Home-made Liposomal EGCG — a cost effective therapy for Autism, Parkinson’s, and Alzheimer’s? Plus alternative antioxidants — Whey protein and Liposomal vitamin C

A $30 ultrasonic jewellery cleaner can be repurposed to make inexpensive liposomal supplements

 

Today’s post is really one for those who prefer not to use prescription drugs to treat autism, or those that are just unable to access them. It is also one our longtime reader Ling might regard as MacGyver-esque (from the TV series following the adventures of Angus MacGyver, a secret agent armed with remarkable scientific resourcefulness to solve any problem out in the field using any materials at hand).

It is about increasing the bioavailability of OTC supplements (EGCG in today’s case, but applicable to many others) to get closer to achieving their often elusive health benefits in autism.

There are some effective OTC autism therapies, but most are not. This is why repurposing existing prescription drugs is likely necessary.

 

Liposomal

One of the big things in the supplement world at the moment is to call products “liposomal” and triple the price. The theory is that a preparation contains the active drug/supplement inside very tiny, fat-like particles. This form is easier for the body to absorb and allows more drug/supplement to get to the target area of the body, such as the brain. Liposomal drugs may have fewer side effects and should, in theory, work better than other forms of the drug.

This fatty encapsulation helps protect the active compound from degradation in the digestive system and improves its absorption through the gut. It can also enhance delivery to target tissues (like the brain) because liposomes can sometimes cross biological barriers more easily.

This should mean higher effectiveness with lower doses and potentially fewer side effects compared to non-encapsulated forms.

 

If you are interested in the details:

https://en.wikipedia.org/wiki/Liposome

“A liposome is a small artificial vesicle, spherical in shape, having at least one lipid bilayer. Due to their hydrophobicity and/or hydrophilicity, biocompatibility, particle size and many other properties, liposomes can be used as drug delivery vehicles for administration of pharmaceutical drugs and nutrients, such as lipid nanoparticles in mRNA vaccines, and DNA vaccines. Liposomes can be prepared by disrupting biological membranes (such as by sonication).

Liposomes are most often composed of phospholipids, especially phosphatidylcholine, and cholesterol, but may also include other lipids, such as those found in egg and phosphatidylethanolamine, as long as they are compatible with lipid bilayer structure. A liposome design may employ surface ligands for attaching to desired cells or tissues.”

 

 

  

By making your own liposomal supplements you will save a lot of money, compared to commercial ones and have access to an undegraded product. If you customize the recipe/ingredients thoughtfully, and carefully control the processing, the result might replicate some of the benefits seen in university studies. You might wonder why compounding pharmacies are not already doing this - maybe some are.

You can pretty much buy everything you need on Amazon. Once you have figured out your ingredients and decided how big a batch to make, it is no more complex than baking a cake.

 

Liposomal vitamin C and whey protein as therapies for oxidative stress

Oxidative stress is a core feature of most autism, particularly in the early years, and a feature of aging for everyone. Vitamin C is a natural antioxidant, but it is a water soluble vitamin that your body automatically regulates and excretes via urine. If you take mega-doses of a standard supplement it just goes down the toilet, it does not reach the bloodstream.

Intravenous vitamin C causes a large increase in levels in the blood. This can be used to treat sepsis and even mast cell activation syndrome (MCAS). It has potential in oncology (cancer treatment) because at high concentrations, vitamin C can act as a pro-oxidant, generating hydrogen peroxide that is selectively toxic to tumor cells.  

It has also been used for Ehlers-Danlos syndrome, fibromyalgia and other conditions

Some practitioners consider IV vitamin C for autism because of its: 

• Antioxidant effects – reducing oxidative stress, which is elevated in many children with autism.
• Anti-inflammatory properties – calming neuroinflammation and microglial activation.
• Support for neurotransmitter synthesis – vitamin C is a cofactor in dopamine and norepinephrine production.
• Possible mast cell stabilization – relevant in children with autism and comorbid mast cell activation syndrome (MCAS).
• Histamine degradation support – helps recycle tetrahydrobiopterin (BH4), indirectly involved in histamine metabolism.

 

It has been found that liposomal vitamin C can achieve levels in the blood somewhere in between IV-vitamin C and regular vitamin C by food or supplements. 

High levels of vitamin C can cause side effects such as kidney stones.

Liposomal vitamin C is better tolerated than very high doses of standard vitamin C. It looks like things are likely to start going wrong above 3,000mg a day of liposomal.

Healthy people just need a good diet. If they have a poor diet then take a multivitamin.

Liposomal or IV therapy is only for people with real health issues.

People with MCAS plus autism certainly do have health issues.

Ehlers–Danlos syndrome (and milder subclinical versions) is linked to MCAS, ADHD, autism and Tourette’s. So that is another group to consider.

Fibromyalgia was put forward (by me) as a step towards autism in some females, in subsequent levels of their family tree.

So overall the idea of liposomal vitamin C has much more merit than a natural sceptic would have first thought. (There are loads of YouTube videos of people doing this, and likely many did not really need it.)

 

Whey protein as an antioxidant 

This topic was recently highlighted by our reader Stephen and it naturally fits into this post.

Back in 2013 when I was developing my son’s therapy I had to choose between NAC and whey protein to boost glutathione (GSH), the body’s key antioxidant. I chose NAC.

Here is a great paper to support the use of whey protein.

 

Improving Antioxidant Capacity in Children With Autism: A Randomized, Double-Blind Controlled Study With Cysteine-Rich Whey Protein 

Previous studies indicate that children with autism spectrum disorder (ASD) have lower levels of glutathione. Nutritional interventions aim to increase glutathione levels suggest a positive effect on ASD behaviors, but findings are mixed or non-significant. A commercially available nutritional supplement comprising a cysteine-rich whey protein isolate (CRWP), a potent precursor of glutathione, was previously found to be safe and effective at raising glutathione in several conditions associated with low antioxidant capacity. Therefore, we investigated the effectiveness of a 90-day CRWP intervention in children with ASD and examined whether intracellular reduced and oxidized glutathione improvements correlated with behavioral changes. We enrolled 46 (of 81 screened) 3-5-year-old preschool children with confirmed ASD. Using a double-blind, randomized, placebo-controlled design, we evaluated the effectiveness of daily CRWP (powder form: 0.5 g/kg for children <20 kg or a 10-g dose for those >20 kg), compared with placebo (rice protein mimicking the protein load in the intervention group), on glutathione levels and ASD behaviors assessed using different behavioral scales such as Childhood Autism Rated Scale, Preschool Language Scale, Social Communication Questionnaire, Childhood Behavioral Checklist and the parent-rated Vineland Adaptive Behavior Scale, 2nd edition (VABS-II). Forty children (CRWP, 21; placebo, 19) completed the 90-day treatment period. Improvements observed in some behavioral scales were comparable. However, the VABS-II behavioral assessment, demonstrated significant changes only in children receiving CRWP compared to those observed in the placebo group in the composite score (effect size 0.98; 95% confidence intervals 1.42-4.02; p = 0.03). Further, several VABS-II domain scores such as adaptive behavior (p = 0.03), socialization (p = 0.03), maladaptive behavior (p = 0.04) and internalizing behavior (p = 0.02) also indicated significant changes. Children assigned to the CRWP group showed significant increases in glutathione levels (p = 0.04) compared to those in the placebo group. A subanalysis of the VABS-II scale results comparing responders (>1 SD change from baseline to follow up) and non-responders in the CRWP group identified older age and higher levels of total and reduced glutathione as factors associated with a response. CRWP nutritional intervention in children with ASD significantly improved both glutathione levels and some behaviors associated with ASD. Further studies are needed to confirm these results.

 

This study used a special commercial product called Immunocal, a cysteine-rich whey protein isolate (CRWP) that serves as a potent glutathione precursor.

There are less expensive alternatives to Immunocal that still offer high-quality, undenatured, cysteine-rich whey protein, especially if your goal is to support glutathione production without paying premium prices. These products are typically marketed as cold-processed, non-denatured whey protein concentrates or isolates, and some are even made from the same raw material sources as Immunocal.

If you want to further increase absorption you can even make a liposomal version of a cysteine-rich whey protein!! 

Regular body builders’ whey protein is great to help build muscles and to maintain muscle mass in seniors, but it is not the ideal source of cysteine. It has degraded during the production process, that why there are fancy ones available.

I think Stephen would indeed be well advised to add a scoop of cysteine-rich whey protein isolate (CRWP) to his sons’ diets. It should have a more prolonged effect than NAC. For young children with autism NAC really needs to be given 3-4 times a day.

You can have too much cysteine. You do not need high dose of both NAC and CRWP.

 

Back to liposomal EGCG

If you read the reviews many people find commercial liposomal supplements no more effective than the much cheaper, regular ones. I wonder why. Most likely they were not well formulated, or they degraded by the time they were used. These products are not heat or light stable.

Many manufactured products like fish oil supplements no longer maintain the health benefit of the genuine article (fish, in this case). This is because the product degraded and sometimes can even have a negative behavioral effect. 

 

Many healthy natural products like catechins or curcuma have very low bioavailability

There is a long list of healthy products that should be therapeutic in autism including:

·        Green tea catechins like EGCG

·        Turmeric/Curcuma

·        Resveratrol

·        Cocoa

·        Many herbs (sage, oregano, rosemary, Bacopa monnieri, ginseng, lions mane, etc)

They generally have very low bioavailability and so they work great in the lab, but much less so in humans; unless you consume very large amounts, for example turmeric in an Indian diet.

 

EGCG

I have written about EGCG in the past and have highlighted the research from Spain, more specifically from the beautiful city of Barcelona (just avoid visiting during the peak summer months). The research showed a benefit in Fragile X and Rett syndrome. As usual, no customized intervention has yet been brought to the market.

https://www.epiphanyasd.com/search/label/EGCG?max-results=20

Yet another study showing the potential benefit of EGCG, was published recently, this time in Pakistan.

 

Cross-linking catechins with neuro-regulatory model for autism spectrum disorder: A management in rats’ experiment 

We found that BDNF levels returned to normal levels within the groups who received Catechins treatment at III, IV, and V concentrations (compared to Group II), showing Catechins could potentially treat autism-like symptoms. The BDNF values measured in nano-grams per millilitre were Group I (13.1±0.3), followed by Group II (5.1±0.2) and Group III (9.8±0.3), Group IV (8.0±0.3), and then Group V (10.1±0.3). The BDNF concentration measured in Groups III, IV and V surpassed the BDNF level of Group II (PPA-induced) per results from a post-hoc Tukey's test at p 

Catechins successfully decreased neuroinflammatory markers throughout the brain and establish protective brain mechanisms that potentially improve ASD-associated behavioral symptoms. Rats given 100, 200, and 400 mg/kg of various catechins showed increases in BDNF levels of up to 75%, 61%, and 77%, respectively, as opposed to only 39% for rats that received no treatment. The findings of a study suggested a continuous and expandable neuroprotective effect based on dose strength. The experimental results demonstrated that in ASD models, catechins offer a potent and dosage-dependent defense against neuroinflammatory injuries.

  

This study confirms that epigallocatechin gallate (EGCG), among catechins, shows great promise for managing neuroinflammation in ASD patients. The results indicate that catechins deliver substantial reductions in neuroinflammatory markers, as they serve as protective element that improves behavioral and cognitive manifestations of ASD. Future investigations must explore mechanisms of effect and find best-use dosages for catechins while establishing their safety and lasting effect durations.

 

Then I came across this paper where the university made their own liposomal version of EGCG and tried it on their model of Parkinsons’ disease. It also worked very well. Autism is not Parkinsons’ but both conditions feature activated microglia, the brain’s immune cells that are also tasked with synaptic pruning housekeeping duties.

 

Epigallocatechin-3-Gallate-Loaded Liposomes Favor Anti-Inflammation of Microglia Cells and Promote Neuroprotection

Microglia-mediated neuroinflammation is recognized to mainly contribute to the progression of neurodegenerative diseases. Epigallocatechin-3-gallate (EGCG), known as a natural antioxidant in green tea, can inhibit microglia-mediated inflammation and protect neurons but has disadvantages such as high instability and low bioavailability. We developed an EGCG liposomal formulation to improve its bioavailability and evaluated the neuroprotective activity in in vitro and in vivo neuroinflammation models. EGCG-loaded liposomes have been prepared from phosphatidylcholine (PC) or phosphatidylserine (PS) coated with or without vitamin E (VE) by hydration and membrane extrusion method. The anti-inflammatory effect has been evaluated against lipopolysaccharide (LPS)-induced BV-2 microglial cells activation and the inflammation in the substantia nigra of Sprague Dawley rats. In the cellular inflammation model, murine BV-2 microglial cells changed their morphology from normal spheroid to activated spindle shape after 24 h of induction of LPS. In the in vitro free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, EGCG scavenged 80% of DPPH within 3 min. EGCG-loaded liposomes could be phagocytized by BV-2 cells after 1 h of cell culture from cell uptake experiments. EGCG-loaded liposomes improved the production of BV-2 microglia-derived nitric oxide and TNF-α following LPS. In the in vivo Parkinsonian syndrome rat model, simultaneous intra-nigral injection of EGCG-loaded liposomes attenuated LPS-induced pro-inflammatory cytokines and restored motor impairment. We demonstrated that EGCG-loaded liposomes exert a neuroprotective effect by modulating microglia activation. EGCG extracted from green tea and loaded liposomes could be a valuable candidate for disease-modifying therapy for Parkinson’s disease (PD).

 

Looks great, but you cannot buy their product. It then appeared that people are already making liposomal supplements at home.

Dig a little deeper to see what other clever ideas exist in the university research world that might make DIY versions better. 

 

Liposomal Formulations for an Efficient Encapsulation of Epigallocatechin-3-Gallate: An In-Silico/Experimental Approach

As a part of research project aimed to optimize antioxidant delivery, here we studied the influence of both salts and lipid matrix composition on the interaction of epigallocatechin-3-gallate (EGCG) with bilayer leaflets. Thus, we combined in silico and experimental methods to study the ability of neutral and anionic vesicles to encapsulate EGCG in the presence of Ca2+ and Mg2+ divalent salts. Experimental and in silico results show a very high correlation, thus confirming the efficiency of the developed methodology. In particular, we found out that the presence of calcium ions hinders the insertion of EGCG in the liposome bilayer in both neutral and anionic systems. On the contrary, the presence of MgCl2 improves the insertion degree of EGCG molecules respect to the liposomes without divalent salts. The best and most efficient salt concentration is that corresponding to a 5:1 molar ratio between Mg2+ and EGCG, in both neutral and anionic vesicles. Concerning the lipid matrix composition, the anionic one results in better promotion of the catechin insertion within the bilayer since experimentally we achieved 100% EGCG encapsulation in the lipid carrier in the presence of a 5:1 molar ratio of magnesium. Thus, the combination of this anionic liposomal formulation with magnesium chloride, avoids time-consuming separation steps of unentrapped active principle and appears particularly suitable for EGCG delivery applications.

 

The Mozafari method for Liposomal delivery

The latest methods used in universities to make liposomal products cannot be entirely replicated at home, but there is a well-known method developed by Dr Mohammad Mozafari that has been proved to increase bioavailability 2 to 8 times. The Mozafari method is used today by biohackers at home. Often they seem to skip some important steps.

We can fine tune his method, for example by noting the research showing that magnesium ions can help stabilize the liposomes and improve encapsulation of EGCG. Calcium ions have a very negative effect and so make sure no calcium (for example, from hard water) enters the process. YouTubers just use tap water. So use high-quality deionized (DI) water and add Magnesium Chloride (MgCl₂).

Anionic liposomes (negatively charged phospholipids) promote better EGCG insertion compared to neutral liposomes. With Mg²⁺, anionic liposomes reached 100% encapsulation efficiency experimentally. So it was actually perfect.

Magnesium chloride (MgCl₂) at about a 5:1 molar ratio relative to EGCG

(Example: for 500 mg EGCG ≈ 1.1 mmol, add ~5.5 mmol MgCl₂ — roughly 670 mg MgCl₂·6H₂O)

Both pH and temperature control are important and seem to get ignored by YouTubers.

Choose the right lipid. Here are the choices:

Most DIYers are using Lecithin (sunflower or soy), which contains phosphatidylcholine (PC), plus other substances you do not want. It is cheaper than pure PC.

If you are making liposomal vitamin C, glutathione, DHA or EGCG for therapeutic use (e.g., autism, MCAS, oxidative stress), pure PC gives superior performance.

Lecithin is zwitterionic, meaning it contains both positive and negative charges, but is overall electrically neutral. This dual nature is what makes lecithin perfect for encapsulating both water-soluble (like vitamin C) and fat-soluble (like curcumin) compounds in liposomes.

For closer to University-grade work we need to look at pure chemicals.

·        Phosphatidylcholine (PC) — neutral

·        Phosphatidylserine (PS) — anionic (negative charged)

·        CHEMS (Cholesteryl Hemisuccinate), a negatively charged cholesterol derivative.

·        Cholesterol

 

Component             Role  

PC                               Bilayer structure & fluidity         

PS                               Anionic charge, Mg²⁺ interaction          

Cholesterol               Stabilization (optional)    

CHEMS                      Additional anionic charge (optional)

 

Phosphatidylserine (PS) is itself therapeutic

PS naturally concentrates in the brain, especially in neuronal membranes.

It is known to support memory, attention, synaptic function, and neuroplasticity — ideal for neurodegenerative and developmental conditions.

PS is negatively charged (anionic), which helps form stable liposomes and can improve encapsulation of positively charged or hydrophilic molecules like EGCG.

PS has functional activity, beyond just being a carrier, PS itself may synergize with EGCG and other cognitive-enhancing compounds.

Adding cholesterol makes the liposome less leaky and more resistant to degradation. Without cholesterol, liposomes are more prone to oxidation, fusion, or breakdown over time

  

Example for 2 g Total Lipids:

Lipid Component	Weight (grams)	Percentage
PC	1.2 g	60%
PS	0.4 g	20%
Cholesterol	0.4 g	20%

 

• PC provides a stable bilayer and good liposome formation.
• PS introduces a negative charge that enhances electrostatic interaction with Mg²⁺ and EGCG.
• Cholesterol improves membrane rigidity and stability, helping prevent leakage.
• You can adjust cholesterol slightly depending on how rigid you want the membrane.
• Maintain MgCl₂ at ~5:1 molar ratio to EGCG in the aqueous buffer for optimal encapsulation, as per references.

EGCG is highly oxidation-sensitive.

Both vitamin C (ascorbic acid) and vitamin E (tocopherol) protect:

·        the lipids in the liposome from peroxidation,

·        the EGCG itself from degradation.

So it is wise to add both vitamin C and E.

• Vitamin E is lipid-soluble and embeds in the bilayer.
• Vitamin C is water-soluble and protects the aqueous core.

 

Here is the home version.

 

 

Equipment

• Glass beaker or jar
• Ultrasonic cleaner (sold to clean jewellery)
• Stirring rod
• pH strips or meter
• Dark glass storage bottle

 

Method

1.     Mix Vitamin E with PS

o    Combine PS powder and vitamin E oil or powder thoroughly in a small container.

2.     Prepare aqueous phase

o    Dissolve EGCG powder and magnesium chloride in ~20 mL PBS or distilled water with buffer salts.

o    Add vitamin C to this aqueous solution last and stir gently until dissolved.

3.     Hydrate lipids

o    Slowly add the aqueous phase (EGCG + MgCl₂ + vitamin C) to the PS + vitamin E mix.

o    Stir or vortex gently to disperse.

4.     Sonicate

o    Place the mixture in an ultrasonic cleaner bath for 20–30 minutes, stirring occasionally.

o    Solution should become milky/opalescent, indicating liposome formation.

 

How to Use Ultrasonic Cleaner for Liposomal EGCG

1.     Prepare your liposome suspension in a suitable sealed container—usually a small glass vial or bottle with a tight lid (e.g., amber glass bottle or glass vial).

2.     Fill the ultrasonic cleaner tank with clean water—enough so that when you place your container in it, the water level reaches just below the lid or about 2/3 up the container’s height. The water must not overflow into your liposome container

3.     Place your sealed bottle/vial into the ultrasonic bath, making sure it sits upright and stable.

4.     Turn on the ultrasonic cleaner for the recommended at medium power.

5.     During the process, keep an eye on the temperature—if the water or sample gets too warm (>40°C), pause and let it cool, since heat will degrade EGCG.

6.     After sonication, remove the bottle and store the liposomal EGCG in a dark, refrigerated place.

 

Important Tips

• Use sealed containers to avoid contamination or water ingress.
• Never put the liposomal suspension directly into the ultrasonic cleaner’s water bath.
• If your ultrasonic cleaner has a temperature control or timer, use those settings to protect the sample.
• Clean the ultrasonic tank well before and after use.

The final product will be stable for 7 days in the fridge.

You can freeze portion sized doses in a silicone ice cube tray. Later store in the freezer in a zip lock bag for 2-3 months. Defrost in the fridge, one by one, as you need it.

Keeping the temperature below 40°C is essential when sonifying delicate compounds like EGCG, vitamin C, and phospholipids (especially phosphatidylserine). They degrade or oxidize easily when exposed to excessive heat.

 

 1. Use a Cold Water Bath

• Fill the ultrasonic cleaner with cold water (4–10°C).
• Add ice cubes to keep it cold.
• Replenish ice as needed during sonication.

 

2. Monitor Temperature

• Use a probe thermometer or an infrared laser thermometer.
• Check the temperature of your sample, not just the water bath.

 

Ultrasonic waves create cavitation — rapid formation and collapse of microbubbles — which:

• Generates localized heat (tiny hot spots in the solution)
• Transfers energy into the liquid, raising the overall temperature gradually
• Can increase your solution temperature from room temp to 50–60°C in 10–15 minutes if not managed

Even if the water bath feels lukewarm, the inside of your beaker can be much hotter, especially in the center.

 

 Sensitive Ingredients at Risk

• EGCG starts to degrade above ~40°C
• Phospholipids can oxidize or become unstable
• Vitamin C rapidly degrades in heat and light

 

Best Practices to Avoid Overheating

Tip	Why
Use an ice-cold bath	Keeps temp under control during sonication
Sonicate in short bursts (30s ON, 30s OFF)	Prevents buildup of heat
Stir gently before and after sonication	Helps distribute heat and emulsify
Monitor temperature regularly	Keeps you in the safe zone (under 40°C)
Work with small batches (≤100 mL)	Easier to cool and control

 

Sonication is the process of using high-frequency sound waves—usually ultrasound—to agitate particles in a liquid. In making liposomes, sonication helps break down large lipid aggregates into smaller, uniform liposomes by creating tiny bubbles that collapse and generate energy, mixing and dispersing the components more thoroughly.

In simpler terms, sonication is like using sound waves to gently shake and break up the mixture so that the fat-like particles (liposomes) form smaller, more consistent droplets, which can improve how well the active ingredient (like EGCG) gets absorbed by your body.

  

Conclusion

Immunocal, a cysteine-rich whey protein isolate (CRWP) does show a benefit in autism. It is an OTC product that is sold online.

EGCG is the most abundant catechin in green tea.  When taken orally, EGCG has poor absorption even at daily intake equivalent to 12 cups of green tea.

EGCG does show merit in autism research studies, but to be effective in humans the bioavailability needs to be improved. Using liposomes to encase the EGCG is a promising approach.

Will the method proposed in this post be effective?

I asked Gemini AI to comment on the method and it replied: 

"The EGCG method you described is a fantastic example of a DIY approach that is grounded in sound scientific principles. By using specific lipids, optimizing the formulation with magnesium, and strictly controlling the process, it goes far beyond the simplistic and often ineffective methods seen online.

While it won't produce a pharmaceutical-grade product, it is highly likely to create a solution that contains a significant number of genuine, stable, and correctly sized liposomes. This makes it far more likely to be an effective delivery system than the typical DIY liposomal vitamin C, which is often just an unverified emulsion."

Some people do grow their own broccoli sprouts to make sulforaphane, others grow wheat sprouts for spermidine. Some people grow their own probiotic bacteria. Making tumeric balls is a simple way to get the benefits of tumeric. There are many home-made options, and I think the parent almost certainly benefits. 

You would think that some enterprising pharmacist in Barcelona would start producing small batches of liposomal EGCG, using research grade equipment. I think Rett syndrome and Fragile X syndrome parents would buy it. Not to mention those who have parents diagnosed with Parkinson's or Alzheimer's.

   

  

Arginine and its Derivatives in Cognitive Impairment

Source: Epiphany ASD Blog

Today’s post is very relevant to dementia, relevant to schizophrenia and diabetes and I believe some autism, including that of my son; agmatine is part of his Polypill therapy.

Arginine is highly versatile amino acid and you need the arginine metabolism to be working correctly, particularly in your brain.

Arginine is a widely available from diet and can be produced from citrulline and indirectly from glutamine; so you are unlikely to be deficient in arginine, except in your brain and particularly if you have Alzheimer’s.

In Alzheimer’s it has been shown that the microglia in effect destroy arginine in the brain and this may play a role in what initiates the disease.

Research has suggested that a deficiency in polyamines, another derivative of Arginine, is a feature of dementia.

A deficiency of arginine in the brain will likely cause a deficiency of polyamines.

Your body needs nitric oxide to maintain a healthy blood pressure and this requires arginine to follow the blue line in the above chart towards citrulline and be converted by eNOS.  In most older people this does not happen and oxidative stress appears to be a big part of the problem.

Agmatine – good 

Agmatine has been shown in research to have a benefit in Alzheimer’s.  

This could be due to increased eNOS improving blood flow, an increase in Polyamines, or by reducing insulin resistance in the brain. Recall those studies of intranasal insulin? We had "type 3 diabetes", which was a brain-specific blunting of insulin.

https://www.ncbi.nlm.nih.gov/pubmed/27810390 

"Agmatine administration rescued the reduction in insulin signalling, which in turn reduced the accumulation of Aβ and p-tau in the brain. Furthermore, agmatine treatment also reduced cognitive decline. Agmatine attenuated the occurrence of AD in T2DM mice via the activation of the blunted insulin signal"

Methylarginines – not good

Two by-products of arginine are bad for you in the way Agmatine is good for you.

Nitric Oxide is produced via iNOS, nNos and eNOS. In simple terms we want nitric oxide to be produced in the endothelium, the name for cells that line the interior surface of blood vessels and lymphatic vessels, To achieve this we needs lots of the enzyme eNOS and not much iNOS or nNOS, this is one of Agmatine’s jobs.

Two derivatives of arginine/proteins in the body with very long names are abbreviated to NMMA and ADMA. They both inhibit eNOS and so will restrict blood flow and this will appear as elevated blood pressure.   

The methylarginines NMMA, ADMA, and SDMA are ubiquitous constituents of the main vegetables of human nutrition.

Endogenous methylarginines, N(G),N(G)-dimethyl-L-arginine (asymmetric dimethylarginine, ADMA), N(G)-N('G)-dimethyl-L-arginine (symmetric dimethylarginine; SDMA), and N(G)-monomethyl-L-arginine (monomethyl arginine; NMMA) are supposed to be produced in human body through the methylation of protein arginine residues by protein arginine methyltransferases (PRMT) and released during proteolysis of the methylated proteins. Micromolar concentration of ADMA and NMMA can compete with arginine for nitric oxide synthase (NOS) reducing nitric oxide (NO) formation, whereas SDMA does not. Indeed, increased ADMA and SDMA plasma levels or a decreased arginine/ADMA ratio is related with risk factors for chronic kidney disease and cardiovascular disease. To the best of our knowledge the exogenous presence of methylarginines, like that in fruits and vegetables, has never been described so far. Here, we report the finding that methylarginines are ubiquitous in vegetables which represent an important part of human daily diet. Some of these vegetables contain discrete amounts of ADMA, SDMA, and NMMA. Specifically, among the vegetables examined, soybean, rye, sweet pepper, broad bean, and potato contain the highest ADMA and NMMA mean levels. Our results establish that the three methylarginines, in addition to being produced endogenously, can also be taken daily through the diet in conspicuous amounts. We propose that the contribution of the methylarginines contained in the vegetables of daily diet should be taken into account when the association between vegetable assumption and their levels is evaluated in clinical studies. Furthermore, a comprehensive understanding on the role of the digestive breakdown process and intestinal absorption grade of the methylarginines contained in vegetables is now needed. 

ADMA

Asymmetric dimethylarginine (ADMA) is a naturally occurring chemical found in blood plasma. It is closely related to L-arginine. ADMA interferes with L-arginine in the production of nitric oxide (NO), a key chemical involved in normal endothelial function and, by extension, cardiovascular health. ADMA inhibits eNOS, which in simple terms is the good NOS, the other two being iNOS and nNOS.

ADMA is considered a marker for vascular disease. 

NMMA (NG-monomethyl-l-arginine, or just called Targinine) 

The following study is very interesting for your older relatives. As we already know oxidative stress is a feature of aging. Many people have high blood pressure in old age. Nitric Oxide (NO) is needed keep blood vessels wide open. In old age (>60) oxidative stress reduces NO availability to nothing. 

Since oxidative stress is reversible (in this study vitamin C was used) you wonder why more older people, particularly with high blood pressure, do not take entioxidants. 

Age-Related Reduction of NO Availability and Oxidative Stress in Humans

A novel finding of the present study is that in normotensive subjects, the reduction in endothelial function associated with aging seems to be mediated by a progressive reduction of NO availability, inasmuch as the inhibiting effect of L-NMMA on acetylcholine-induced vasodilation was progressively impaired by advancing age. It is worth noting that after the age of 60 years, the inhibiting effect of L-NMMA on response to acetylcholine was very weak, suggesting that in aged individuals NO availability is almost totally compromised. To assess the possible role exerted by oxidative stress, we tested the antioxidant vitamin C.¹⁹ Up to the age of 60 years, despite the evident decline in endothelium-dependent vasodilation, vitamin C did not modify the response to acetylcholine. In contrast, in the oldest individuals (age >60 years) characterized by a profound alteration in NO availability, vitamin C not only enhanced the response to the endothelial agonist but also restored the inhibiting effect of L-NMMA on vasodilation to acetylcholine. Thus, in the present study, the use of L-NMMA and vitamin C, never tested before in investigating the mechanisms responsible for the previously demonstrated age-related endothelial dysfunction in humans,¹⁷ seems to indicate that the progressive impairment in endothelium-dependent vasodilation is caused by a progressive alteration of the l-arginine-NO pathway. Only in old age (after ≈60 years) does the production of oxidative stress appear, leading to the complete compromise of NO availability.  

Arginase

Arginase is an enzyme that acts as the catalyst for the reaction.

 arginine + H₂O → ornithine + urea 

People with schizophrenia and also people with diabetes tend to have high levels of Arginase. This will affect how arginine is metabolized. If arginase is increased there is less arginine that can go towards creatine, citrulline or agmatine. 

Going towards citrulline involves the production of nitric oxide NO. Now in schizophrenia we see a reduction in the good type of NO, that produced in the endothelium, the cells that line the interior surface of blood vessels and lymphatic vessels. As a result, we vascular dysfunction in schizophrenia.

Agmatine is also elevated in schizophrenia, which may be one of those feedback loops since agmatine will inhibit iNOS, nNOS while increasing eNOS

So where is there a reduction in Arginine in schizophrenia?

Well it looks like it is creatine which takes the hit.

CREATINE ABNORMALITIES IN SCHIZOPHRENIA AND BIPOLAR DISORDER 

“Patients with schizophrenia had a statistically significant reduction in Cr levels as compared with controls; bipolar disorder patients showed no difference in Cr as compared with controls”

In people with elevated arginase a useful strategy might be to use an arginase inhibitor.

Arginase Inhibitors: A Rational Approach Over One Century.

The next paper highlights the arginase inhibitor I favour, which is L-norvaline. The paper is from Kursk university. Kursk gave its name to the nuclear-powered submarine that was lost in the Barents Sea in 2000 and triggered a new international cooperation to rescue stricken submarines. The Battle of Kursk was the largest tank battle of all time and the final major offensive by the Germans against the Russians in World War 2, where Hitler wanted to cut off a large bulge in the front line and trap a lot of Russians. Thanks to some clever English mathematicians, encrypted German communications were readable and the Russians repositioned their forces in advance, allowing them to counter attack. The Allies then invaded Sicily and that was the end for the Germans in Russia. 

Arginase Inhibitor in the Pharmacological Correction of Endothelial Dysfunction

The present research shows expressed endothelium-protective property of arginase inhibitor, L-norvaline, characterized by decrease of coefficient of endothelial dysfunction and the approached its application to a group of intact animals. In other words, L-norvaline prevents the development of systemic endothelial dysfunctions in L-NAME- and methionine-induced NO deficiency.

Age-induced memory impairment (AMI)

Now we move to Polyamines that are on the bottom left my graphic at the start of this post. Spermidine and Spermine are very beneficial derivatives of arginine that most older people will be lacking. Autophagy is the cellular garbage disposal service that is dysfunction in many neurological disorders. We generally want more autophagy.

Spermidine-triggered autophagy ameliorates memory during aging 

The aging process drives the progressive deterioration of an organism and is thus subject to a complex interplay of regulatory and executing mechanisms. Our understanding of this process eventually aims at the delay and/or prevention of age-related pathologies, among them the age-dependent decrease in cognitive performance (e.g., learning and memory). Using the fruit fly Drosophila melanogaster, which combines a generally high mechanistic conservation with an efficient experimental access regarding aging and memory studies, we have recently unveiled a protective function of polyamines (including spermidine) against age-induced memory impairment (AMI). The flies’ age-dependent decline of aversive olfactory memory, an established model for AMI, can be rescued by both pharmacological treatment with spermidine and genetic modulation that increases endogenous polyamine levels. Notably, we find that this effect strictly depends on autophagy, which is remarkable in light of the fact that autophagy is considered a key regulator of aging in other contexts. Given that polyamines in general and spermidine in particular are endogenous metabolites, our findings place them as candidate target substances for AMI treatment.  

Spermine and spermidine reversed age-related cardiac deterioration in rats

Aging is the most important risk factor for cardiovascular disease (CVD). Slowing or reversing the physiological impact of heart aging may reduce morbidity and mortality associated with age-related CVD. The polyamines, spermine (SP) and spermidine (SPD) are essential for cell growth, differentiation and apoptosis, and levels of both decline with age. To explore the effects of these polyamines on heart aging, we administered SP or SPD intraperitoneally to 22- to 24-month-old rats for 6 weeks. Both treatments reversed and inhibited age-related myocardial morphology alterations, myocardial fibrosis, and cell apoptosis. Using combined proteomics and metabolomics analyses, we identified proteins and metabolites up- or downregulated by SP and SPD in aging rat hearts. SP upregulated 51 proteins and 28 metabolites while downregulating 80 proteins and 29 metabolites. SPD upregulated 44 proteins and 24 metabolites and downregulated 84 proteins and 176 metabolites. These molecules were mainly associated with immune responses, blood coagulation, lipid metabolism, and glutathione metabolism pathways. Our study provides novel molecular information on the cardioprotective effects of polyamines in the aging heart, and supports the notion that SP and SPD are potential clinical therapeutics targeting heart disease                                                               

Spermidine boosts autophagy to protect from synapse aging 

Figure 1. summarizes the suggestion that spermidine-triggered restoration of autophagy protects synapses from age-induced changes, and thus delays the normally occurring decline of memory formation. Given that spermidine is a physiologic, easy administrable substance, future research may consider its supplementation to counter age-dependent dementia.

Spermidine operates directly at presynaptic active zone scaffolds (composed of Brp/bruchpilot protein) to allow for an autophagy-dependent homeostatic regulation of these specializations. In effect, spermidine protects learning efficacy from aging-induced decline.                                      

 Spermidine in health and disease

 Having your longevity and eating too

Although caloric restriction has clear benefits for maximizing health span and life span, it is sufficiently unpleasant that few humans stick to it. Madeo et al. review evidence that increased intake of the polyamine spermidine appears to reproduce many of the healthful effects of caloric restriction, and they explain its cellular actions, which include enhancement of autophagy and protein deacetylation. Spermidine is found in foods such as wheat germ, soybeans, nuts, and some fruits and vegetables and produced by the microbiota. Increased uptake of spermidine has protective effects against cancer, metabolic disease, heart disease, and neurodegeneration. 

Although spermidine induces autophagy and autophagy inhibition curtails many of the health-promoting effects of spermidine, additional mechanisms have been proposed to explain the beneficial effects of spermidine on aging. These potentially autophagy-independent mechanisms include direct antioxidant and metabolic effects on arginine bioavailability and nitric oxide (NO) production. However, it has not been formally determined whether these routes act in a completely autophagy-independent manner or are interrelated with autophagy (in an additive or synergistic way) (see the figure), and it will be important to define actionable molecular targets that explain the beneficial effects of spermidine in diverse pathophysiological settings. In this sense, it will also be of interest to explore synergisms of spermidine with other CRMs that initially act through different mechanisms.

It is a surprise that those long-lived Japanese eat Natto? Also, it is a good source of vitamin K2 and importantly it is an estrogen and so an ERβ agonist.

Not all probiotics are helpful to produce polyamines and one well known probiotic, VSL#3, has been shown to reduce their level. Choose your bacteria very carefully. 

Here the probiotic strain Bifidobacterium animalis subsp. lactis LKM512 is used to increase polyamine production

Longevity in Mice IsPromoted by Probiotic-Induced Suppression of Colonic Senescence Dependent on Upregulation of Gut Bacterial Polyamine Production

Alzheimer’s and Arginine

In a fairly recent study it was suggested that the immune system in the brain is being suppressed and the microglia are slightly mutated along with the over-expression of arginase. Arginase is the enzyme that coverts arginine to ornithine plus urea.

So, in Alzheimer’s there will be a lack of arginine available for its other purposes. 

So, we would expect a lack of creatine, agmatine and citrulline. Along the way we should see less Nitric Oxide.

Based on my graphic above, it would seem that L-Norvaline should improve the outcome in Alzheimer’s mice.

We already know that Agmatine improves Alzheimer’s mice, as we now should expect.

So, my cocktail for an aging mouse would be: - 

·        L-Norvaline (used by body builders)

·        Agmatine (used by body builders)

·        Creatine (used by body builders)

·        Natto/wheatgerm/ LKM512 probiotic

·        Vitamin C or NAC

·        Citrulline (used by body builders)

·        Betanin (an approved food colour additive, see below)

Served with cheese, naturally.

A New Potential Cause for Alzheimer’s: Arginine DeprivatiON

Alzheimer’s study suggests immune cells chew up an important amino acid 

Increasingly, evidence supports the idea that the immune system, which protects our bodies from foreign invaders, plays a part in Alzheimer’s disease. But the exact role of immunity in the disease is still a mystery. A new Duke University study in mice suggests that in Alzheimer’s disease, certain immune cells that normally protect the brain begin to abnormally consume an important nutrient: arginine. Blocking this process with a small-molecule drug prevented the characteristic brain plaques and memory loss in a mouse model of the disease. Published April 15 in the Journal of Neuroscience, the new research not only points to a new potential cause of Alzheimer’s but also may eventually lead to a new treatment strategy. “If indeed arginine consumption is so important to the disease process, maybe we could block it and reverse the disease,” said senior author Carol Colton, professor of neurology at the Duke University School of Medicine, and a member of the Duke Institute for Brain Sciences. The brains of people with Alzheimer’s disease show two hallmarks -- ‘plaques’ and ‘tangles’ -- that researchers have puzzled over for some time. Plaques are the build-up of sticky proteins called beta amyloid, and tangles are twisted strands of a protein called tau. In the study, the scientists used a type of mouse, called CVN-AD, that they had created several years ago by swapping out a handful of important genes to make the animal’s immune system more similar to a human’s. Compared with other mice used in Alzheimer’s research, the CVN-AD mouse has it all: plaques and tangles, behaviour changes, and neuron loss. In addition, the gradual onset of these symptoms in the CVN-AD mouse gave researchers a chance to study its brain over time and to focus on how the disease begins, said the study’s first author Matthew Kan, an MD/PhD student in Colton’s lab. Looking for immune abnormalities throughout the lifespan of the mice, the group found that most immune system components stayed the same in number, but a type of brain-resident immune cells called microglia that are known first responders to infection begin to divide and change early in the disease. The microglia express a molecule, CD11c, on their surface. Isolating these cells and analyzing their patterns of gene activity, the scientists found heightened expression of genes associated with suppression of the immune system. They also found dampened expression of genes that work to ramp up the immune system. “It’s surprising, because [suppression of the immune system is] not what the field has been thinking is happening in AD,” Kan said. Instead, scientists have previously assumed that the brain releases molecules involved in ramping up the immune system, that supposedly damage the brain. The group did find CD11c microglia and arginase, an enzyme that breaks down arginine, are highly expressed in regions of the brain involved in memory, in the same regions where neurons had died. Blocking arginase using the small drug difluoromethylornithine (DFMO) before the start of symptoms in the mice, the scientists saw fewer CD11c microglia and plaques develop in their brains. These mice performed better on memory tests. “All of this suggests to us that if you can block this local process of amino acid deprivation, then you can protect -- the mouse, at least -- from Alzheimer’s disease,” Kan said. DFMO is being investigated in human clinical trials to treat some types of cancer, but it hasn’t been tested as a potential therapy for Alzheimer’s. In the new study, Colton’s group administered it before the onset of symptoms; now they are investigating whether DFMO can treat features of Alzheimer’s after they appear. Does the study suggest that people should eat more arginine or take dietary supplements? The answer is ‘no,’ Colton said, partly because a dense mesh of cells and blood vessels called the blood-brain barrier determines how much arginine will enter the brain. Eating more arginine may not help more get into the sites of the brain that need it. Besides, if the scientists’ theory is correct, then the enzyme arginase, unless it’s blocked, would still break down the arginine. “We see this study opening the doors to thinking about Alzheimer’s in a completely different way, to break the stalemate of ideas in AD," Colton said. "The field has been driven by amyloid for the past 15, 20 years and we have to look at other things because we still do not understand the mechanism of disease or how to develop effective therapeutics

The full study: -

Arginine deprivation and immune suppression in a mouse model of Alzheimer's disease.

The pathogenesis of Alzheimer's disease (AD) is a critical unsolved question; and although recent studies have demonstrated a strong association between altered brain immune responses and disease progression, the mechanistic cause of neuronal dysfunction and death is unknown. We have previously described the unique CVN-AD mouse model of AD, in which immune-mediated nitric oxide is lowered to mimic human levels, resulting in a mouse model that demonstrates the cardinal features of AD, including amyloid deposition, hyperphosphorylated and aggregated tau, behavioral changes, and age-dependent hippocampal neuronal loss. Using this mouse model, we studied longitudinal changes in brain immunity in relation to neuronal loss and, contrary to the predominant view that AD pathology is driven by proinflammatory factors, we find that the pathology in CVN-AD mice is driven by local immune suppression. Areas of hippocampal neuronal death are associated with the presence of immunosuppressive CD11c(+) microglia and extracellular arginase, resulting in arginine catabolism and reduced levels of total brain arginine. Pharmacologic disruption of the arginine utilization pathway by an inhibitor of arginase and ornithine decarboxylase protected the mice from AD-like pathology and significantly decreased CD11c expression. Our findings strongly implicate local immune-mediated amino acid catabolism as a novel and potentially critical mechanism mediating the age-dependent and regional loss of neurons in humans with AD.

So Arginine for Alzheimer’s? Not so simple

Eating more arginine is not an effective way to increase the level of arginine in your brain and also the high level of arginase might just soak it all up anyway.

Other science does suggest that there are other ways to increase the amount of arginine in your brain, such as L-citrulline.  We have already seen that we can inhibit arginase with L-norvaline among other things.

Betanin for Alzheimer’s

Since we are on Alzheimer’s, we might as well include another clever idea.

Our reader Tyler highlighted another interesting Alzheimer’s study, which suggests preventing/treating Alzheimer’s with Betanin, the pigment in beet root.

This might sound mad, but is deadly serious. The research showed that Betanin inhibits the formation of the trademark beta-amyloid plaques that define Alzheimer’s. No plaques, no Alzheimer’s.

Vegetable compound could have a key role in ‘beeting’ Alzheimer’s disease

Beetroot has already been featured in this blog; it has numerous health benefits.

To lower blood pressure and increase exercise endurance it is the nitrates that are helpful, but beetroot has numerous other effects; it even increases insulin sensitivity, so is a good choice for diabetics and pre-diabetics.

The Potential Benefits of Red Beetroot Supplementation in Health and Disease

Betanin without the beetroot?

Betanin has such a strong colour it is used commercially as a food colourant, it appears as E162 on the label. In Europe it is called Beetroot red E162 and is inexpensive.

Personally, I take my betanin with the rest of the beetroot. 

Vascular Dementia - before I forget

Vascular dementia is the easiest type of cognitive impairment to understand. Reduced blood flow to the brain, most likely due to reasons including a loss of endothelial nitric oxide, effectively starves the brain. We saw how cocoa flavanols improve blood flow and hence mild cognitive impairment, this is via an NO-dependent mechanism that nobody fully understands. In autism things get more complicated and we saw in earlier posts that we seem to have unstable blood flow rather than just reduced blood flow. Nonetheless, improving cerebral blood flow may well be useful for some people with autism; so more eNOS and not too much arginase, cocoa flavanols may well be beneficial. Antioxidants are hopefully already being taken.

Conclusion

I was surprised just how much in the post can be implemented today with no prescription medication.

It is no surprise that certain diets (Mediterranean/Okinawan) promote not only longevity but also an extended healthy life expectancy.

I think there are some tips here for fine tuning out of balance brains found in autism, schizophrenia and bipolar.

I hope someone trials my cocktail on an Alzheimer’s mouse and a regular older mouse. 

·        L-Norvaline and Citrulline

·        Agmatine

·        Creatine

·        Natto/wheatgerm/ LKM512 probiotic

·        Vitamin C or NAC

·        Betanin


I suspect this cocktail would be more effective than Donepezil or Memantine, neither of which address the underlying cause of Alzheimer's disease. In reality some of the above might not even be needed (e.g. creatine and citrulline).

Agmatine as an alternative for some people who respond to intranasal insulin is an interesting idea. Research seems to have stalled because the preservative in the insulin causes irritation inside the nose.

Note: Creatine deficiency is a known cause of MR/ID/Autism and some types are treatable  https://creatineinfo.org/. It is detectable by Magnetic Resonance Spectroscopy or by measuring creatine levels in plasma and urine. Babies born with creatine deficiency may exhibit hypotonia (floppy baby syndrome) due to weak muscles.