Semi related topics show imuno – The Solution actions angiogenesis, disease removal, & toxicity movement

These are semi related topics.  What is similar to all of them is angiogenesis and disease removal.  The keys to similarity of all of these topics us that these topics don’t make money for the pharmaceutical industry so no research.  Which means no ability to talk professionally about them.

-Blood vessel video on angiogenisis 

Ted talk about how our bodies can go wrong by not trimming or growing blood vessels and capillaries as we need them.  

William Li presents a new way to think about cancer treatment: angiogenesis, targeting the blood vessels that feed a tumor. The crucial first (and best) step: Eating cancer-fighting foods that beat cancer at its own game.  (This is much bigger than cancer.  Anytime something goes wrong, it can either be deficient or excessive.

https://www.youtube.com/watch?v=B9bDZ5-zPtY                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          

-fighting cancer with veterinarian remedies – deworming medicine from animal studies.

Next we explore how about this same problem of angiogenesis and the capillaries with a drug that makes it easier for our bodies to manage keeping them in line.  (We are not surprised that this leads to cancer free.)

https://www.mycancerstory.rocks/single-post/2016/08/22/Shake-up-your-life-how-to-change-your-own-perspective

-The use of melatonin for cancer.  

This is really important to people who are restless.

If cancer has something to do with capillaries getting out of hand like a garden full of weeds, then this naturally occurring hormone can help us manage all of these issues as well.  What Is most important about this research to me is that REM sleep is essential for the brain’s system to do it’s daily clean & drain.  https://www.youtube.com/watch?v=Roh4lQXneQg&feature=youtu.be.  then see the neurologist’s long ramble below from Stasha Gominak about how important it is to sleep.  

-Restoration of the liver

Burt Berksonm MD is able to rehabilitate livers that the AMA says should be transplanted.    He restores livers easily and quickly with some antioxidants.  Alpha lipoic acid regrows organs and stops cancer.  Rapid liver regrowth, including vascular regrowth. (There is the angiogenesis again…). Hepatitis D, Reduces inflammation, Kills cancer, Lupis, autoimmune hepititus, Oxidative stress 

Books on PDF link here http://simplymimi.net/archives/1477

Mimi also put the right products together in a capsule called Liver-Alive.  Care must be taken to get the right quality and type of ALA.  Berkson is clear about the type.  I have complied, tested and realize that this is true.  My products reflect that.  Warning from the Doctor:   imported less expensive Chinese product is not sufficient and could do damage to the liver instead of repair it.

-Video of Neurologist speaking about hibernation and biome sources D B etc.

Vitamin D, Deep sleep & Gut Bacteria w/ Dr. Stasha Gominak

1 hour and 25 mins.  It kind of rambles as she feels her way through her 

discovery but I find it really interesting.

https://www.youtube.com/watch?v=74F22bjBmqE&feature=youtu.be

My theoretical explorations about angiogenisis causes;

  • Maybe we are holding onto pain and those blood vessels change?  Perhaps we abandon something inside us and heavy metals are the result?  Then the heavy metals or the DNA dis-regulation causes us to not be able to hear and respond to the command to clear the blood vessels, clean the area, remove the problems.  
  • Perhaps were over-use our brains, nerves, etc. or they have viruses in them and the signal to regulate them gets cut?
  • Perhaps our microbiome helps to regulate things and when the heart & metabolism slow down the body creates infection etc. and we end up on antibiotics.

Imuno and how we might understand it a bit more

Chondroitin Sulfate in imuno is more than capable of supplying the usable sulfate ions, (not necessarily the metabolic enzyme itself. )  A see it at www.imunothesolution.com

LEARN ABOUT HOW IT MIGHT BE WORKING   http://simplymimi.net/archives/1555

Autism is the deficiency in a key detoxification pathway that needs sulfur

Autism is the deficiency in a key detoxification pathway that needs sulfur

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How other people use imuno

 

 

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Using imuno – The Cream with neuro-endocrine points

I had a sinus infection so I started with the places that were most tense for me. Although my training (Masanaga) does not believe in treating the tension (Jitsu).  Instead, find the void. (Kyo) treat that.  My greater intuition told me to stay with the tense points for now.

In this figure, you see a point on the eye brow.  You might notice that this area has a very tight muscle.  That’s what to target, on both sides and allow the imuno to get into it.

The ones right to the side of the nose are large intestine 20, also known as Welcome Smell.  I used this point but I added one directly inside the nose at the bottom and I told my imuno cream to penetrate into the tight condition there.

I also used the last point near the nose but I pressed the face into the cheek bone and put the imuno cream here.

It is important to note that these points might be the same for you but they may also be different!  Be flexible and allow them to present themselves.

 

There are a few other key points that I found myself at.  You should not be put off by how technical this appears.  It is not.  If you put your hands on your face to comfort and massage yourself, where do you go to?  Pay attention to that.  That’s pretty much all I do but I notice where I was brought so I can check it out if I need it again.

What I found on my forehead was the one directly over the eyebrow.  It is very tense and indeed made me feel like I had a headache.  I went up and down that tight muscle and looked at both ends and both sides of the muscle until I could identify where it was most triggered or tense.  I put the imuno there.  (this type of thing can be anywhere, not just at this point. Ask your masseuse.)

 

This picture shows you the last point on my head.  right in front of the ear.  But mine was up a bit different.  I felt where the fleshy fold comes down, then turns into the ear.  I further explored the fleshy part in front of the ear and I found a little opening that goes right inside the skull.  That’s where I put my imuno.  EXPLORE.  It’s not hard to do.  I am just sharing the end result.  I am not over thinking this.

 

lastly, I put some imuno into the ear.  As you can see, the ear covers lots of ground.  Most of the time, I would look for a place that is more direct but… since I am looking for the void, and my ears hadn’t cleared yet and everything sounded like I had my earplugs in, I put some imuno inside my ears.  I remembered blowing my nose and the ears popped, hurt a whole bunch and then I could hear.  As I write this, I realize, they never went back nor needed another hit.  And once it was fixed, I stopped being aware of it.

Auricular acupuncture is not 2000 years old like it’s older sibling.  But China has gotten great results with it in their hospitals for back pain, PTSD, etc.  Now, you don’t need to know how to place the needles or the  seeds.  You can simply use some imuno.

In auricular acupuncture, they tape seeds onto your ear abscess to stimulate the tissue underneath it which is connected to the points.  What I love about auricular is you can see the organs being messed up inside the ear.  Literally!  If you have the guts to look at problems google “aricular abscess” and select images.

What my ear is telling me as I massage it is that my occiput is really tight.   It might be  out of alignment.  I will do some yoga, get some much needed exercise and do some ear massage.  This will give it a chance to re-align.  Of course, some imuno cream in case it can clean the mis-alignment as well.  I am loving my imuno cream as a “soft chiropractic tool”.

 

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Bravo Yogurt and Non-Dairy Drink eliminate toxins from weight loss

From Dr. Ruggiero:

Attached, please find a short presentation explaining the risk of toxicity associated with weight loss in today’s polluted world and the benefits of Bravo as a means to detoxify while loosing weight.  Please feel free to use, share and distribute this presentation.

This presentation was given, booked as a “premiere event” at the  “Toxicity and Longevity Summit” in Chicago on October 26.    The lead researcher,  Dr. Jerry Blythe and myself (Marco Ruggiero) demonstrated how weight loss protocols accompanied by Bravo lead to elimination of toxins (lipophilic chemical toxicants) from the body.

  • Based on recent scientific evidence, weight loss is accompanied by release of toxic, carcinogenic, neurotoxic substances that, in today’s polluted world, accumulate in our fat cells over the years.
  • Dr Ruggiero refers to a paper where the Authors demonstrated that weight loss is accompanied by increase of polychlorinated biphenyls (PCBs) in serum.
    • PCBs are carcinogenic, neurotoxic substances that also disrupt the endocrine system.
    • The Summit creators (who did not use Bravo) demonstrated that lipophilic chemicals like PCBs increased in serum during weight loss.
    • Dr. Ruggiero demonstrated through scientific references that adding Bravo increases the elimination of lipophilic chemicals that include phthalates, dioxin and many other.
    • Bravo demonstrated that it helps elimination of harmful toxicants that accumulate in fat cells over the years and are released in the bloodstream as consequence of weight loss.

Further, The most recent papers of ours clarify the mechanism of action of this formidable effect of Bravo at the genetic (DNA) level.

  • We demonstrated that in Bravo there are hundreds of different probiotic species and, unlike any other probiotic, there are phages and plasmids that confer unique properties.
  • Among these properties, we focused on the role of phages and plasmids in detoxification.
  • We feel that these results on the molecular characterization of Bravo cast a new light on the uniqueness of the product and explain its efficacy.

In particular, we are grateful to provide a safe and effective way to release weight and have the toxins managed as well.  This is also true of Lyme where toxins are liberated as the person begins to detox the condition.

In some cases, the only way to detoxify a weight loss program in today’s polluted world, is to accompany the weight loss program with an efficient detoxification program.  We know that Bravo works thanks to its unique composition.   It proves inimitable for this scope.

I am certain that you realize that the need for stress free, scientific weight loss is enormous.  You may want to convey the scientifically solid message that weight loss without detoxification can be harmful and Bravo is a valid solution.

Very truly yours,

Marco Ruggiero

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Notes from the “Toxicity and Longevity Summit”

Frustrated with traditional approaches to improving health, energy, and the immune system? Searching for ways to turn back the clock, yet nothing seems to work for you? Then don’t miss out on the Toxicity and Longevity Summit! (Full Flyer- in Link) We are lucky to have Marco Ruggier MD, Ph.D., an expert on Autism, Cancer, and the Microbiome with us to educate on not only the best diet to follow- but how to address one of the biggest roadblocks to overall health–TOXINS.

Dr. Ruggiero will present a simple, 2-phase protocol that helped Dr. Jerry Blythe regain optimal health without expensive procedures or significant side-effects. Together, Dr. Ruggiero, Dr. Stefania Paccini, and Dr. Blythe published a well-received, peer-reviewed study on the protocol and its findings.

Blythe Study Who Should Attend? **Anyone looking for cutting edge strategies to improve health and longevity. **Physicians who are looking for effective holistic solutions for their patients.

Additional Summit Speakers Peter Greenlaw,  Summit Host is a leading expert on toxins, weight loss, and longevity. Peter is the author of Why Diets are Failing Us (with Dr. Dennis Harper), TDOS Syndrome and Solutions (with Dr. Nicholas Messina), and Your Third Brain (with Dr. Marco Ruggiero) Jerry Blythe, MD, is a renowned speaker on toxins and healthy aging. A 72- year old marathon runner, Dry Blythe will share his life-changing experience from following this 2-phase protocol.

Can’t Make it in person? Register for the Broadcast and get the recording.  Hi there, You are invited to a Zoom meeting. When: Oct 26, 2019, 09:30 AM Central Time (the US and Canada) Register in advance for this meeting:https://zoom.us/meeting/register/91d9adf424085e2f7510d14dfea9e911 After registering, you will receive a confirmation email containing information about joining the meeting.

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Article from 2017

Case report: intermittent fasting and probiotic yogurt consumption are associated with reduction of serum alpha-N-acetylgalactosaminidase and increased urinary excretion of lipophilic toxicants

Jerry Blythe1 and Stefania Pacini2

1Retired Medical Doctor, Indianapolis, IN, USA 2Silver Spring Sagl, Arzo-Mendrisio, Switzerland

Corresponding author

Running title: Key words:

Stefania Pacini, MD, PhD. Silver Spring Sagl. Via Raimondo Rossi 24, Arzo-Mendrisio 6864. Switzerland.
E-mail: info@bravo-europe.com

Intermittent fasting and lipophilic toxicants

intermittent fasting; probiotic yogurt; nagalase; detoxification; thyroid

Abbreviations: N-acetyl(2-hydroxypropyl)cysteine (NAHP); 3-Phenoxybenzoic acid (3PBA), N- acetyl phenyl cysteine (NAP), Phenylglycoxylic acid (PGO), Monoethylphthalate (MEP); 2- Hydroxyisobutyric Acid (2HIB); Thyroid Stimulating Hormone (TSH); Dr. Jerry Blythe (Dr. JB); chronic lymphocytic leukemia (CLL); Institutional Review Board (IRB); Gc protein-derived Macrophage Activating Factor (GcMAF).

Abstract

In this study, we describe the changes associated with three months of intermittent fasting and probiotic yogurt consumption in a 72-year-old marathon runner with chronic lymphocytic leukemia for a number of years. Serum alpha-N-acetylgalactosaminidase (nagalase), a marker of inflammation and cancer cell proliferation, was significantly decreased at the end of a three- month observation. These results are consistent with immune modulating properties of certain probiotics based on the fermentation of milk and colostrum. Urinary excretion of non-metal toxicants that accumulate in adipose tissue such as Perchlorate, N-acetyl(2- hydroxypropyl)cysteine (NAHP), 2,4-Dichlorophenoxyacetic acid, 3-Phenoxybenzoic acid (3PBA), N-acetyl phenyl cysteine (NAP), Phenylglycoxylic acid (PGO), Monoethylphthalate (MEP) and 2- Hydroxyisobutyric Acid (2HIB) was significantly increased. These results are consistent with the weight loss (5 Kg) associated with intermittent fasting and with the known features of probiotics as detoxification tools. Consistent with certain toxicants acting as endocrine disruptors, we observed an increased elimination of toxicants and a 33% decrease of serum Thyroid Stimulating Hormone (TSH), suggesting a trend toward normalization of thyroid function. These results support the hypothesis that a combination of intermittent fasting with the consumption of specific probiotic yogurts may lead to immune modulation, detoxification and other improvements.

Introduction

A recent review of the metabolic effects of intermittent fasting published in the July 2017 Annual Review of Nutrition suggests that nutritional regimens may “offer promising nonpharmacological approaches to improving health at the population level, with multiple public health benefits”.(1) This statement is consistent with the conclusions of a study published in 2013 describing how “Intermittent fasting is reported to improve the lipid profile; to decrease inflammatory responses, reflected by changes in serum adipokine levels; and to change the expression of genes related to inflammatory response and other factors”.(2) The interest in intermittent fasting as a nutritional approach to weight loss and a number of health issues, is demonstrated by the number of peer-reviewed papers on this topic. A PubMed query for “intermittent fasting” performed in July 2017, yielded, yielded 755 results with a quasi- exponential increase in the number of papers in the past 5 years.

Intermittent fasting appears particularly efficient in achieving sustained weight loss and enhancing body composition by reducing fat mass.(3) And, a reduction of fat mass is often further associated with a release of lipophilic toxicants that may have accumulated in adipocytes(3); such a release may pose a health danger if it is not accompanied by appropriate detoxification procedures. Among the different detoxification approaches, interventions involving modulation of the microbiota appear particularly promising as they target a system, in this instance, the microbiota that is involved in all aspects of human physiology. Such interventions have received a great deal of attention in the past few years.(4,5)

Thus, it has been known for more than 10 years that probiotic microbial strains have properties that enable them to bind metals and toxins from food and water; interestingly, binding of toxicants by probiotics appears to be instantaneous, thus leading to the proposal of exploiting these features for decontamination in food and intestinal models.(6)

Based on these premises, Dr. JB, a retired Medical Doctor, embarked on a three-month experience of intermittent fasting and probiotic yogurt consumption with the goal of weight loss and detoxification. In this study, we describe the changes associated with such a nutritional approach.

Subject and methods

Dr. JB was born through Cesarean section in 1945, was fed with formulas, and suffered several early childhood infections, including pneumonia at the age of 5, treated with chloromycetin (one of the first commercial uses of chloramphenicol was chloromycetin in 1949). The drug is seldom used today because of potential toxicity and bone marrow depression. At the age of 6, he developed a passion for running that continues to this day. He kept logs of his mileage and calculates that he has run approximately 112,000 miles (180,246 Km) including a number of marathons; his remarkable achievements have recently been described in the journal, The Socionomist.(7)

He has been healthy and energetic throughout his adult life without illness other than Typhoid Fever acquired outside the United States in 1970 and treated with chloramphenicol. His bone marrow suffered a residual leukopenia and lymphocytosis which appear to have not affected his daily activities. In 2012, he was diagnosed with chronic lymphocytic leukemia (CLL) at an Indianapolis Cancer Center, and found to have a 13q14.3 deletion. Retrospective study of blood analyses, however, suggests that the condition had been evolving over a number of years and diagnosable as CLL since 2008 when for the first time he developed a leukocytosis along with his lymphocytosis. After careful consideration, Dr. JB opted for complementary/alternative approaches for his condition with particular focus on healthy nutrition and exercise, avoiding refined sugars, alcohol, carbonated drinks (sodas), cookies, pies, candy or cakes; he maintained his consistent training program that he is still implementing, running on average 6 miles per day at a comfortable pace.

In March 2017, JB embarked on an intermittent fasting program patterned on the weight loss program described by He et al.(3) Shortly thereafter, he introduced daily consumption of 100 ml of probiotic yogurt aimed at supporting the immune system and helping detoxification, choosing a product whose properties have been previously described.(8,9)

Blood and urine analyses were performed immediately before and after the three-month experience. Analysis of serum alpha-N-acetylgalactosaminidase (nagalase) was performed at the European Laboratory of Nutrients, (Bunnik, The Netherlands); the results are expressed as Units = nmol/min/mg. Blood analyses were performed at Lab Corp, Laboratory Corporation of America. Analyses of toxicants in early morning urine samples were performed at The Great

Plains Laboratory Inc. (Lenexa, KS, USA) and are expressed as μg/g creatinine. Copies of the original records are conserved at the office of Dr. JB.

Since this is a single case report that does not produce generalizable knowledge, nor is it an investigation of an FDA regulated product, Institutional Review Board (IRB) review is not required for this activity.(10) Likewise, since this case report described the experience of one of the Authors, Dr. JB, MD, the concept of “informed consent” is implicit in the authorship of the study.

Results

After three months of intermittent fasting and probiotic yogurt consumption, Dr. JB’s body weight decreased from 70.7 to 66.2 Kg (6.4%). Serum nagalase values significantly decreased from 1.92 to 1.06 U (44.8%), a value that is very close to the upper normal value set by the laboratory (0.95). Serum TSH values also significantly decreased from 7.83 to 5.2 μIU/ml (33.6%). Leucocyte count decreased from 58 to 54.9 (cells x 103/μL) (5.3%) and the percentage of lymphocytes decreased from 94 to 90% (4.4%). The number of platelets favorably increased from 93 to 125 (cells x 103/μL) (34.4%).

Concomitant with these changes, urinary excretion of a number of lipophilic toxicants significantly increased (Table 1).

______________________________________________________________________________

Table 1
Non-metal toxicants in urine______________________________________________________________________________

Compound Before After

______________________________________________________________________________

Monoethylphthalate (MEP) 2-Hydroxyisobutyric acid (2HIB) Phenylglycoxylic acid (PGO) Perchlorate

1,925 3,353 4,364 7,349 36 184 3.5 13 N.D. 0.11 70 135 0.18 1.7 N.D. 0.27

N-acetyl phenyl cysteine (NAP)
N-acetyl(2-hydroxypropyl)cysteine (NAHP)
2,4-Dichlorophenoxyacetic acid
3-Phenoxybenzoic acid (3PBA) ______________________________________________________________________________ Values are expressed as μg/g creatinine; the first term (before) refers to the value observed before intermittent fasting and probiotic yogurt consumption; the second term (after) refers to the value observed after the three-month experience.______________________________________________________________________________

It is worth noticing that the toxicants whose excretion was increased concomitantly with implementation of intermittent fasting and probiotic yogurt consumption, are among the most common environmental pollutants. It may be argued that elevated initial levels of phthalates were associated with Dr. JB’s consistent running and consumption of water from years of soft, plastic water bottles possibly containing phthalates. Interpretation of initial elevated level of 2HIB is more complex. Thus, 2HIB is formed endogenously as a product of branched-chain amino acid degradation in patients with ketoacidosis.(11) However, 2HIB is also a metabolite of gasoline additives(12), and elevated levels of 2HIB can indicate exposure to environmental pollution, exposure that could be associated with activities such as running in urban environments. Since Dr. JB had no clinical or laboratory signs of ketoacidosis, we interpret the elevated levels of 2HIB as consequence of environmental exposure.

Discussion

The changes observed during a three-month experience of intermittent fasting and probiotic yogurt consumption seem to support the hypothesis that such a nutritional approach provides health benefits ranging from weight loss to detoxification and immune system support.

Decrease of nagalase can be interpreted as the result of modulatory activities of the probiotic yogurt on the immune system. It is known that fermentation of milk leads to formation of immune-modulatory and anti-oxidant molecules, and as such, may play a role in decreasing the inflammatory status responsible for the elevated nagalase levels.(13) In addition, the probiotic yogurt used in this study contains bovine colostrum, a supplement known to have intrinsic immune-modulating properties(14) and to be rich in vitamin D-binding protein that is the precursor of Gc protein-derived Macrophage Activating Factor (GcMAF).(15) It has been proposed that nagalase is a marker of cancer cell proliferation (16) as well as of systemic inflammation.(17) Therefore, the observed decrease of serum nagalase activity, together with the decrease of lymphocytes accompanied by an increase of platelets, may reflect a positive impact of the nutritional approach described herein on the underlying pathologic condition of Dr. JB.

Such a positive impact may also be consistent with the observed decrease of serum TSH values. We interpret this change as a consequence of the increased excretion of toxicants known to act as endocrine disruptors with particular reference to thyroid function. For example, phthalates bind to thyroid hormone receptors altering the signaling of thyroid hormones (18), and perchlorate interferes with iodine uptake with resulting increased production of TSH as compensatory mechanism.(19) Thus, it can be hypothesized that increased elimination of phthalates, perchlorate and other endocrine disruptors may be responsible for the trend toward normalization of thyroid function.

The observed increased urinary excretion of lipophilic toxicants is consistent with recent report describing how such a nutritional plan followed by Dr. JB is associated with an increase of serum concentrations of lipophilic pollutants such as polychlorinated biphenyls, possibly mobilized from adipose tissue.(3) We attribute the increased urinary excretion of lipophilic

chemicals to the effects of the cleansing procedures associated with the plan described in He et al.(3) as well as to the known detoxifying effect of probiotics. For example, Lactobacillus acidophilus, a component of the probiotic yogurt used in this study, is endowed with antioxidant effects, decreasing malondialdehyde and increasing the levels of the antioxidants, glutathione reductase, superoxide dismutase, and glutathione peroxidase.(20) Other probiotic strains provide significant protection against metal toxicity by decreasing the level of toxicants in the liver and kidney, and by preventing alterations in the levels of glutathione peroxidase and superoxide dismutase.(21) Another mechanism of action responsible for the detoxifying effects attributable to probiotic yogurt consumption lays in the observation that lactic acid bacterial strains remove toxins from liquid media by physical binding(22) , and protect against toxins such as polycyclic aromatic hydrocarbons, heterocyclic aromatic amines, amino acid pyrolysates and mycotoxins.(23) In addition, it has been recently demonstrated that probiotics such as those represented in the probiotic yogurt used in this study may improve kidney function thus contributing to the overall detoxifying effect. (24)

The authors wish to emphasize that the data in this case report are from one individual, and not part of a larger study. Like Aronson who encourages reports of single cases in medicine(25) , the authors believe the history and observations may benefit those addressing the impact of environment on health.

Conclusions

The experience of Dr. JB suggests that intermittent fasting associated with probiotic yogurt consumption may lead to body weight reduction, increased excretion of toxicants and modulation of the immune system with overall positive effects on health.

Acknowledgements

The Authors wish to thank Mr. Peter Greenlaw, author of books on healthy nutrition, who introduced Dr. Blythe to the nutritional plan described in this study, and connected him with Dr. Pacini after completion of the three-month experience.

References

1. Patterson, R.E., Sears, D.D. Metabolic effects of intermittent fasting.
Annu. Rev. Nutr., Jul 17. doi: 10.1146/annurev-nutr-071816-064634. [Epub ahead of print].

2. Azevedo, F.R., Ikeoka, D. and Caramelli, B. Effects of intermittent fasting on metabolism in men. Rev. Assoc. Med. Bras. (1992). 2013; 59:167-173.
doi: 10.1016/j.ramb.2012.09.003.

3. He, F., Zuo, L., Ward, E., and Arciero, P.J. Serum polychlorinated biphenyls increase and oxidative stress decreases with a protein-pacing caloric restriction diet in obese men and women. Int. J. Environ. Res. Public Health; 2017 14: 59. doi:10.3390/ijerph14010059.

4. NIH HMP Working Group, Peterson, J., Garges, S., Giovanni, M. et al. The NIH Human Microbiome Project. Genome Res. 2009; 19:2317-2323.
doi: 10.1101/gr.096651.109.

5. Kieffer, D.A., Martin, R.J. and Adams, S.H. Impact of dietary fibers on nutrient management and detoxification organs: gut, liver, and kidneys. Adv. Nutr. 2016; 7:1111-1121. doi: 10.3945/an.116.013219.

6. Ibrahim, F., Halttunen, T., Tahvonen, R. and Salminen, S. Probiotic bacteria as potential detoxification tools: assessing their heavy metal binding isotherms.
Can. J. Microbiol. 2006; 52:877-885.

7. Blythe, J., Mailbag@. The Socionomist. March 2017, 10-11.

8. Pacini, S., Punzi, T, Morucci, G. and Ruggiero, M. Macrophages of the mucosa-associated lymphoid tissue (malt) as key elements of the immune response to vitamin d binding protein- macrophage activating factor. It. J. Anat. Embryol. 2011; 116:136.

9. Schwalb, M., Taubmann, M., Hines, S., Reinwald, H., and Ruggiero, M.
Clinical observation of a novel, complementary, immunotherapeutic approach based on ketogenic diet, chondroitin sulfate, vitamin D3, oleic acid and a fermented milk and colostrum product. Am. J. Immunol. 2016; 12:91-98. doi:10.3844/ajisp.2016.91.98.

10. John Hopkins Medicine. 102.3 Organization Policy on Single Case Reports and Case Series. http://www.hopkinsmedicine.org/institutional_review_board/guidelines_policies/organization _policies/102_3.html
Accessed August 16, 2017.

11. Konkoľová, J., Chandoga, J., Kováčik, J., et al. Severe child form of primary hyperoxaluria type 2 – a case report revealing consequence of GRHPR deficiency on metabolism. BMC Med. Genet. 2017; 18: 59. doi:10.1186/s12881-017-0421-8.

12. Benson, J.M., Tibbetts, B.M. and Barr, E.B. The uptake, distribution, metabolism, and excretion of methyl tertiary-butyl ether inhaled alone and in combination with gasoline vapor. J. Toxicol. Environ. Health A. 2003; 66:1029-1052.

13. Ebner, J., Aşçı Arslan, A., Fedorova, M., Hoffmann, R., Küçükçetin, A. and Pischetsrieder, M. Peptide profiling of bovine kefir reveals 236 unique peptides released from caseins during its production by starter culture or kefir grains.
J. Proteomics. 2015; 117:41-57. doi: 10.1016/j.jprot.2015.01.005.

14. Shing, C.M., Hunter, D.C. and Stevenson, L.M. Bovine colostrum supplementation and exercise performance: potential mechanisms. Sports Med. 2009; 39:1033-1054. doi:10.2165/11317860-000000000-00000.

15. Senda, A., Fukuda, K., Ishii, T. and Urashima, T. Changes in the bovine whey proteome during the early lactation period. Anim. Sci J. 2011; 82:698-706. doi:10.1111/j.1740-0929.2011.00886.x.

16. Thyer, L., Ward, E., Smith, R., et al. GC protein-derived macrophage-activating factor decreases α-N-acetylgalactosaminidase levels in advanced cancer patients. Oncoimmunology. 2013. 2:e25769. doi: 10.4161/onci.25769.

17. Suh, M.J., Tovchigrechko, A., Thovarai, V. et al. Quantitative differences in the urinary proteome of siblings discordant for type 1 diabetes include lysosomal enzymes.
J. Proteome Res. 2015; 14:3123-3135. doi: 10.1021/acs.jproteome.5b00052.

18. Zoeller, R.T., Environmental chemicals as thyroid hormone analogues: new studies indicate that thyroid hormone receptors are targets of industrial chemicals?
Mol. Cell. Endocrinol. 2005; 242:10-15.

19. Leung, A.M., Pearce, E.N. and Braverman, L.E. Perchlorate, iodine and the thyroid. Best Pract. Res. Clin. Endocrinol. Metab. 2010; 24:133-141. doi:10.1016/j.beem.2009.08.009.

20. Bagherpour Shamloo, H., Golkari, S., Faghfoori, Z. et al. Lactobacillus Casei decreases organophosphorus pesticide diazinon cytotoxicity in human HUVEC cell line.
Adv. Pharm. Bull. 2016; 6: 201–210. doi:10.15171/apb.2016.028.

21. Majlesi, M., Shekarforoush, S.S., Ghaisari, H.R., Nazifi, S., Sajedianfard, J. and Eskandari, M.H. Effect of probiotic Bacillus coagulans and Lactobacillus plantarum on alleviation of mercury toxicity in rat. Probiotics Antimicrob. Proteins. 2017; Jan 13. doi:10.1007/s12602-016- 9250-x.

22. Haskard, C.A., El Nezami, H. S., Kankaanpää, P. E., Salminen, S. and Ahokas, J.T.
Surface binding of Aflatoxin B1 by lactic acid bacteria. Appl. Environ. Microbiol. 2001; 67:3086– 3091. doi: 10.1128/AEM.67.7.3086–3091.2001.

23. Zoghi, A., Khosravi-Darani, K. and Sohrabvandi, S. Surface binding of toxins and heavy metals by probiotics. Mini Reviews in Med. Chem. 2014; 14:84-98.

24. Abbasi, B., Ghiasvand, R. and Mirlohi, M. Kidney Function Improvement by Soy Milk Containing Lactobacillus plantarum A7 in Type 2 Diabetic Patients With Nephropathy: a Double- Blinded Randomized Controlled Trial. Iran J. Kidney Dis. 2017; 11:36-43.

25. Aronson, J. K. Unity from diversity: the evidential use of anecdotal reports of adverse drug reactions and interactions. J. Eval. Clin. Pract. 2005; 11:195–208.

Authors’ Contributions

Jerry Blythe, MD: performed the experience described in this study, provided critical input and assisted in revising and improving the paper.

Stefania Pacini, MD, PhD: wrote the first draft of this paper, provided critical input and assisted in revising and improving the paper.

Disclosures

Jerry Blythe discloses no conflict of interest. He bought all the foods and supplements used during his experience and paid for the analyses reported in this study.

Stefania Pacini works as external consultant for Silver Spring Sagl, the company producing the probiotic yogurt used in this experience. She had no prior knowledge of the nutritional plan followed by Dr. Blythe nor of the results of the analyses that were communicated only after completion of the experience.

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Autism is the deficiency in a key detoxification pathway that needs sulfur

Autism is the deficiency in a key detoxification pathway that needs sulfur.

___________

Also see this part of the article

Sulfate and Sulfation

___________

Thus Chondroitin Sulfate in imuno is supplying the usable sulfate ions, not the metabolic enzyme itself.  see it at www.imunothesolution.com

http://www.enzymestuff.com/epsomsalts.htm

Why Use Epsom Salts

Dr Rosemary Waring found most people with autism conditions have a deficiency in a key detoxification pathway. The pathway involves using sulfur in the form of sulfate (known as sulfation). The enzyme involved is phenol sulfur-transferase (PST), but the problem is thought to hinge on an inadequate supply of usable sulfate ions, not the metabolic enzyme itself.

Dr Waring found most children on the autism spectrum are very low in sulfate and may be as low as 15 percent of the amount in neurologically typical people. 

People with low or no ability to convert compounds to sulfate have problems handling environmental chemicals, some medications, and even some chemicals produced within the body. They include people with other conditions such as Alzheimer’s disease, Parkinson’s disease, rheumatoid arthritis, and chemical sensitivities.

Epsom Salts
copyright Kd. 2002 last updated 8.25.05•Why Use Epsom Salts•What are Epsom salts and how do they work

•How to give Epsom salts

•Phenolic foods, additives, and enzymes

•Which enzyme products do not contain fruit-derived enzymes?


Why Use Epsom Salts

Dr Rosemary Waring found that most people with autism conditions have a deficiency in a key detoxification pathway. The pathway involves using sulfur in the form of sulfate (known as sulfation). The enzyme involved is phenol sulfur-transferase (PST), but the problem is thought to hinge on an inadequate supply of usable sulfate ions, not the metabolic enzyme itself.

Dr Waring found that most children on the autism spectrum are very low in sulfate and may be as low as 15 percent of the amount in neurologically typical people. People with low or no ability to convert compounds to sulfate have problems handling environmental chemicals, some medications, and even some chemicals produced within the body. They include people with other conditions such as Alzheimer’s disease, Parkinson’s disease, rheumatoid arthritis, and chemical sensitivities. 

The PST sulfation pathway is necessary for the breakdown and removal of certain toxins in the body. This includes the processing of a type of chemical called a phenol. Phenols are a regular and necessary part of life. All foods contain some phenolic compounds. However, some foods have a much higher content than others do. If the sulfation pathway is not functioning well, a person may not be able to process out the phenolic compounds as fast as they consume them. There is a cumulative effect. When the phenols start backing up in the system, it can cause a myriad of negative reactions. Symptoms of phenol intolerance include night waking, night sweats, irritability, eczema, and other skin conditions. The symptoms of phenol intolerance and yeast may be very similar because they both involve the body trying to deal with toxins.

This detoxification pathway processes other phenolic compounds including salicylates (salicylates are a subset of phenols), artificial food colorings, artificial flavorings, and some preservatives. Besides requiring PST, research has found the salicylates further suppress the activity of any PST enzyme present, making matters worse. Food dyes also have been shown to inhibit the PST enzyme.

First Part

You can unclog this ‘bottleneck’ in one of two ways. One is reducing the amount of phenols and toxins entering the body. This is the basis of the Feingold Program or diet. The Feingold Program removes the hard-to-process artificial colorings, flavorings, and three preservatives. It also removes the most problematic of the salicylate foods at the beginning of the program. Later in the program, you may be able to add these salicylate foods back after testing them one at a time. The foods targeted by the Feingold Program and their effects on hyperactivity in children have been extensively studied. Eliminating these chemicals has been effectively helping many children with all sorts of behavior problems for many years, although the reasons why are just now beginning to be understood. 

Feingold/Failsafe Programs

There is an abundance of studies in the references that specifically show that eliminating these types of chemicals significantly improve neurological problems in children. 

www.feingold.org

note: look in the Research section at the top of the page at the above link. This site contains information on the possible symptoms from various food and environmental chemicals also. 

A literature review by Kidd (2000) concludes that although the exact cause of attention deficit conditions is unknown, the current consensus is that genetics plays a role. Other major contributors include adverse responses to food additives, intolerances to foods, sensitivities to environmental chemicals, nutrient deficiencies, and exposures to neurodevelopmental toxins such as heavy metals. This sounds exactly like the factors contributing to autism, migraines, sensory integration issues and other related conditions.

Second Part

The second method of enhancing the detoxification process is to supply more sulfate. This increases the amount of toxins processed out. Sulfate ions may not be absorbed well from the gut, so simply giving more sulfur directly by swallowing supplements may not produce satisfactory results. Some people have seen improvements by supplementing with the sulfur-containing amino acids cysteine and taurine, or MSM (methysulfonylmethane), or by using one of the many commercially available MSM creams. However, others have not found this tolerable. This may be because their body is unable to convert the sulfur to the needed sulfate form.

Most people do see improvement with Epsom salts because the form of sulfur in the Epsom salts is already sulfate and readily available to the body.

What are Epsom salts and how do they work?

Epsom salts are magnesium sulfate. Salts are just molecules that form because the parts have opposite electrical charges that bind together. Magnesium has a positive charge. Sulfate has a negative charge, and performs all sorts of unique biological functions. The two elements dissociate in solution (English translation: break apart and separate in liquid). Epsom salts are available at most local grocers or health food stores, or inexpensively in bulk at agricultural supply stores. 

The magnesium and sulfate in the salts are absorbed into the body through the skin. Because the sulfur is already in the sulfate form, it does not need to be converted like other forms of sulfur do. Sulfate is thought to circulate in the body up to about nine hours. Any Epsom salts left on the skin may continue to be absorbed as long as it is still on the skin, offering continuous ‘timed-released’ input into the bloodstream – like medications given through skin patches. Many people on a typical ‘modern’ processed diet are very deficient in magnesium as well, which Epsom salts also supply in a highly available form. Main effects of insufficient magnesium are hyperness, irritability, anxiety, and muscle twitching or spasms. So the salts may provide two-way assistance.

see Magnesium

How to give Epsom salts

Here are several methods for giving Epsom salts. The ratio is not exact, just what seems to get the salts dissolved and on the skin.

Epsom salt baths – Most people use about one to two cups per tub. Dissolve the salts in hot water first and then fill the tub to about waist deep, as warm as possible. The amount of salts you may find works best will depend on the individual tolerance, the temperature of the water, and the size of the tub. The warmer the water and larger the tub, the more salts will dissolve. If you see negative reactions, such as irritability or hyperactivity, then decrease the amount of salts. You may need to start with as little as one tablespoon of salts, and work up gradually. Epsom salts baths are very calming for most people. This works well just before bedtime. Most guides say to soak for about 20 minutes or more. It is okay to let the salts dry on the skin. You may notice a dry clear-white powder. If it is too itchy or irritating, just rinse it off. If the skin feels too dry, use lotion or oils to moisturize. Diarrhea or loose stools may result if children drink the bath water.

Spray – Mix one part salts and one part water (add more water if the salts are not dissolved) and put in a spray-squirt bottle. Mist the person’s chest and/or back and let it dry on the skin. This method works well in the summer.

Footbath – Mix one part salts to two parts water (or more so the salts dissolve) and let the person soak their feet in it. My boys would soak their feet about 30 minutes while they did reading or homework.

Homemade lotion – This is my favorite at the moment. Cheap and easy.

Recipe 1 from Karen D: Heat some Epsom salts with a little water to dissolve them. I put about one teaspoon of water in three tablespoons of salts and microwave for a minute or so. Add more water if necessary. Then mix this into around four ounces of any lotion or cream you like. I have used suntan lotion, handcream, cocoa butter, body lotion, aloe vera cream, whatever I find that is on sale or inexpensive without the chemicals I am trying to avoid. This seems to work better if the cream or lotion is water-based rather than oil-based. Good buys are at the local grocer in the lotion section. Apply to skin anywhere as often as desired. Some new commercially prepared Epsom salt creams are available but can be very expensive and may contain chemicals that are not tolerated.

Recipe 2 from Rubby: Well – my recipe for the Epsom salt cream is quite unscientific. I don’t really measure my ingredients – I just add a bit of everything until I have the consistency I like.

Ingredients:

Hot water – approximately 50ml

Epsom Salt – approximately 4-5 tablespoonfuls (I keep on adding the salt to the water for as long as it dissolves – usually 5 tblsp)

White Petroleum Jelly – 5-6 tblsp (or more ??)

Natural Cocoa Butter Cream – 2-3 tblsp

I start by adding the salt into the hot water and boiling it for a few minures (make sure the salt is dissolved), then I add the Petroleum jelly and mix it all with a hand mixer (one you would use to whipp cream); once I get a white, creamy mixture, I add some cocoa butter cream and mix again.And that’s it. I get approximately 250 – 350 ml cream. I use it only once a day, on days when we don’t do a bath. I use it to massage my daughter’s back, her chest and her legs (with a focus on her feet – she loves it). Somethimes, I add in a few drops of Lavander Oil.

The cost – minimal. I buy my local pharmacy brand (in Toronto – Shoppers Drug Mart – “Life”) Petroleum Jelly (500 gr.) $3 (CAD), Coca Butter Cream (400 ml) $3 and Epsom Salt (1kg) $3.5 (CAD). I think that the two creams I use will make at least 3 Epsom salt mixtures, which means that my cream costs me approximately $2-3 (CAD). And it lasts me a long time – even though I try to put on my daughter as much as possible.

Epsom salt oil – Neither of my sons nor I liked the salty film left on the skin after a bath (felt itchy). I mixed some coconut oil in with the salts and water. Actually, it is more oil than water. Three tablespoons water plus four tablespoons salts plus 12 tablespoons coconut oil. The coconut oil is good for the skin anyway and it seems to counter the drying effect of the salts. I found that just mixing the salts and oil did not dissolve the salts, so I needed to add some water. I apply this liberally on the skin and it soaks in plus leaves the skin smooth and soft. Adjust the quantity of salts to your liking.

Sponge – A solution of one part salts to four parts water works well. Dampen a sponge in the mixture and apply to any part of the body.

Poultice or skin patch – You can mix some Epsom salts and whatever kind of lotion the person can tolerate into a paste. Put this paste on a large bandaid and apply to the skin. The salts will soak into the skin.

High phenolic foods, chemical additives, and enzymes

Phenols and salicylates do not occur in the same intensity in all foods. Those parents on the Feingold diet point out some studies rank foods by the quantity of phenols present in a food as very low, low, medium, high, and very high. These are not absolute values or correspond with exact toxicities or reactions to the foods. Rather it is included only as a guide. The Feingold literature also notes that salicylates are cumulative in the body, and may only be processed out at a certain rate. So, if you consume more than the body can process out, you get a reaction. 

Regarding the other chemicals, even small amounts of coloring or other chemicals may cause a reaction, which indicates some sort of pharmacological effect as well. For people who are sensitive to phenols, a strong broad-spectrum enzyme product may help somewhat with phenolic foods. Several parents found they could give low quantities of some phenols, but needed to keep track of the total phenol load for the day, or week. Enzymes may be helping some by breaking down a wide array of foods, or by releasing more sulfur, magnesium, and molybdenum which are helpful in processing phenols. 

In April 2002, No-Fenol became available. It is a very unique enzyme mixture just for assisting with the digestion of highly phenolic foods, including fruits, chemicals, and artificial additives. No-Fenol performed very well in months of preliminary tests with phenolic-sensitive children. Since its release, it continues to give excellent results with these foods.

The exact reason No-Fenol helps is not precisely understood. The phenol metabolism, sulfation, and detoxification issues are rather complex. It may not be due so much to the presence of phenols as to the specific structure of these phenols. The research literature indicates that some phenolic compounds are modified by the addition of carbohydrate groups to their structures, which may inhibit their crossing into cells and being metabolized properly. A current hypothesis for why No-Fenol helps may be because the enzymes in this product are able to remove certain carbohydrate groups from the phenols, or otherwise modify their structure, thus allowing normal processing by the detoxification pathways.

see The No-Fenol File

Since fruit-derived enzymes may contribute some phenols into the system, products without the fruit-derived proteases (bromelain, papain, actinidin) may help those concerned about phenols. Enzymedica is one of several companies that makes enzyme products without fruit-derived enzymes or fillers.

Many parents giving these enzymes have said how wonderful it is to be able to give even a low amount of phenols again. Just being able to add foods containing a little bit of fruit greatly expands their child’s menu. Enzymes may help protect against hidden sources of the unwanted phenol compounds. 

Fruits and vegetables are very beneficial in maintaining good health. Flavonoids, beta-carotenes, and other phenolic compounds have been specifically identified as important in preventing an number of illnesses, such as various cancers, and have an important role as effective antioxidants.

see Phenols in the Diet

Sometimes a food may appear to give a ‘phenolic’ or unwanted reaction. It may be because the food actually contains a phenolic-based preservative. At times this turns out to be the case with dairy. Vitamin A palmitate is often added to low-fat or skim milks. The palmitate may be preserved with a phenolic compound. When people switch to a whole milk or milk product without this preservative (or artifical colorings/flavorings) they no longer have a problem with dairy. The same may be true of commercial breads. Often the shortening or pan sprays used in baked goods contain artificial preservatives that are the cause of the problem and not the grains in themselves. This may be the same with other foods as well.

see Feingold Program
see Dairy – the Multi-faceted Substance

Which enzyme products to not contain fruit-derived enzymes?

Enzymes in general can be great! However, a particular person may not tolerate certain fillers, added herbs, particular enzyme ingredient, or even a particular blend.

The fruit-derived enzymes are perfectly fine enzymes which are well-studied and work great for many people. But it is also know that the can be a problem for those that are phenol sensitive, salicylate sensitive, or have detoxification problems. If you are not sensitive to fruit-derived enzymes, the fruit-derived enzymes are not a problem.

If you are having difficulting tolerating enzymes, check to see if it contains fruit-derived enzymes. If so, try a product without the fruit-derived enzymes…not many out there but there are some good choices. Personally, I like Lacto as a starter enzyme, particularly if you have problems with dairy or serious gut injury. But just about any product without the fruit-derived enzymes may work…just start at a lower dose and gradually increase the amount to higher doses over the course of 4-5 days. After a little gut healing, higher levels of proteases aren’t a problem and you can switch around to other products, or add more enzymes in, if you want.

Here is a list of what the products without fruit-derived enzymes I are aware of. Please note that even if a product does not have fruity-derived enzymes, you still need to check to see if it fits your needs or purpose. If anyone knows of other products without fruit-derived enzymes, please let me know and I’ll add it to this reference list:

Enzymedica (the Thera-blends do not contain fruit-derived enzymes):

•Acid Soothe – between or with meals to help with upset or acid stomach

•Allerase – between meals to help with allergies, not intended for food digestion

•Candidase – between meals to help with candida yeast, not intended for food digestion

•Digest – broad-spectrum intended for food digestion

•Digest Gold- broad-spectrum intended for food digestion; 3 times more potency than Digest

•Enhance – intended to help with supplement digestion and absorption

•Lypo – broad-spectrum, no cellulases, high fat digestion; note: this works well if you are taking a time-released medication containing cellulose

•Lacto – broad-spectrum, low proteases, high dairy digestion including casein, lactose and fats in dairy

•MucoStop – between meals to help with excess mucos, helps with respiratory problems, not intended for food digestion

•pH-Basic – intended for perceived pH issues, not intended for food digestion

•V-gest – higher in diets with lots of fiber foods such as roughage, fruits and vegetables; intended for food digestion; some reports of it helping with phenol sensitivities

•Virastop – between meals to help with viruses and systemic cleaning, not intended for food digestion

Houston Nutraceuticals:

•AFP Peptizyde – helps with protease needs, intended for casein, gluten, soy protein problems and other protein foods

•Zyme Prime for SCD – broad-spectrum for food digestion

•No-Fenol – helps with fiber foods, helps with phenol sensitivities

Klaire Labs:

•Vital-Zymes Complete – broad-spectrum for food digestion, higher in proteases for casein, gluten, soy and other proteins

Kirkman:

•Carb-Digest with Isogest™- semi-broad-spectrum intended to help with carb and fiber foods ONLY, no proteases; the n*zymes blend does not contain fruit-derived enzymes

•DPP-IV Forte – boosts DDP IV activity in the presence of other enzymes, helps with a particular bond in casein, gluten, soy; note: DPP IV enzyme activity does not breakdown the entire gluten/casein protein by itself but is essential nonetheless

•Phenol Assist – for fiber foods, intended to help with phenol sensitivities

Pure Essence:

•Candex – between meals to help with candida yeast, not intended for food digestion

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Sulfate and Sulfation

Sulfate and Sulfation

Mimi says:

This really works for me.
I have some water in a water bottle with about a teaspoon in it.
Go by the directions on the package for a guideline and cut it way back.
My tummy rumbles and bloating goes away.

see this important post as well…

Autism is the deficiency in a key detoxification pathway that needs sulfur

page1image1422403232

R.H. Waring, School of Biosciences, University of Birmingham, Birmingham. B15 2TT U.K.

https://www.epsomsaltcouncil.org/wp-content/uploads/2015/10/sulfation_benefits.pdf

Summary

  1.  Sulfate is essential for many biological processes.
  2.  Sulfate is needed for formation of proteins in joints; low levels of sulfate are found in   plasma and synovial fluid from patients with rheumatoid arthritis.
  3.  Sulfate is needed to start the cascade of digestive enzymes released from the pancreas. Without proteases, lipases and amylases, food is not digested efficiently.
  4. Sulfate is essential in forming the mucin proteins which line the gut walls. These have main functions- they stop the gut contents from ‘sticking’ and they block transport of toxins from the gut to the bloodstream. Low plasma sulfate has been found in patients with irritable bowel disease.
  5. Sulfate is necessary for formation of brain tissue. Before birth, the functional units of the brain, ‘neurons’, are laid down on a scaffolding network of sulfated carbohydrate chains. Reduced sulfation can lead to faulty neuronal connections and later dysfunction.
  6. Sulfation is a major pathway in detoxifying drugs and environmental contaminants.
  7. Sulfate is not easily absorbed across the gut wall. Recent research has shown that it can be absorbed across the skin. It is also formed in the body by oxidation of the aminoacids cysteine and methionine. However, this pathway is often sub-optimal and many people benefit from sulfate supplementation.

Background

The process of sulfation involves adding on a sulfate residue, which has two negative charges, to a biological molecule. This leads to a number of ‘knock-on’ effects because the addition of a highly charged polar group can fundamentally alter the molecular configuration and properties. Sulfation can activate or inactivate a wide range of biological compounds and any aberration in the supply of sulfate can have potentially serious consequences.

Principally, sulfation is a major inactivation pathway for catecholamines such as the neurotransmitter dopamine, about 80% of which is sulfated in man (1). Usually, when chemical neurotransmitters are released in the central nervous system, they act at receptor proteins and are then inactivated by sulfation or by FAD-linked mono-oxygenases or alternatively are carried by transporter proteins back into the initiating neurone. Failure of a major pathway such as sulfation will lead to a neurotransmitter imbalance and this can have effects on behavior, mood and function, as can be seen in migraine where many patients have reduced sulfation capacity. The process of sulfation also affects the functioning of peptides and proteins. Mucin proteins, which line the gastrointestinal tract, are sulfated glyco-proteins which control adhesion/lubrication of gut contents and absorption of nutrients (2). They have long peptide backbones with repeating sub-units and also peptide side-chains, rather similar to a ‘bottle-brush’. These amino acid sequences are glycosylated with a range of sugars and both these carbohydrate residues and the peptides themselves are sulfated. As the addition of sulfate residues imparts net negative charges, the proteins adopt an extended configuration to minimise their interactions, since the negative charges repel each other . Removal of the sulfate residues leads to a protein which has a more globular structure and provides less protection for the tissues from the intestinal contents. Reduced sulfation has been linked with gut dysfunction in irritable bowel disease (3) and lower levels of sulfation of the ileal mucins have recently been shown to occur in children with autism (4) and in irritable bowel. This correlates with the finding that gut permeability is increased in a large percentage of autistic children (5). The effects of mucin modification can also be seen in the colonisation of the gut by microflora as sulfation increases resistance to colonisation by pathogenic bacteria (and viruses). It is interesting that Helicobacter pylori, which can colonise the stomach, only does so when it has produced a sulfatase enzyme to de-sulphate the gastric mucins (6). This reduced sulfation of mucin proteins may underlie the relatively common finding of Candida infections in the gut, since the slight negative charges on Candida cells would lead to their repulsion by the negatively charged sulfate groups on normal mucins. The reduction in mucin sulfation is probably an indirect cause of the alterations in gut flora which are often found in gastrointestinal inflammation.

Peptides can also be sulfated, usually on tyrosine residues,by the enzyme tyrosyl protein sulfotransferase (TPST); the gastric hormones gastrin and cholecystokinin are good examples of this pathway. Both are involved in the digestive process and both are activated by sulfation. In a complex cascade, gastrin is sulfated and, with hydrochloric acid from the stomach, causes release of cholecystokinin, which also requires sulfation. Together with peptide fragments released from gastric proteolysis (mediated by the hydrochloric acid), this acts with the peptide hormone secretin on pancreatic tissue to induce the secretion of a range of proteolytic enzymes and also amylase and lipases. Lower levels of pancreatic amylase alter the digestibility of starch-based foods and allow increased fermentation of pathogenic bacteria while the decreased pancreatic lipase activity promotes formation of foul-smelling fatty stools which contain undigested triglycerides. Without the sulfation process to trigger the release of pancreatic proteases such as trypsin and chymotrypsin, the complete digestion of proteins to their amino acid building blocks (proteolysis) cannot take place, so that peptides, rather than amino acids, are found in the gastrointestinal tract. As reduced sulphation of mucins may have made the gut more permeable, the stage is set to allow peptides to penetrate into the blood stream. Some peptides, particularly those derived from casein and gluten, have been found to be neuroactive with effects on the brain where they can act at opioid receptors, affecting behaviour, mood and responses to physical stimuli such as pain. This ‘leaky gut’ hypothesis therefore links with the opioid theory to explain why peptides from casein and gluten in particular seem to cause problems for some people. Although the blood-brain barrier is usually seen as being non-permeable to many compounds it may, like the gut, be ‘leaky’ in individuals with previous head injuries or who are living in stressful conditions. Several studies have reported the presence of brain-derived proteins and antibodies, such as those from myelin, within the peripheral circulation. If relatively large proteins can cross from the brain, it seems possible that peptides and proteins could potentially be transported into the brain, although the mechanisms involved are not known. Simple diffusion across ‘leaky’ gap junctions may be all that is necessary.

The supply of sulfate in man is rarely sufficient to allow synthesis of all the required sulfated biocomponents. There are two main sources of sulfate in vivo. Unlike other anions, such as chloride (Cl-), which seem to be readily and easily absorbed, sulfate is carried into the body from the g.i. tract via a sodium ion-linked sulfate transporter (7). This is easily saturated, so that ingestion of many small divided doses of sulfate over a period of time leads to higher blood sulfate concentrations than when a single large dose is taken. Anaerobic bacteria can convert sulfate (SO42-) into sulfide (S2-); individuals with bowel disease may have a range of pathogenic species of bacteria in the gastrointestinal tract which readily carry out this reduction so any ingested sulfate may be reduced to sulfide and therefore become unavailable. Magnesium sulfate is readily absorbed across the skin so topical applications or bathing may be more successful in raising plasma sulfate levels. Sulfate is also produced in vivo by oxidation of methionine or cysteine, both sulfur – containing amino acids which are provided from dietary proteins, and this pathway probably provides ∼80% of the sulfate required in man. The first stage in this process involves the enzyme cysteine dioxygenase (CDO); cysteine sulfinic acid is formed and undergoes fission to provide sulfite (SO32-) ions which are then further oxidised to sulfate (SO42-) ions by the enzyme sulfite oxidase (SOX). As can be seen, if CDO or SOX have reduced activity, the provision of sulfate will also be decreased. The human CDO gene has 5 exons and 4 introns and is localised to chromosome 5 (5q22-23); it is similar to the rat gene and proteins from the 2 genes have about 90% homology. The gene has a Ptx-3 response element, with a range of cis-acting elements (expressed in the CNS exclusively in dopaminergic neurones). The CDO protein is found in heart, thyroid and kidney, as well as brain and the liver. There seem to be variations in the properties of CDO enzymes from different tissues which may reflect differences in degree of glycosylation. CDO is known to be polymorphic in human populations and there are sub-sets with lower activity (~ 30% of the population) or nul activity (~ 3% of the population) (8). The nul S-oxidisers are heavily over-represented in chronic disease states with an auto-immune component such as rheumatoid arthritis and primary biliary cirrhosis (9,10). Even within ‘normal’ populations there is a wide variation in the extent of conversion of dietary sulfur amino acids to sulfate. In experiments carried out over decades in this department in practical classes with medical students, it was found that increased levels of protein in the diet produced proportional increases in urinary sulfate excretion, as would be expected. However, about 30% of the students reached a plateau in sulfate excretion, despite increasing ingestion of protein, suggesting that some pathway or pathways had become saturated. Provision of sulfate for in vivo metabolism is therefore a rate-limiting step for many people.

In some conditions, large amounts of sulfate are lost from the body in urine; the kidney (as well as the gut) may be ‘leaky’, so that sulfate is not resorbed as would be expected. Sometimes this is linked with an increased excretion of protein/peptides in urine , suggesting that kidney function is impaired. There may, of course, be dysregulation of the sulfate excretion system. On the apical or brush border side of the cells of the renal tubules, the sodium-dependent NaSi-1 sulfate co- transporter removes sulfate from the filtered fluid and this is exchanged for other anions, usually bicarbonate so that sulfate is transported into the blood from the baso-lateral side (11). Another renal sulfate transporter, Sat-1, is a sulfate/bicarbonate/oxalate exchanger and has been found in the brain and is highly expressed in the cerebellum and hippocampus (12). This complicated process suggests that conservation of sulfate anions is critical for human function. It has however been suggested that the kidney transporters respond to the levels of sulfate in the gastrointestinal tract so that sulfated glycosaminoglycans(GAGs) lost from the intestine during chronic infection could release sulfate and so provide a signal to the renal transporter to excrete the anion (13). The sulfate transporters NaSi-1 and Sat-1 are both subject to regulation by a member of hormonal and dietary changes (14). Renal re-absorption of inorganic sulfate is increased in growth and development (15) and regulated by vitamin D, dietary sulfate, glucocorticoids and thyroid hormones (16), also metabolic acidosis (17) and non-steroidal anti-inflammatory drugs (NSAIDS) (18) and chronic potassium depletion (19). To add to a complex interaction, the renal sulfate transporter proteins are themselves sulfated and the brush border contains a matrix of sulfated GAGs, so reduced levels of sulfate in vivo could have the effect of altering the structure of the systems required to retain sulfate in the body. This would of course result in a spiral of increasing dysfunction and it is unclear which are the controlling factors. Further, most of the work on sulfate metabolism has been done with the rat and should be invoked with caution since this species uses different pathways from those seen in human beings.

Not only is there an impaired level of sulfate in many disease states, there is also often a corresponding lack of sulfotransferase activity. These are the enzymes which carry out sulfation of a wide range of substrates. They belong to a super-family which uses PAPS (3′-phosphoadenosine – 5′-phosphosulfate) as co-factor and are widely distributed throughout the body (20). This process is critical for life as a defect in PAPS synthesis is lethal in humans (21) reflecting the wide range of biosubstrates which are sulfated. Sulfotransferases with endogenous substrates are important in tissue development and mice with defects in the Golgi-membrane enzyme which sulfates glycosaminoglycans are non-viable as these polysaccharides cannot be converted to the unique binding sites which are recognised as signals for growth (22, 23). Other mutant mice without the heparan sulphate 20-sulfotransferase gene die from defective kidney development (24) suggesting that the sulfate/sulfation axis is vital in kidney development and function. These enzymes with endogenous substrates are membrane-bound while those which sulfate signal molecules such as steroids, thyroid hormones and neurotransmitters are cytosolic (25). The same enzymes also carry out the second stage in metabolism of drugs and related compounds to give water-soluble derivatives which are more readily excreted. All sulfotransferases have common motifs, as they contain a five-stranded parallel β-sheet flanked by α-helices which contains both the PAPS binding site and also the centre of the catalytic site, structures which are highly conserved throughout evolution. The substrate-binding sites, however, are totally different in the membrane-bound and cytosolic enzymes (26).

The major enzymes responsible for the sulfation of phenols and catecholamines (SULT1A1 and 1A3 respectively) have been mainly studied in blood platelets where activity is approximately co-regulated with other tissues in the body, such as the gut and brain. SULT1A1 and 1A3 have 93% amino acid sequence identity while a third more recently discovered isoform, SULT1A2, differs by only 3 residues from SULT1A1. This is, however, sufficient to give it slightly different properties with regard to substrate repertoire, inhibitor sensitivity and thermal stability. All three genes are located close together on Chromosome 16. SULT1A1 is present in high concentration in the liver, brain, blood platelets and many other tissues while SULT1A3 is also found in platelets and brain but tends to be concentrated in the gastrointestinal tract rather than the liver. Only limited information is available on the distribution of SULT1A2 but it has been detected in the liver (by RNA) and some bladder tumours. There is a wide variation in SULT1A1 activity in human populations, as much as 50-fold having been reported by one group of workers (27). The level of enzyme activity in platelets correlates with the level of protein as detected by immunoblotting. A number of different alleles for SULT1A1 have been found and an alloenzyme with His 213 is less active than one with arginine at the same site. A range of different SULT1A2 alleles are also known and are in a linkage disequilibrium with the SULT1A1 alleles. Polymorphisms do not appear to have been reported for SULT1A3 although investigations on the platelet enzyme activity in families suggest that they may exist, since there is a wide range of activity which is highly heritable (28).

Sulfotransferase activity is known to be altered in some dysfunctional states, for example most patients with migraine have low SULT1A1 and sometimes low SULT1A3 activity (29). They are therefore less able to sulfate dietary phenols and catecholamines in the g.i. tract and in the bloodstream via the platelets as the SULT enzyme is co-regulated in both tissues. Sulfation results in inactivation of amines and these individuals are susceptible to foods which contain substrates for the enzymes (cheese/tyramine, chocolate/phenylethylamine, bananas/serotonin). The increased blood levels of compounds with neurotransmitter activity are thought to ‘trigger’ migraine headaches in those who are already susceptible. It has been shown that individuals who are susceptible to migraine are metabolically unstable (with raised excitotoxic amino acid levels) so that very small changes in blood and brain catecholamine levels are sufficient to provoke a migraine attack. SULT1A1 and 1A3 can also be inhibited by flavonoids (30) and by foods containing these compounds which typically occur in fruit and vegetables (31). The incubation of cytosolic preparations from 35 different fruits and vegetables with platelet sulfotransferases produced a wide range of results . Some cytosols inhibited the sulfation of both test substrates although most appeared to more potently inhibit 4-nitrophenol sulfation than dopamine. Ingestion of citrus fruit, especially oranges, and red wine is often reported as being a migraine ‘trigger’ and the component flavonoids (naringin and resveratrol) are inhibitors of both SULT1A1 and SULT1A3. Further studies with purified flavonoids have shown that some can virtually abolish SULT1A1 activity at very low (less than 100nM) concentrations but at higher concentrations (over 1μM) they appear to act as substrates for SULT1A3. The inhibitory effect can be partially overcome by the presence of magnesium ions. These not only appear to enhance enzyme activity, but also to decrease enzyme sensitivity to flavonoids like quercetin by a factor of about five.

Other sulfotransferases can also be affected. The enzyme tyrosylproteinsulfotransferase (TPST) is membrane-bound and found in most tissues of the body, including the platelet and the gastrointestinal tract, where it is co-regulated (32). The TPST enzyme is a 50-54 kDa integral membrane glycoprotein of the trans-Golgi network. At least 2 forms are known, TPST-1 and TPST-2. The latter is a type II transmembrane protein of 377 amino acid residues, encoded by m- RNA originating from seven exons of a gene located on chromosome 22. A 304- residue segment in the terminal domain of TPST-2 has 75% amino acid homology to the corresponding segment of TPST-1 including conservation of the residues involved in binding PAPS. The enzyme and requires the presence of acidic residues on the amino-terminal side of the target tyrosine (33). The TPST family is highly conserved from the evolutionary standpoint, presumably because the post- translation modification of tyrosine residues in peptides and proteins has been required for millennia. A number of substrates are known and these include the leukocyte adhesion molecule P- Selectin glycoprotein ligand 1 (PSGL-1) which is required for binding to P-selectin on activated endothelium and IgA, IgM, IgCT and complement C4 (34). TPST is the enzyme responsible for sulfation of gastrin and cholecystokinin as well as the sulfation of proto-mucin monomers. Sulfated cholecystokinin (CS) has receptors in the brain as well as the gut and is required for release of the peptide hormone oxytocin. Vitamin B6 (pyridoxal phosphate) has been reported to inhibit both SULT1A1 and TPST. However, studies in this laboratory have shown that, as with quercetin, this inhibition can be blocked by magnesium ions and that a ratio of at least 1:1 magnesium:B6 will restore enzyme activity.

Carbohydrates are also substrates for sulfation, particularly the polysaccharide chains attached to glycosaminoglycans (GAGs). Here, there are specific patterns of sulfation which control a range of functions and vary by cell type. These sulfated GAGs are involved in G-protein signalling, calcium signalling, gap junctions (cell-cell signalling) and in differentiation and growth (35). As the enzymes involved in carbohydrate sulfation have relatively high Michaelis constants (compared with SULT 1A1/2, for example), low levels of sulfate will affect this pathway preferentially. Because sulfated polysaccharides and GAGs are so important in the development of the foetal and neonatal brain, any alteration in their structure may have deleterious effects on their function. Sulfate transport across the placenta increases dramatically around the time of birth when most of the glial cells are being formed and these increased levels of sulfate are associated with formation of astrocytes and oligodendrocytes from progenitor cells (36). It is of interest that children have higher levels of plasma sulfate than adults (0.47 nmol/l at birth decreasing to 0.33 nmol/l at 36 months; adults levels are around 0.27 nmol/l although there can be a wide range). This relatively high level of sulfate, as compared with the adult state and with, for instance, laboratory rats, may be associated with a requirement for sulfate patterning of carbohydrates in neuronal development. Recent studies on perineuronal nets have found these structures coating the proximal dendrites of certain neurons including those in 5 areas of the brain involved in autism (37). It seems probable that the nets play a role in signal modulation of the GABA-ergic neurons; they contain chondroitin sulfate and so are distinctive from other structures. In rat studies, these perineural nets have been found in the thalamus, which controls sensory integration and emotions and also in the hypothalamus, controlling hormonal and autonomic regulation neurons. Neurones in the human cerebral cortex which are ensheathed by perineuronal nets rarely undergo cytoskeletal changes in Alzheimer’s disease, suggesting a neuro- protective effect of extracellular matrix components (38). Other proteoglycans with sulphated GAG chains also have a role in brain development, particularly the syndecan family which regulates the maturation of dendritic spines and also glypican which is expressed during axon growth. Neurocan is a chondroitin sulfate proteoglycan of the lectican family, mainly expressed during modeling and remodeling stages in the CNS. It binds to a wide range of cell surface molecules and to structural extracellular matrix components and has been suggested as being responsible for the ‘fine-tuning’ of neural networks as it modulates cell-binding and neurite outgrowth (39).

Sulfation of proteins and carbohydrate chains is also important for the function of joints in the body. A special protein/carbohydrate complex exists to allow the surfaces of the different bones in eg wrist, knee and ankle to slide over one another as the joint is used. This slippery lining to the joints is called the ‘synovium’; the surfaces of the bones are held slightly apart by the lubrication of synovial fluid. Rather like the mucin proteins in the gut walls, the synovium is heavily sulfated on proteins and associated GAGs to give a structure which is both very resistant to breakdown in use and also able to protect the bones in the joint from rubbing against each other and causing damage. Patients with rheumatoid arthritis have been shown to have very low levels of sulfate in plasma, synovial fluid and also in the synovium proteins (40) and this loss of sulfate is linked with the disease progression, showing that sulfation is vital for joint function.

Conclusion

As more research is done, the importance of the sulfate supply becomes more apparent, since sulfation plays such a major part in so many biochemical processes while sulfate generation in the body is often sub-optimal.

References

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  2. Sulfomucins in the human body. A.V. Nieuw Amerongen, J.G. Bolsher, E. Bloemena, E.C. Veerman, Biological Chemistry 1998 379(1), 1-18.
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  1. High incidence of poor sulphoxidation in patients with primary biliary cirrhosis. A.B. Olomu, C.R. Vickers, R.H. Waring, D. Clements, C. Babbs, T.W. Warnes and E. Elias, New Engl. J. Med. 1988 318, 1089-1092.
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  4. Blood sulfur-amino acid concentration reflects an impairment of liver trans-sulfonation pathway in patients with acute abdominal inflammatory processes. J.R. Viva, A. Gimenez, A. Corbucho, I.R. Puertes, E. Borras, C. Garcia, T. Barber, Brit. J. Nutr. 2001 85(2), 173- 178.
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  6. Ontogeny of renal sulfate transporters: postnatal mRNA and protein expression. D. Markovich and T.S. Fogelis, Pediatric Nephrology 1999 13(9), 806-811.
  7. Molecular aspects of renal tubular handling and regulation of inorganic sulfate. L. Bech, C. Silve, Kidney International 2001, 59(3), 835-845.
  8. Metabolic acidosis regulates rate renal Na-Si co-transport activity. K. Puttaparthi, D. Markovich, N. Halaihel, P. Wilson, H.K. Zajcek, H. Wang, J. Biber, H. Murer, T. Rogers, M. Levi, Am. J. Physiol. 1999 276, (6Pt1) C1398-1404.
  9. Ibuprofen – induced changes in sulfate renal transport. K. Sagawa, L.J. Benincosa, H. Murer, M.E. Morris, J. Pharm. Exptl. Therap. 1998 287(3), 1092-7.
  10. Chronic K depletion inhibits renal brush border membrane Na/sulfate co-transport. D. Markovich, H. Wang, K. Puttaparthi, H. Zajiceh, T. Rogers, H. Murer, J. Biber, M. Levi, Kidney International 1999 55(1), 244-251.
  11. Human 3’-phosphoadenosine – 5’-phephosulfate synthetase 2 (PAPS S2) pharmacogenetics gene resequencing, genetic polymorphisms and functional characterisation of variant allozymes. Z.H. Xu, R.R. Freimuth, B. Eckloff, E. Wieben, R.M. Weinshilboum, Pharmacogenetics 2002 12(1), 11-21.
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  13. Dynamic biosynthesis of heparan sulphate sequences in developing mouse brain: a potential regulatory mechanism during development. S. Guimond, K. Turner, M. Kita, M. Ford- Periss and J. Turnbull, Biochem. SR. Tans. 2001 29, (pt2) 177-181.
  14. Defective heparan sulfate biosynthesis and neonatal lethality in mice lacking N- deacetylase/N-sulfotransferase-1. M. Ringrall, J. Ledin, K. Holmborn, T. van Kupperelt, F. Ellia, I. Eriksson, A. Olofsson, L. Kijellen and E. Forsberg, J. Biol. Chem. 2000 275(2) 5926-25930.
  1. Renal agenesis in mice homozygous for a gene trap mutation in the gene encoding heparan sulfate 20-sulfotransferase. S.L. Bullock, J.M. Fletcher, R.S. Beddington and V.A. Wilson, Genes Development 1998 12, 1894-1906.
  2. Human cytosolic sulphotransferases: genetics, characteristics and toxicological aspects. H. Glatt, H. Boeing, C.E.H. Engelke, L. Ma A. Kuhlow, U. Pabel, D. Pamplun, W. Teubner, W. Meinl, Mutation Res. 2001 482, 27-40.
  3. Structure and function of sulfotransferases. M. Negishi, L.G. Pedersen, E. Petrotchenho, S. Shevtsov, A. Gorokhov, Y. Kakura, L.C. Pedersen, Arch. Biochem. Biophys. 2001 390(2), 149-157.
  4. Phenol sulfotransferase pharmacogenetics in humans: association of common SULT 1A1 alleles with TS-PST phenotype. R.B. Raftogianis, T.C. Wood, D.M. Otterness, J.A. van Loon, R.M. Weinshilborum, Biochem. Biophys. Res. Common, 1997 239, 298-304.
  5. Inheritance of human platelet thermolabile phenol sulphotransferase activity. R.A. Price, N.J. Cox, R.S. Spielman, J.A. van Loon, B.L. Maidak, R.M. Weinshilborum, Genet. Epidemiol 1988 5, 1-15.
  6. Platelet sulphotransferase activity, plasma sulphate levels and sulphation capacity in patients with migraine and tension headache. Z. Alam, N. Coombes, R.H. Waring, A.C. Williams and G.B. Steventon, Cephalalgia 1997 17, 761-764.
  7. The effects of flavonoids on human phenol sulphotransferase activity. R.A. Ghazali and R.H. Waring, Life Sciences 1999 65(16), 1625-1632.
  8. Dietary modulation of human platelet phenol sulphotransferase activity. R.M. Harris and R.H. Waring, Xenobiotica 1996 12, 1241-1247.
  9. Human platelets possess Tyrosyl protein sulfotransferase (TPST) activity. D.C. Sane and M.S. Baker, Thrombosis and Haemostasis, 1993 69(3), 272-275.
  10. Isolation of tyrosylprotein sulfotransferase from rat liver. P. Ramaprasad and C. Kasinathan, Gen. Pharmacol. 1998 30(4), 555-9.
  11. Existence of distinct tyrosylprotein sulfotransferase genes: molecular characterisation of tyrosyl proteinsulfotransferase-2. R. Beisswanger, D. Corbeil, C. Vannier, C. Thiele, U. Dohrmann, R. Kellner, K. Ashman, C. Niehrs, W.B. Huttner, Proc. Natl. Acad. Sci. USA 1998 95(19), 1134-1139.
  12. Structural modification of fibroblast growth factor-binding heparan sulfate at a determinable stage of neural development. Y.G. Brichmann, M.D. Ford, J.T. Gallagher, V. Nurcombe, P.F. Bartlelt and J.E. Turnbull, J. Biol. Chem. 1998 273(6), 4350-4359.
  13. Specificities of heparan sulfate proteoglycans in developmental processes. N. Perriman and M. Bernfield, Nature 2000 404, 725-728.
  14. Role of proteoglycans in neural development, regeneration and the ageing brain. D.H. Small, S.S. Mok, T.G. Wilkinson & V.H. Nurcombe, J. Neurochem. 1996 67, 889-899.
  1. Perineuronal nets in the rhesus monkey and human basal forebrain including basal ganglia. I. Adams, K. Rauner, C. Arelin, W. Hartig, A. Fine, M. Mader, T. Arendt, G. Brucliner, Neuroscience 2001 108(2), 285-298.
  2. Neurocan: a brain chondroitin sulfate proteoglycan. U. Rauch, K. Feng, X. H. Zhoru, Cellular and Molecular Life Sciences 2001 58(12-13), 1842-1856.
  3. Sulfate metabolism is abnormal in patients with rheumatoid arthritis. Confirmation by in vivo biochemical findings. H Bradley, A Gough, R Sokhi, A Hassell, R H Waring, P Emery J Rheumatol 21 (7) 1192-1196
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imuno – The Cream

imuno® – The Cream

imuno 50ml jar, front and rear label

imuno® THE CREAM – for a Better Flow

  • For external use only. Store in a cool, dry place (Refrigerate for long term storage). Keep out of direct sunlight.
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imuno® is designed by Molecular Biologist, Dr Marco Ruggiero, MD. PhD. Author of peer-reviewed scientific papers on immunotherapy and quantum biology.

imuno® is produced under licence in a pharmaceutical facility which is audited by Medsafe, a division of the Ministry of Health of New Zealand, to both GMP and Pharmacy Standards NZ.

imuno® is the elegant solution. It is the revolutionary supra-molecular structure that reproduces the self-assembly of cell components. This delivers the combined molecules, at the same time, at the required dose, to their biologic targets, and produces powerful support. Also, the manufacturing process for imuno® is meticulous and time-consuming, and it is not technically possible to get the same result by simply mixing the active ingredients together since the procedure follows a proprietary algorithm based on biologic negentropy.

 

INGREDIENTS

  • Certified Organic Shea Butter – Fair Trade.certified organic
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  • Chondroitin Sulfate – vegan, from microbial fermentation.
  • Phosphatidylcholine – ultra pure.
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imuno® – GcMAF made better

imuno® – The Cream contains all the benefits of imuno®– The Solution.

imuno® – The Cream’s active ingredients are nanosized for deep delivery through the skin.

imuno® is produced under licence in a pharmaceutical facility which is audited by Medsafe, a division of the Ministry of Health of New Zealand, to both GMP and Pharmacy Standards NZ.

Instructions

  • For external use only. Store in a cool, dry place (Refrigerate for long term storage). Keep out of direct sunlight.
  • Use the supplied spatula (or your finger) to remove some imuno® Cream from the jar.
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Color Doppler Evaluation of Isovolumetric Relaxation Time and of Signals Arising from Axons of the Median Nerve as a Means to Evaluate Mitochondrial Functionality in the Context of Immunotherapy of Cancer and Chronic Conditions Associated with Mitochondrial Dysfunction

imuno-Method-to-Evaluate Mitochondrial-Functionality-in-Cancer-Immunotherapy-12Oct19

Color Doppler Evaluation of Isovolumetric Relaxation Time and of Signals Arising from Axons of the Median Nerve as a Means to Evaluate Mitochondrial Functionality in the Context of Immunotherapy of Cancer and Chronic Conditions Associated with Mitochondrial Dysfunction

Stefania Pacini and Marco Ruggiero

Silver Spring Sagl, Arzo-Mendrisio, Switzerland

Article history

Received: 11-09-2019 Revised: 01-10-2019 Accepted: 11-10-2019

Corresponding Author: Stefania Pacini
Silver Spring Sagl,
Via Raimondo Rossi 24, Arzo- Mendrisio 6864, Switzerland Email: info@bravo-europe.com

Abstract: Reactive Oxygen Species (ROS) arising from the disruption of mitochondrial respiration act as endogenous mutagens and tumor promoters. When production of ROS exceeds the capacity of DNA repair mechanisms, random mutations and aneuploidy ensue, thousands of genes become unbalanced and genetic/epigenetic chain reactions lead to progressive aneuploidy with only two outcomes: karyotypes so altered that are not viable or karyotypes of autonomous, immortal, cancer cells. Consistent with the concept that abnormalities of cellular respiration in mitochondria precede DNA alterations in the nucleus, transplant of normal mitochondria suppresses tumorigenesis and metastases in vitro and in vivo. However, the transplant of mitochondria is an experimental procedure that cannot be easily applied to clinical practice. In order to overcome this limitation, we designed a novel formula based on microbial chondroitin sulfate, vitamin D3 and ultrapure phosphatidylcholine, molecules that are known to restore mitochondrial functionality and suppress ROS production. Here we describe how such an approach can be evaluated by color Doppler ultrasonography of the radial artery and measure of the Isovolumetric Relaxation Time (IVRT). In addition, we show for the first time color Doppler signals originating from axons of the median nerve that may be indicative of quantum phenomena at the level of mitochondria. We propose the use of the original ultrasonographic techniques here described for evaluating the effectiveness of substances or strategies aimed at restoring mitochondrial functionality at the macroscopic and quantum levels.

Keywords: Cancer, Mitochondria, Reactive Oxygen Species, Chondroitin Sulfate, Vitamin D, Phosphatidylcholine, Isovolumetric Relaxation Time, Median Nerve, Quantum Entanglement

American Journal of Immunology

Introduction

Cancer is a complex disease where metabolic, genetic and epigenetic factors are closely intertwined and produce such a variety of aberrations in all aspects of cellular physiology that is difficult to ascertain which is the cause and which is the effect. According to decade- old theories that have received experimental confirmation in the past few years, initiation of cancer can be found in metabolic alterations at the level of mitochondria with consequent production of Reactive Oxygen Species (ROS) that act as endogenous mutagens

and tumor promoters (Seyfried, 2015). When prolonged mitochondrial damage is of such an extent that production of ROS exceeds the capacity of DNA repair mechanisms, random mutations occur with the risk of inducing aneuploidy, a condition that leads to the imbalance of thousands of genes at a time. Such an occurrence leads to genetic and epigenetic chain reactions that either lead to non-viable karyotypes, or in relatively rare cases that develop over time, to immortal cancer cells in a process that resembles speciation (Hirpara et al., 2018). Chemicals or physical treatments such as ionizing radiations, that are defined mutagens for their ability to interact with DNA, are also known disruptors of mitochondrial respiration; defective mitochondrial respiration associated with compensatory fermentation is then responsible for the deluge of mutations that culminate in aneuploidy and, ultimately, in speciation (Seyfried, 2015; Hirpara et al., 2018). Pervasive genetic and epigenetic instability culminating in aneuploidy and speciation further disrupts mitochondrial respiration up to the point that the process becomes autonomous and self-sustaining. In addition, disruption of mitochondrial respiration by carcinogens with consequent compensatory fermentation leads to tissue acidification that, in turn, destabilizes the cancer microenvironment and favors local cancer growth as well as metastatization, processes that are further sustained by acidification- induced neo-angiogenesis.

If the primum movens of cancer lays in alterations of mitochondrial functionality rather than in mutations of nuclear DNA in somatic cells, it may be argued that replacing defective mitochondria with functional ones should prevent or neutralize tumorigenesis. This concept is consistent with the decade-old theory of Nobel Laureate Otto Warburg postulating that respiratory insufficiency is at the origin of cancer (Warburg, 1956). Plenty of evidence gathered in the past 30 years demonstrates that transplant of normal mitochondria- often together with normal cytoplasm-suppresses tumorigenesis in vitro and in vivo (Seyfried, 2015). Normal mitochondria, by restoring cellular respiration would restore normal energy homeostasis, thus leading to cancer cell differentiation. In this context, transplant of normal mitochondria would be effective even in advanced stages of tumor progression and also in the presence of aneuploidy as demonstrated by studies describing how normal mitochondria inhibit tumorigenicity even in the presence of extensive mutations in the nucleus of cancer cells (Seyfried, 2015).

As far as clinical applications are concerned, mitochondrial transplant has recently been considered a promising approach for mitochondrial diseases and cancer (McCully et al., 2016; Chang et al., 2019) and mitochondrial transfer has been patented under the name “Autologous Germline Mitochondrial Energy Transfer” for human assisted reproduction (Woods and Tilly, 2015). However, it is difficult to conceive the application of this type of protocols to conditions that are characterized by mitochondrial dysfunction such as cancer, neurodegenerative diseases, autism or aging and we are not aware of studies reporting successful application of mitochondrial transplant in these conditions. A recent study performed on human breast cancer cells in vitro demonstrated that the anti-cancer effects of mitochondrial transplant are associated with significantly decreased ROS production and shift in energy production with less energy deriving from glycolysis (Chang et al., 2019). Transplant of normal mitochondria into human breast cancer cells, in addition

to direct anti-cancer effects, also increased sensitivity to known chemotherapeutic drugs such as Doxorubicin and Paclitaxel (Chang et al., 2019). Since these drugs have different mechanisms of action-Doxorubicin inhibits transcription and replication of nuclear DNA and increases DNA damage by inhibiting topoisomerase II; Paclitaxel is a cytoskeletal drug that inhibits mitosis by stabilizing microtubules-it may be hypothesized that restoring mitochondrial activity in cancer cells not only induces cancer cell apoptosis per se but also makes cancer cells more sensitive to different, possibly all, types of chemotherapy.

Based on the studies and considerations mentioned above, we developed a formula that has the goal of restoring mitochondrial function without the need of a mitochondrial transplant. Such an approach is based on combining the effects of known regulators and restorers of mitochondrial function, the glycosaminoglycan chondroitin sulfate, vitamin D3 and a major constituent of mitochondrial membrane, the phospholipid phosphatidylcholine. This approach and its constituents have been thoroughly described in two recent papers (Ruggiero and Pacini, 2018a; 2018b) and clinical results associated with this approach have also been recently described (Antonucci et al., 2018; 2019a; 2019b). Here, we propose a rapid and simple method to assess the efficacy of such an approach in modulating energy production at the level of mitochondria. This method is based on the evaluation of Isovolumetric Relaxation Time (IVRT) by color Doppler ultrasonography of the radial artery. We also discuss the implications arising from the original observation of Doppler signals originating from within the axons of the median nerve.

Materials and Methods

IVRT, often designated also isovolumic relaxation time, is used in cardiology as an indicator of diastolic physiology or dysfunction. It is the time comprised between the closure of the aortic valve and the opening of the mitral valve. During this time, the fibers of the myocardium relax while the volume of the left ventricle remains constant until the opening of the mitral valve. The length of IVRT is dependent on the amount of energy generated by mitochondria; during this interval, cardiomyocytes extrude calcium and this is achieved by using all the energy, under the form of ATP, generated by mitochondria. Therefore, in the presence of defective energy production at the level of mitochondria, IVRT is longer; on the other hand, substances or treatments that increase energy production at the level of mitochondria decrease IVRT. In other words, IVRT is inversely proportional to the energy generated by mitochondria in cardiomyocytes. In cardiology, IVRT is measured by combining trans-thoracic Doppler echocardiography and M-mode sonography or phonocardiogram and transmitral Doppler echocardiography. Using these techniques, it was demonstrated that normal IVRT is around 60 ms (Brecker et al., 1992). It is worth noticing that the evaluation of IVRT is a rather complex procedure that is usually performed in clinics or departments dedicated to cardiology. Here, we describe a simplified method to evaluate IVRT by color Doppler ultrasonography of the radial artery using commonly available generic ultrasound systems. The ultrasounds used for evaluating IVRT, also known as sub-thermal ultrasounds, are considered harmless for the human body up to the point that they have been and are being, used in obstetrics and gynecology for decades to obtain images in utero. Any modern ultrasound system with a linear probe for muscle-skeletal examination and color Doppler application can be used. The first step of the procedure consists in finding the pulse of the radial artery by

palpation of the volar surface of the wrist. The second step consists in visualizing the radial artery in an axial section at the level of the wrist in B-mode; this appears as a pulsating black circle (Fig. 1).

The radial artery can be measured and studied for anomalies. Once the radial artery has been visualized, the color Doppler is turned on and the flow of blood can be visualized (Fig. 2).

At first, the probe has to be tilted towards the elbow of the subject so to have a homogeneous image of the flow of blood coming towards the probe and being recorded as “red” according to the known rule of Doppler ultrasonography “Blue Away Red Towards” (BART). At this point, the probe can be tilted towards the hand of the subject so that the flow in the radial artery appears blue and the application to measure velocities and times can be turned on (Fig. 3).

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Fig. 1: Ultrasonography of the volar side of the wrist, axial section. The radial artery is visualized as a black circle and has a diameter of 1.7 mm

Fig. 2: Color Doppler ultrasonography of the radial artery. The probe is tilted towards the elbow of the subject ■■

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Stefania Pacini and Marco Ruggiero / American Journal of Immunology 2019, ■■ (■): ■■■.■■■ DOI: 10.3844/ajisp.2019.■■■.■■■

Fig. 3: Color Doppler ultrasonography of the radial artery. The probe is tilted towards the hand of the subject and the cursor to measure velocities and times is applied

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Results and Discussion

According to our knowledge, this is the first description of a technique to evaluate IVRT at the level of the radial artery. The measure of IVRT by color Doppler ultrasonography of the radial artery following the procedure described above is shown in Fig. 4.

IVRT is evaluated by measuring the time interval corresponding to the negative curve following the systolic peak. Figure 4 shows an example of IVRT measured with this procedure with a resulting value of 118+3.2 ms. This value appears longer than those measured by trans- thoracic ultrasonography and this difference is due to the measure at the periphery. Although this measure is indirect in comparison to that obtained by trans-thoracic ultrasonography, the procedure has the convenience that it can be performed in a few minutes, with no discomfort for the subject and no need to undress, with obvious advantages. It is worth noticing that this procedure has not the goal to diagnose diastolic dysfunction but to evaluate the effectiveness of strategies aimed at increasing mitochondrial energy production with the ultimate goal of restoring mitochondrial functionality in cancer cells as well as in conditions associated with mitochondrial dysfunction. In addition, the procedure described above allows simple and rapid visualization of the median nerve in the volar side of the forearm by sliding the probe towards the elbow and placing it at 90 degrees in order to obtain an axial image as in the example of Fig. 5.

The median nerve has an ovoidal shape and is surrounded by the epineurium that appears as a relatively thick (0.4-0.5 mm) hyper-echoic-white-linear structure. The perineurium surrounding the fascicula appears as a thinner (0.3 mm) hyper-echoic linear structure. After having visualized the nerve, the resulting spectral curve

is shown in Fig. 6 and probe is tilted towards the elbow so to capture any flow moving towards the probe. Application of color Doppler to the inside of the median nerve and analysis of the resulting spectral curve is shown in Fig. 6 and 7.

The pattern evidenced in Fig. 7 appears peculiar and we are not aware of any description of these color Doppler signals originating from inside the median nerve. We attribute these signals to mitochondria trafficking-flow-inside the axons of the nerve. It is well assessed that energy provided by mitochondria is required for synaptic function and, more in general, for all neurologic functions (Sajic et al., 2013; Nunn et al., 2016) and it has been demonstrated that mitochondria move at a speed between 0.3 and 0.7 μm/s in peripheral nerves (Sajic et al., 2013). Peripheral nerve stimulation increases the number and the speed of mitochondria moving towards synapses that are areas of high energy requirements. It can be therefore hypothesized that the Doppler signals observed in Fig. 7 are associated with trains of mitochondria moving inside the axons of the median nerve. Such a flow of mitochondria and the possibility to study it as proposed here for the first time, implies interesting consequences as far as cancer and other diseases associated with mitochondrial dysfunction are concerned. Evolution of mitochondria has favored the generation of energy at the so-called “redox sweet spot” (Nunn et al., 2016), where energy production with minimal ROS generation is optimized for health and longevity. Such optimization is achieved through quantum phenomena that involve electron tunneling, prolonged quantum coherence and entanglement. The strong electromagnetic fields generated by electron transport inside mitochondria (Guo et al., 2018) could be responsible for protection against de-coherence and there is evidence supporting the hypothesis that in complex biological systems quantum coherence can be sustained for times orders of magnitude longer than those achievable in simplified quantum systems at room temperature (Nunn et al., 2016). This type of biological quantum coherence is deemed responsible for consciousness and its intrinsic role in the universe (Hameroff and Penrose, 2014). The observation that mitochondria move at measurable speeds, thus generating further, stronger, electromagnetic fields, adds a layer of complexity that may lead to an augmented state of quantum coherence. We are not aware as to whether the signals originating from the median nerve that we have captured using the original procedure described above are associated with this augmented state of quantum coherence or are macroscopic evidence of entanglement. It is tempting to speculate that the signatures of these signals correspond to a level of information that we have just begun to decipher. For example, the two signalscircled in red in Fig. 7, designated with the letter “A”, appear to be identical and may represent the macroscopic signature of a phenomenon of entanglement or synchronization occurring in the electron clouds of fast- moving mitochondria. Similar, albeit not identical in size, couples can be observed for the signals circled in “B” and “C”, whereas the signals in “D” and “E” do not show repetitive patterns. It is also interesting to notice that, if the signals in “A” are associated with mitochondrial quantum entanglement, they may also be indicative of the occurrence of a wormhole; this is a structure enabling non-conventional transfer of information between separate points in time and space, independently of spatial contiguity or direction of the arrow of time as we recently discussed in the context of virus-associated information transfer (Pacini and Ruggiero, 2019). The signals evidenced for the first time in Fig. 7 may also serve to study in real-time the effects

of hormesis in mitochondria and, more specifically, the effects of substances or strategies aimed at improving mitochondrial functionality. It is well assessed that mitochondria-generated energy has an important role in hormesis since mild stressors elicit an adaptive response that increases mitochondrial efficiency (Tapia, 2006). Hormesis, by favoring the evolutionary selection of more efficient electron transport chains in mitochondria would then prevent and/or counteract mitochondrial dysfunction with associated excessive ROS production. Such a concept of hormesis in the context of radiations and cancer is well documented (Luckey, 2008; Shibamoto and Nakamura, 2018) and it can be hypothesized that the effects observed in a recent study on multiple myeloma can be ascribed to phenomena of hormesis leading to enhanced mitochondrial efficiency possibly through enhancement of coherent states at quantum level (Antonucci et al., 2019b). Whatever the case, the procedures described above represent a rapid and simple method to study mitochondrial functionality at the macroscopic and possibly at the quantum level, with a number of practical applications. The rationale for suggesting implementation of the ultrasonographic procedures described here in oncology and in other conditions associated with low energy production at the level of mitochondria is the following; if a substance or a treatment is able to increase energy production at the level of cardiomyocytes and enhance quantum coherence at the level of moving mitochondria, it may also restore mitochondria functionality in other tissues including cancerous tissues. Consistent with this concept, we have previously demonstrated that immunotherapeutic agents successfully used in cancer, in particular vitamin D receptor (VDR) agonists complexed with amphipathic macromolecules and lipophilic compounds, are able to increase IVRT (Pacini et al., 2013).

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Fig. 4: Evaluation of isovolumetric relaxation times (IVRT) by color Doppler ultrasonography

Fig. 5: Ultrasonography of the median nerve

Fig. 6: Ultrasonography of the median nerve, positioning of the cursor of the color Doppler

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Fig 7: Ultrasonography of the median nerve, signals from color Doppler. Please notice, the lower panel is an enlarged section of the image from Fig. 6 that has been reduced in size

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Since it can be hypothesized that this effect on the mitochondria of cardiomyocytes is occurring also in cancer mitochondria, it can be deduced that restoration of mitochondria functionality might have been contributed to the clinical efficacy of these compounds (Ruggiero et al., 2014). We also published evidence supporting the hypothesis that the results observed in vitro and in vivo with VDR agonists complexed with amphipathic macromolecules and lipophilic compounds were due to the formation of supramolecular complexes of a glycosaminoglycan, chondroitin sulfate, with vitamin D3 and lipophilic molecules (Ruggiero et al., 2016). We recently described the molecular and functional characteristics of a supramolecular structure formed by microbial chondroitin sulfate, vitamin D3 and ultra-pure phosphatidylcholine (Ruggiero and Pacini, 2018a; 2018b) Below, we describe how each one of these components, has a role in restoring mitochondrial functionality and how these effects may occur at the quantum level.

Microbial chondroitin sulfate. It has been observed since 1969 that chondroitin sulfate dramatically reduces morbidity, i.e., coronary episodes, in patients with ischemic heart disease (Morrison, 1969). It has also been known since 1988 that chondroitin sulfate is responsible for the anti-cancer effects of cartilage preparations (Rosen et al., 1988; Prudden, 1985). It has been recently demonstrated that chondroitin sulfate improves mitochondrial activity in human cells by increasing the efficiency of metabolic processes, with particular reference to the generation of energy and reduction of ROS levels (Montell et al., 2015). Therefore, it can be deduced that the heart-supporting and anti-cancer effects of chondroitin sulfate are due to the restoration of mitochondrial energy generation and the decreased production of ROS. It is worth noticing that the observations quoted above refer to animal-derived chondroitin sulfate that is a heterogeneous mixture of high- and low-molecular-weight species (Ruggiero and Pacini, 2018a). The recent development of microbial chondroitin sulfate represents an evolution that may further improve the efficacy of chondroitin sulfate both in cardiology and oncology as well as in other conditions associated with mitochondrial dysfunction. Microbial chondroitin sulfate, at variance with its animal-derived counterpart, is characterized by over 99% of purity and it is homogeneous since it is constituted by low-molecular- weight species. These characteristics are responsible for higher biological activity demonstrated by preclinical and clinical studies (Ruggiero and Pacini, 2018a). Such increased efficacy is not surprising as it is well known that low-molecular-weight glycosaminoglycans are much more effective than mixtures of high- and low-

molecular-weight species (Ruggiero and Pacini, 2018a). In addition, it is worth mentioning that ROS production in mitochondria is influenced by quantum phenomena, more precisely, coherent electron spin dynamics (Usselman et al., 2016).

Therefore, the effects of chondroitin sulfate on mitochondrial efficiency could be ascribed to the phenomena of enhanced coherence and entanglement that can be studied with the original ultrasonographic procedures described above. Chondroitin sulfate and in particular microbial chondroitin sulfate, carries plenty of quantum information associated with phenomena of entanglement between the electron clouds surrounding the highly charged glycosaminoglycan and the electron clouds of DNAs (Rieper et al., 2011), where the plural stands for mitochondrial and nuclear DNA. Thanks to its quantum properties, chondroitin sulfate could be considered the trait d’union that harmonizes information in the two cell compartments containing DNA. It is known that such a harmonization is essential for optimal health and longevity; alterations affecting the balance between genetic information contained in the nucleus and that in the mitochondrion leads to decreased efficiency of the electron transport chain that in turn leads to decreased energy generation and increased ROS production, two hallmarks of cancer and other conditions associated with mitochondrial dysfunction that include aging (Nunn et al., 2016). If mitochondria work as sensors balanced between the two realms of quantum and classical worlds where the relationship between the de-coherent macroscopic world and the coherent quantum world is responsible for mitochondrial function or dysfunction (Nunn et al., 2016), then the protocellular structures that we described in 2018 (Ruggiero and Pacini, 2018a) may act as quantum harmonizers for mitochondria (see below). It is also worth mentioning that microbial chondroitin sulfate is synthesized by non- genetically modified bacteria and, therefore, carries the entangled information originating from bacterial DNA. Genetic information in mitochondria shares many similarities with that of bacteria since “… genome evolution models now indicates that 97% of modern mitochondrial protein domains as well their homologues in bacteria and archaea were present in the universal common ancestor of the modern tree of life” (Harisha and Kurlandb, 2017). This consideration further strengthens the concept that microbial chondroitin sulfate may act as a quantic trait d’union able to restore, in dysfunctional mitochondria, the primeval information at the basis of life on this planet as a sort of reboot that brings the mitochondrion to an unadulterated state.

Vitamin D3. The inotropic effects of vitamin D3 are well assessed (Witte et al., 2016) just like its anti-cancer effects that can be summarized as cancer prevention and adjuvant in cancer treatment (Chakraborti, 2011). As with chondroitin sulfate, co-existence of inotropic and anti-cancer effects leads to hypothesize that an influence on mitochondria is at the base of both phenomena. The actions of vitamin D3 at the level of mitochondria have been demonstrated in many cell types and it is well known that they are mediated by VDR. In particular, it was demonstrated that stimulation of VDR is associated with reduced formation of ROS in mitochondria (Ricca et al., 2018). These recent results are consistent with our hypothesis, first elaborated in 2013, that activation of VDR by a multi-molecular complex of VDR agonists, amphipathic macromolecules and lipophilic compounds was responsible for anti-cancer and inotropic effects (Pacini et al., 2013; Ruggiero et al., 2014; Thyer et al., 2013). In the design of the formula described (Ruggiero and Pacini, 2018a), vitamin D3 was chosen also because of its quantum properties that arecomplementary to those of chondroitin sulfate and trace their origins to the emergence of life on Earth. According to researchers from Lithuania and Norway “Life on Earth or elsewhere could have emerged in the form of prebiotic kernels and self-reproducing photoactive fatty acid micelles, which gradually evolved into nucleotide-containing micelles due to the enhanced ability of nucleotide-coupled sensitizer molecules to absorb visible light” (Tamulis and Grigalavicius, 2014). The Authors, Tamulis and Grigalavicius, describe how provitamin D molecules emerged from a quantum mechanical origin and favored harvesting of light in what they define prebiotic kernels that are structures similar to the protocellular structures we described in 2018 (Ruggiero and Pacini, 2018a). As far as the quantum properties of vitamin D3 are concerned, Tamulis and Grigalavicius state “Our discovered phenomenon of the quantum entanglement in the provitamin D prebiotic system enhance of photosynthesis in the system because simultaneously excite two prebiotic kernels in the system by appearance of two additional quantum entangled excited states. We can propose that quantum entanglement enhanced the emergence of photosynthetic prebiotic kernels and accelerated the evolution of photosynthetic life because of more absorbed light energy”. (Tamulis and Grigalavicius, 2014). The similarity between the prebiotic kernels described by Tamulis and Grigalavicius (2014) and the protocellular structures made of chondroitin sulfate, phosphatidylcholine and vitamin D3 (Ruggiero and Pacini, 2018a) is further highlighted by the role played by fatty acids in establishing self- assembling micelles. In the formula described in 2018 (Ruggiero and Pacini, 2018a; 2018b), fatty acids are provided by ultrapure phosphatidylcholine following a molecular model that we described for the first time in

1985 (Vannucchi et al., 1985). The positively charged quaternary ammonium groups of the polar head of phosphatidylcholine bind, via electrostatic interactions, with the negatively charged carboxylic and sulfate groups of microbial chondroitin sulfate. From our paper of 1985, it can be calculated that each molecule of microbial chondroitin sulfate is encircled by approximately 50 molecules of phosphatidylcholine that expose their hydrophilic tails constituted by fatty acids to the aqueous environment. Such a molecular arrangement induces the self-assembly of bilayers of phosphatidylcholine leading to cell membrane-like structures similar in principle to the prebiotic kernels described by Tamulis and Grigalavicius (2014) whose self-assembly, proceeding uniquely according to the laws of quantum physics, can be calculated and visualized by quantum mechanical time-dependent density functional theory. There is, however, a significant difference between the prebiotic kernels of Tamulis and Grigalavicius and the protocellular structures that we described in 2018 (Ruggiero and Pacini, 2018a; 2018b); the latter are homogeneous, single-phospholipid, bilayers that encircle low-molecular- weight microbial chondroitin sulfate instead of nucleic acids. We chose a glycosaminoglycan rather than a nucleic acid or a protein because the sequence of the former is repetitive and does not reveal biochemical or genetic information under the form of a sequence of four nitrogenous bases or twenty amino acids, thus avoiding recognition as “not-self” by the immune system.

However, as discussed above, microbial chondroitin sulfate carries plenty of information at the quantum level and this information is exploited to restore mitochondrial functionality by enhancing coherence thus increasing the generation of energy and decreasing the production of ROS. This noteworthy difference notwithstanding, there are no doubts that vitamin D3 plays an essential role both in the prebiotic kernels of Tamulis and Grigalavicius and in our protocellular structures acting as an intermediate evolved to harvest the energy of the sun at the chemical and quantum levels.

Phosphatidylcholine. It is well known that phosphatidylcholine is essential for mitochondrial functionality as it is one of the major constituents of the membrane of the organelle (Cigana Schenkel and Bakovic, 2014). It also known that phospholipids of the mitochondrial membrane are not only architectural components, but participate in the activity of respiratory proteins. Phosphatidylcholine is essential for protein transport into the inner membrane of mitochondria and into the matrix and depletion of phosphatidylcholine affects the integrity of the TIM23 translocase and precursor transfer to the TIM23 complex (Schuler et al., 2016). TIM23 complex is the translocase that transports precursor proteins with a cleavable pre-sequence into the inner membrane and the matrix. Phosphatidylcholine is also essential for the function of the Sorting and Assembly Machinery (SAM complex) of the outer membrane of the mitochondria (Schuler et al., 2016). In the approach described (Ruggiero and Pacini, 2018a), ultrapure phosphatidylcholine was used in order to exploit the properties of the well-described spontaneous association of circulating glycosaminoglycans and phosphatidylcholine (Vannucchi et al., 1985) and the resulting effects on negentropy as discussed (Ruggiero and Pacini, 2018a). In addition, phosphatidylcholine proves instrumental in determining the kinetics of absorption and intracellular delivery of the supramolecular complexes (Ruggiero and Pacini, 2018a). As far as the quantum properties of phosphatidylcholine are concerned, it is worth noticing that they are complementary to those of chondroitin sulfate and vitamin D3 and contribute to the overall efficiency of the formula described (Ruggiero and Pacini, 2018a). In addition to participating to the formation of the protocellular structures described above, phosphatidylcholine contributes to the process of geometry optimization for the supramolecular complexes constituted by the phospholipid, vitamin D3 and microbial chondroitin sulfate, thus introducing a further level of quantum information. In the example provided by Tamulis and Grigalavicius (2014), the prebiotic kernel is assembled with the tail of provitamin D oriented at 0 or 180 degrees to the opposite side of the tail of precursor fatty acid in order to optimize hydrophobic and electrostatic interactions. As mentioned before, according to Tamulis and Grigalavicius, such an assembly proceeds exclusively according to the laws of quantum physics. In the formula described (Ruggiero and Pacini, 2018a), these orientations are maintained as far as the interactions between vitamin D3 and phosphatidylcholine are concerned. However, there is a further layer of complexity due to electrostatic interactions between the –OH group of vitamin D3 and the negatively charged carboxylic and sulfate groups of microbial chondroitin sulfate that are not occupied by binding the polar head of phosphatidylcholine.

Because of these interactions, vitamin D3 lays on both sides of the membrane-like bilayer constituted by phosphatidylcholine self-assembled around microbial chondroitin sulfate. Physical interaction between the electron clouds of the three molecules gives rise to a complex form of entanglement that is probably responsible for sustaining augmented coherence, ultimately contributing to the observed biological effects of the formula.

From the functional point of view, the formula described by Ruggiero and Pacini is based on three natural compounds each one endowed with the ability to restore mitochondrial physiology both at the macroscopic and the quantum levels. The efficacy of each compound is enhanced by the formation of non-covalent bonds with the other two so to form supramolecular complexes able

to target different aspects of mitochondrial metabolism simultaneously, at the same time exhibiting an augmented state of quantum entanglement and coherence. These effects on mitochondrial functionality are probably crucial in determining the responses observed in autism and cancer (Antonucci et al., 2018; 2019a; 2019b). It is well established that autism is associated with mitochondrial dysfunction and defective energy production (Siddiqui et al., 2016) and it is therefore not surprising that an approach based on restoring mitochondrial functionality may help in this condition. As far as the case described by Antonucci et al. (2019b) is concerned, it is worth considering that this neoplasia is among those that accumulate iron, a metal essential to promote cancer cell growth and resistance to chemotherapy. High levels of iron in the cytosol react with ROS produced by defective mitochondria in cancer cells and this leads to further oxidative damage (Zhan et al., 2017). It is therefore plausible that the encouraging results reported by (Antonucci et al., 2019b) may be ascribed also to the restoration of mitochondrial functionality associated with detoxification of metals as also described in (Blythe et al., 2017).

Conclusion

The procedures described for the first time in this study are consistent with the novel applications of ultrasonography in diagnostics and therapy that we first proposed with the Ruggiero-Klinghardt Protocol in 2017 (Antonucci et al., 2018; Klinghardt and Ruggiero, 2017). Color Doppler ultrasonography of the radial artery to measure IRVT and study of Doppler signals arising from within axons of peripheral nerves, will prove instrumental in preventively assessing the efficacy of approaches aimed at restoring mitochondrial functionality in all conditions associated with their dysfunction, from cancer to autism, neurodegenerative diseases, heart conditions, myalgic encephalomyelitis/chronic fatigue syndrome and aging.

Acknowledgement

The Authors wish to thank imuno Corporation, Vanuatu, for development of the formula described by Ruggiero and Pacini and for inspiring discussion.

Authors’ Contribution

Stefania Pacini, MD, PhD and Marco Ruggiero, MD, PhD, contributed equally to this paper.

Disclosures

Marco Ruggiero is the founder of Silver Spring Sagl, a Swiss company producing probiotics and other supplements that are described in some of the references.

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Stefania Pacini works as for Silver Spring Sagl. No product of Silver Spring Sagl is mentioned in the present study. Marco Ruggiero and Stefania Pacini also consult for other companies and have invented/developed several supplements and protocols that are described in some of the references. Marco Ruggiero is member of the Editorial Board of the American Journal of Immunology and is waived from the Article Processing fee for this contribution; he receives no remuneration for his editorial work.

Advisory

No information in this paper is intended or implied to be a substitute for professional medical advice, diagnosis or treatment.

Ethics

This article is original and contains material that has not been published in any journal. All images are from the professional archive of Marco Ruggiero, MD, radiologist, do not refer to any patient or member of the public and are presented solely as examples of applications of ultrasonography.

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cognition table

go to the Table of Cognition Supplement Recommendations

Use applied kinesiology to determine if any of these substances would support your cognition,

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Melatonin contraindications

Classification of Melatonin

Uses of Melatonin

Contraindications

  • Lupron injections

 

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