Warning: There may be unidentified risks in trying parts of this protocol. Anyone who tries part of the protocol needs to monitor their biomarkers and if any harm is being caused stop using it. No medical advice is being given people need to do their own calculations and assessments and take medical advice from a healthcare professional where appropriate. People take their own decisions at their own risk.
- Get a baseline.
Before doing anything it really is worth having a baseline. This really has to include high sensitivity CRP. There are costs to testing and the costs of testing probably exceed the costs of the protocol. A baseline test before starting anything is really helpful.
- The principle
The principle behind this protocol is that if you improve how cells work then you improve how the body works. Cells function by generating energy and producing proteins. If a cell is unable to produce the full range of proteins then it will not function properly. The theory underpinning this protocol is that there are problems both in transcribing genes and creating mRNA, but also in taking mRNA and translating that into proteins.
Cells need to handle varying amounts of nutrients and energy supply. The main cellular currency of energy (ATP) is mainly generated by the mitochondria. However, as the mitochondria take glucose and push it through the Krebs/Citrate/TCA cycle (Citric and TriCarboxylic Acid are the same thing) they generate ATP. The mitochondria also export Citrate/Tricarboxylate through the Citrate Carrier protein (SLC25A1) into the cytosol. That is then used by the cytosol (and nucleus) for various purposes.
An interesting role of TCA in the cytosol is to indicate the energy status of the cell. TCA is converted by the ACLY enzyme into Acetyl-CoA.
The levels of Acetyl-CoA in the cytosol and nucleus control the process of transcription of genes. When genes are transcribed into mRNA an enzyme complex called RNA Polymerase II reads the gene and creates mRNA (using RNA as a substrate). One step of this is to open up the genes by acetylating the histone which is holding the genes. This is done by a HAT which needs Acetyl-CoA to do its job. One acetylation is needed for about every 142 base pairs.
Without the acetylation RNA Pol II cannot do its work. At a later point a Histone Deacetylase enzyme (HDAC) comes along and removes the acetyl group from the histone.
Hence the availability of acetyl-CoA controls the creation of mRNA. A lot of ATP is needed to translate mRNA into proteins so the acetyl-CoA has the effect of limiting the creation of mRNA when energy is scarce so you don’t end up with lots of mRNA hanging around waiting to go into protein creation. ATP is also needed for transcription, but it is the acetyl-CoA that is the main limiting factor.
As creatures develop a burden of senescent cells (and for other reasons) the expression of SLC25A1 (the Citrate carrier) is reduced and citrate is trapped in the mitochondria.
My protocol more widely looks also at translation, but for the purposes of this post I am only going to look at transcription.
- Improving transcription
The main problem is a shortage of acetyl-CoA. However, there is also an issue in the interplay between acetylation and deacetylation. What we don’t want is for the HDAC to deacetylate a histone with a stalled RNA Pol II stuck at it. I have not found anything in pubmed about this, but it is clear that when HDAC is inhibited (there are lots of different HDACs) gene transcription is improved.
Hence I think we can identify a number of routes towards improving transcription
a) More acetyl-CoA
b) More substrate availability
c) Slower deacetylation.
Additionally I think it is worth having Lithium at a roughly 50 micromolar serum concentration for its effects on the WNT pathway.
Dealing with these in reverse
- Slower deacetylation
There are a large number of Histone Deacetylase Inhibitors (HDACi s) around. I use them in quartets for no really good reason save that I wish to minimise the side effects from any one HDACi whilst maximising the HDACi effect. The ones I used tend to have IC50s in the 50 micromolar range (which is not that strong there are nanomolar ones).
In particular my primary quartet is Curcumin, Berberine, Pterostilbene and Quercetin. I have experimented with taking multiple doses in a day (whilst reviewing published research on the maximum sensible dose which has no known negative effects). What I think happens is that a mild HDACi effect can cause cells to function beyond what would normally happen in a younger person with the same genes. With me it caused additional facial hair to grow. However, I am not sure that too much of this is necessary.
- More substrate availablity
There is published research that shows a longevity benefit from eating RNA. Hence I do this. Vitamins B12 and B9 are also needed for transcription. I also take Vitamin B5, but that is really for the acetyl-CoA (because B5 is a coa precursor).
- More Acetyl CoA
There are in fact two enzymes that create Acetyl-CoA in the cytosol. ACLY does this from citrate/TCA and ACCS2 does it from acetate. ACCS2, however, is inhibited by acetylation levels. Hence if the Histone is reasonably acetylated the conversion of acetate to Acetyl-CoA will be limited. Therefore the best route is through additional citrate.
It is possible to increase SLC25A1 by using janus kinase inhibitors, but these have some nasty side effects although the drugs have been licenced for use.
However, what is really interesting is that you can increase cytosolic citrate levels from citrate in the blood.
- Citrate metabolism
The body has an interesting citrate metabolism where a serum level of citrate is maintained at something around the 100 micromolar level. If citrate goes up there is a gene which in Drosophila is called INDY (“I’m not dead yet”) expressed in liver cells that draws citrate out of blood and burns it up to carbonate. The same is the case in humans, but the gene is called mINDY.
However, if you digest citrate it will temporarily increase the serum citrate level and that will go into cells and increase cytosolic citrate giving a boost to nuclear acetyl-CoA levels. Citrate has a sort of half life around 30 mins so this effect does not last long, but it lasts long enough to effect gene expression. It is a bit of a one way valve so the boost to acetyl-CoA levels from a pulse of serum citrate lasts for a while. (until it has been used up)
- Acetyl-CoA - side effects
An increase to cytosolic Acetyl-CoA has side effects in making more acetyl-CoA available for both cellular cholesterol production and cystosolic ROS production.
I think it is worth on a preventative basis topping up melatonin levels though melatonin supplementation during the night. It is worth being aware that cellular cholesterol figures can be increased. That may not affect lipid transport, but it is an issue to keep an eye on.
The availablity of cytosolic ROS does mean that cells have a better tool to fight invaders and may fight viruses that had otherwise been ignored. That’s good for getting rid of warts, but it does mean increasing citrate levels should be done gradually.
- So this citrate thing then what can I do
Well, don’t eat a lot of lemons. Citric Acid does provide citrate, but it also provides protons (acid) and cells don’t like acid. In fact what cells do to move to an alkaline state is to deacetylate the histone (and we don’t want that).
There are quite a few citrate salts available, but I have been working with Sodium, Magnesium, Potassium and Calcium.
I think caution is needed potentially with too much calcium, but supplementing with a mixture of Sodium, Potassium and Magnesium (and potentially calcium) is a good strategy. Sodium in isolation would be a mistake. It is important to maintain a ratio of around 3:1 in Sodium - Potassium and slightly less Magnesium.
- Lots of pills
Well actually what I am now doing is buying the salts as powder and mixing it together that is better than eating lots of pills (which I was doing previously) Sodium, Magnesium and Potassium Citrate are all quite soluble and you can just drink a mixture either in a cordial or simply diluted in water.
However, to start it may be worth taking the pills.
- How much
I think it is important to be careful starting out. The weight I refer to is the Citrate. The calculation of citrate levels from the cation elemental weight varies for each salt. If you buy the powder then sometimes it is hydrated. If you have the elemental mass you can estimate the mass of citrate by tripling it.
I would, however, start small for a week or two with perhaps 0.5 grams or 1 gram of citrate. This should really have no noticeable effect. However, no noticeable effect is a good thing. You can then increase to 2 grams of citrate (if you started with 0.5 grams move to 1 grams first). At this stage it is a question of making sure your systems can cope.
The next step is to move to 2 grams of citrate the first for breakfast and the second for lunch.
I have tried varying amounts of citrate including over 40 grams (divided into 5 g doses over 30 minute intervals). I get a bit of a buzz at that level. However, I strongly advise caution and care. I am currently running a maintenance level of 5g at breakfast and 5g for lunch. I have been supplementing with citrate for over a year and it took quite a time to increase levels to start because of the improved ability of cells to fight infection. If you rush it you may end with lots of spots.
With the cations you are dosing reasonably heavy amounts of the cations hence you need to check that your kidneys are handling this properly. What I have found it that the supplementation does push sodium, potassium and magesium levels up a bit (eg Na 139->142) , but they drop back really quickly and are always in the normal range. However, this may not be the case for everyone and you need to do blood tests to monitor for this.
- Side effects of Citrate
Citrate will cause your urine to become more alkaline. My urinary pH has gone up at times to 9.5. It is worth monitoring urinary pH.