stomach.

 

See Fig. 5-b of this paper, and you will understand the problem with Tru Niagen (nicotinamide riboside).

 

As for food grade stearic acid and mango butter, they have the same problem with digestion. They won't act fast enough for a unitary dose.

 

As for AAKG, that may also have a problem with speed. It certainly won't be as fast as AKG. You will also have to use 4 times as much due to its higher MW.

Thanks! The only brand I could find of AKG in Poland is this (Kirkman Alpha Ketoglutric Acid 300mg + 20mg of Calcium AKG + 10mg of Magnesium AKG). Would this do? If not, I'd have to see how to import from the USA. Ever since I moved to Europe (last month), finding supplements has been a pain.

It is spelled Ketoglutaric Acid not Ketoglutarate though. And description says, it is a buffered product. If it is the same thing, I'd take empty out the capsule and take this with water. Capsule might add extra few minutes to digestion speed.

https://pl.iherb.com...-capsules/58317

Thanks! The only brand I could find of AKG in Poland is this (Kirkman Alpha Ketoglutric Acid 300mg + 20mg of Calcium AKG + 10mg of Magnesium AKG). Would this do? If not, I'd have to see how to import from the USA. Ever since I moved to Europe (last month), finding supplements has been a pain.

It is spelled Ketoglutaric Acid not Ketoglutarate though. And description says, it is a buffered product. If it is the same thing, I'd take empty out the capsule and take this with water. Capsule might add extra few minutes to digestion speed.

https://pl.iherb.com...-capsules/58317

https://www.amazon.e...00MNNSU9U&psc=1

https://www.amazon.e...00MNNSU9U&psc=1

That's AAKG. Turnbuckle pointed that this would digest too slow for this protocol.

That's AAKG. Turnbuckle pointed that this would digest too slow for this protocol.

Adding AKG or AAKG to my SC protocol almost doubled my epigenetic age result, which is now two decades below chronological. Exact words from Turnbuckle.  It works, just not as fast, and AAKG doubled his epigenetic age

Adding AKG or AAKG to my SC protocol almost doubled my epigenetic age result, which is now two decades below chronological. Exact words from Turnbuckle.  It works, just not as fast, and AAKG doubled his epigenetic age

That comment was relevant to the SC protocol, not to this one. Mito biogenesis is much faster. AAKG might work with a unitary mito treatment, but it might be too slow. There's no guarantee. And SC didn't double my epigenetic age, rather, it almost doubled the decrease.

An updated Mito protocol

 

The previous protocol can be found at post #1366

 

 

Background:

 

Previously I posted methods of cycling mitochondrial morphology to clean up defective mtDNA, which eliminated mutations via the PINK1/Parkin QC process. The normal QC process can detect mutated mtDNA genes during fission as all mito genes are critical and thus the mito membrane potential goes to zero if just one is defective. Greatly magnifying fission and fusion with supplements will aid that process. But there is another source of mitochondrial damage that isn’t so easily eliminated — epigenetic damage. Like nDNA, mtDNA also picks up aberrant methylation with age. This methylation degrades ATP production, but the QC process doesn’t catch it unless the problem is addressed at a critical time, like during biogenesis. If a mitochondrion with one loop of methylated mtDNA runs out of enzymes while involved with replication, then membrane potential may dip to zero and it will get labeled for recycling. Thanks to methylation, it won’t have as much enzyme reserves as other mitochondria, so it will be preferentially targeted. Also, biogenesis is the best time to demethylate mtDNA as methyltransferase can’t operate while there is only one strand.

 

Until recently, mtDNA wasn’t even known to have methylation, and researchers are still confused as to why it is there. Some speak of mtDNA hypermethylation like it is bad while normal methylation has some purpose.

 

See, for instance: Hypermethylation of mitochondrial DNA in vascular smooth muscle cells impairs cell contractility

 

I don’t agree. I say all mtDNA methylation is bad. Methylated mtDNA mooches enzymes off other mtDNA, and because they don’t produce as much ATP they don’t produce as much ROS, and thus have a survival advantage as they are less prone to mutation. Eventually the cell will become full of moochers and result in fatigue and many other problems of aging.

 

So I say get rid of them all, mutations and methylation alike.

 

The new protocol:

 

This new procedure is much simplified. It requires only two doses, Mito1 and Mito2, which are alternated on a daily basis.

 

Mito1 (fission)

● NAM+R, 1 g of each

● AKG, 1 g

● PQQ, 20 mg

 

Mito2 (fusion)

● GMS, 1 g

● AKG, 1 g

● PQQ, 20 mg

 

NAM+R (nicotinamide plus ribose) is a fission promoter, GMS (glycerol monostearate) is a fusion promoter, AKG (alpha-ketoglutarate) is a demethylase promoter, and PQQ is a biogenesis promoter. All of these are fast acting.

 

A two week experiment using reps to failure:

 

Warm water was sufficient to dissolve everything, but the PQQ was taken in a capsule to insure that the other ingredients got a slight head start (probably unnecessary).

 

Mito1 and Mito2 were taken on alternating days. Each dose was taken in the evening and reps of dumbbell curls to failure counted first thing in the morning — five or six hours after dosing — using the same arm.

 

My hypothesis was that the number of reps would reflect mito damage. With mito fusion, enzymes are shared, thus ATP production and reps would be maximum. With fission, methylated (or otherwise damaged) mtDNA produce less ATP and reps would be minimum. The difference would reflect average damage, and if the treatment worked, the difference should decline. If all damage was removed, then the difference should go to zero.

 

Which in fact it did. See the plot below. The y-axis shows the reps and % difference, while the x-axis shows days. The curve labeled baseline is without any treatment, and likely reflects the normal intermediate situation with mito morphology in a dynamic state. It is stable at 16 reps. The upper fusion curve is relatively flat and higher than baseline as expected, while the lower fission curve is lower than baseline, but rises to meet the fusion curve after about two weeks, and stays there. Thus the percent difference goes to zero.

 

Results:

 

Improvement in running endurance, reduced hunger, and reduced need for hypertension medication.

Please excuse my ignorance, this thread is really becoming unmanageable. 

Why would 1g NAM after 5hr represent exercising in the state of fission?  

To elaborate, https://www.ncbi.nlm...les/PMC3365962/ showed 5mM NAM resulted in  decreased, then baseline (6hr mark), then increased NAD+ level (figure 1b).  Going on that, exercise should occur +6hr after dosing.  (Curious is the initial drop.)

But, there's also a significant difference in dose.

In PMC3365962, this was a [probably] much higher solution-to-cell-content.  That is, the 5mM would last much longer in the study than a 5mM concentration would last in the blood.  But 1g NAM doesn't get to a 5mM concentration in the blood (assuming around 5l blood), even if the NAM is absorbed instantly into the blood.

On another aspect:  is there anything to show that SIRT causes fission (and not just mitophagy resulting in fragmentation)?

Hi Turnbuckle, 

Do you think AKG is good in the senolytic part of SC protocol?

Thanks!

Hi Turnbuckle, 

 

Do you think AKG is good in the senolytic part of SC protocol?

 

Thanks!

That's a good question, though it's just going to confuse people here on this mito thread. So ask it on the SC thread and I'll answer it. 

Looking at the new protocol, I find it a bit strange that you have PQQ in both the fission and fusion sections.  The reasoning applied throughout most of this thread was to group biogenesis together together with fusion, while emphasizing mitophagy on days of fission.  Taking PQQ with fission seems to run contrary to that. 

Also, do you have any favorite references on the role of AKG in demethylation?

Looking at the new protocol, I find it a bit strange that you have PQQ in both the fission and fusion sections.  The reasoning applied throughout most of this thread was to group biogenesis together together with fusion, while emphasizing mitophagy on days of fission.  Taking PQQ with fission seems to run contrary to that. 

 

Also, do you have any favorite references on the role of AKG in demethylation?

I thought I'd answered that question in post #1739, and the results should speak for themselves. But I'll go over it again: The object of this new method is to get rid of both mutated and epimutated mitochondria. You won't find much research on mito methylation as it is new and still controversial. But it's known that both demethylase and methyltransferase can be found in mitochondria. And it's known that during replication double stranded DNA becomes single stranded. So my thinking was this: If you remove methyl groups from one strand of a double strand of DNA, they will be replaced by methyltransferase copying it from the other strand, but that is not possible during the brief period when the strands are separated during replication. Thus that is the time to hit it with demethylase. And as I mentioned in post #1739, mitochondria being replicated are not making enzymes, so they could run out in a fissioned state, sending membrane potential to zero. Those that run out first will be more likely to get recycled, and since mitochondria with methylated mtDNA will likely run out of enzyme reserves before those with less methylation, they will be removed first. It's a double whammy.

Turnbuckle, can you please provide a link to an AKG product? Is it sold as "Alpha-Ketoglutaric Acid"?

Thanks! The only brand I could find of AKG in Poland is this (Kirkman Alpha Ketoglutric Acid 300mg + 20mg of Calcium AKG + 10mg of Magnesium AKG). Would this do? If not, I'd have to see how to import from the USA. Ever since I moved to Europe (last month), finding supplements has been a pain.

It is spelled Ketoglutaric Acid not Ketoglutarate though. And description says, it is a buffered product. If it is the same thing, I'd take empty out the capsule and take this with water. Capsule might add extra few minutes to digestion speed.

https://pl.iherb.com...-capsules/58317

Yes, it is the same thing: AKG=Alpha Ketoglutarate=Ketoglutaric acid = 2-oxoglutaric acid. This Kirkman product is good for our purposes.

So my thinking was this: If you remove methyl groups from one strand of a double strand of DNA, they will be replaced by methyltransferase copying it from the other strand, but that is not possible during the brief period when the strands are separated during replication. Thus that is the time to hit it with demethylase. And as I mentioned in post #1739, mitochondria being replicated are not making enzymes, so they could run out in a fissioned state, sending membrane potential to zero.

Ah, that makes sense, thanks.  I haven't done a deep dive into epimutations yet, but I will certainly be interested in trying this out.

Still, it seems to me that this approach would decrease the disposal of (regular) mutated mtDNA relative to earlier iterations of the protocol.  If so, it might be ideal to rotate the two versions.  Or, perhaps we could use the older method until we hit a plateau and then switch to this new one for further progress.  I suppose we'll know more once people get a chance to experiment.
 

Ah, that makes sense, thanks.  I haven't done a deep dive into epimutations yet, but I will certainly be interested in trying this out.

 

Still, it seems to me that this approach would decrease the disposal of (regular) mutated mtDNA relative to earlier iterations of the protocol.  If so, it might be ideal to rotate the two versions.  Or, perhaps we could use the older method until we hit a plateau and then switch to this new one for further progress.  I suppose we'll know more once people get a chance to experiment.
 

I've done 15 cycles of the original protocol, so it will be interesting to see how what this new version contributes.

Mitochondrial Remodeling Following Fission Inhibition by 15d-PGJ2 Involves Molecular Changes in Mitochondrial Fusion Protein OPA1

Thiol antioxidant NAC and NMPG which were reported previously to block mitochondrial elongation and stabilization of Drp1 oligomers, also prevented loss of OPA1 isoforms and their synthesis and ubiquitination, which further supports our hypothesis that inactivation of fission protein by any means may subsequently lead to the decreased activity of fusion proteins which would then affect mitochondrial function and dynamics. Our results clearly warn investigators of unintended consequences of using drugs that block mitochondrial fission to prevent apoptosis.

Yes, it is the same thing: AKG=Alpha Ketoglutarate=Ketoglutaric acid = 2-oxoglutaric acid. This Kirkman product is good for our purposes.

Is this the same thing?  AAKG?  Description:  This product is 100% arginine alpha-ketoglutarate with nothing added or removed.  Thanks

Is this the same thing?  AAKG?  Description:  This product is 100% arginine alpha-ketoglutarate with nothing added or removed.  Thanks

No. AAKG is not same as AKG. I ordered Alpha Ketoglutaric Acid (Kirkman brand) which is same as Alpha Ketoglutarate that Turnbuckle mentioned in his protocol. But AAKG has added "arginine" and that's not good for mito protocol because it digests slower.

aribadabar, on 19 Feb 2021 - 12:51 AM, said:

Yes, it is the same thing: AKG=Alpha Ketoglutarate=Ketoglutaric acid = 2-oxoglutaric acid. This Kirkman product is good for our purposes.

 

Thanks!

@userCK: care to share a link? On amazon.de can't find any AKG (except headphones)

@userCK: care to share a link? On amazon.de can't find any AKG (except headphones)

I shared before in the post above. https://pl.iherb.com...-capsules/58317

iherb.eu

Certainly a big step forward in the evolution of this protocol. Thanks Turnbuckle.

With regards to the cycle frequency, how about 4-6 times a year?

Should any of the following common supplements be avoided during Mito1? Mito2?

Quercetin, CoQ10, Curcumin, Berberine, Resveratrol, amino acid mix.

Thanks!

I shared before in the post above. https://pl.iherb.com...-capsules/58317

iherb.eu

The kirkman AKG is in salt form (Calcium and Magnesium AKG).

These products are AKG acid:

https://www.amazon.c...13756044&sr=8-1

https://www.amazon.c...13756044&sr=8-2

https://www.amazon.c...26-bee5153e81cb

The kirkman AKG is in salt form (Calcium and Magnesium AKG).

 

These products are AKG acid:

https://www.amazon.c...13756044&sr=8-1

 

 

https://www.amazon.c...13756044&sr=8-2

 

https://www.amazon.c...26-bee5153e81cb

No. It has 300mg of acid and also Calcium and Magnesium salt based.

No. It has 300mg of acid and also Calcium and Magnesium salt based.

It is a mix of Ca and Mg salts actually netting 300mg of AKG per capsule.

https://www.kirkmang...-acid-caps.html

 

 

By itself, alpha-ketoglutaric acid is a strong organic acid, and taking it by mouth can upset the stomach or irritate the esophagus. Kirkman® has solved this problem by blending alpha-ketoglutaric acid with calcium and magnesium salts to produce a “bi-element buffered alpha-ketoglutaric acid” that will not produce excess acidity.

The kirkman AKG is in salt form (Calcium and Magnesium AKG).

 

These products are AKG acid:

https://www.amazon.c...13756044&sr=8-1

 

 

https://www.amazon.c...13756044&sr=8-2

 

https://www.amazon.c...26-bee5153e81cb

Simplesa's product is also buffered and is only a Ca salt: https://www.simplesa...COA-AKGPlus.pdf

So going with the buffered form is preferred.