Sunday, January 27, 2013

Mitochondria II: Methylene Blue

The Gods of Microbiology and the First Synthetic Drug

A Frenchman and a German are the Janus faces worn by the God of Microbiology. Contemporaries, Pasteur and Koch were bitter rivals. Pasteur delt the coup de grace to spontaneous generation sporting nothing more than a lab receptacle with an innovative down swept opening. Today, a cold glass of pristine 1860s beef broth collects both dust and accolade demonstrating that if the little beasties don't get in, the soup is good. Pasteur also managed to create vaccines for rabies and anthrax. He was lauded the savior of the French Economy via the process known today as pasteurization. Pasteur was a hero, but without any aspirations towards being perceived as humble. While presenting at the Royal Academy of Science he began,” As we all know, Avians
Louis Pasteur
 such as our friends the chickens exhibit a resistance to inoculation with the dread anthrax...” He was interrupted by a senior scientist exhorting that chickens very well could be infected with Anthrax. After a hearty mocking laugh and a good haughty pointing at this gentleman, Pasteur issued a challenge. A duel if you will, not of speed or bronze, but rather of germ culturing and inoculation gun-slingery and chicanery. Pasteur even offered the use of his own Anthrax cultures. After thirty days, the man admitted his defeat and that chicken apparently can t be inoculated with Anthrax. Pasteur then resumed his speech, As we all know, Avians such as our friends the chickens exhibit a resistance to inoculation with the dread anthrax. I have found this to be due to their higher body temperature. Chickens can very easily be infected with anthrax if you bathe them in cool water to lower this temperature. Perhaps I took liberty with the exact dialogue, but illustrated the intended implication quite well: Come at me bro, I am the Hero, nay the God, Pasteur.
Robert Koch
German physician Koch was more willing to listen to his cohorts. He not only isolated and identified the organisms responsible for Cholera, Tuberculosis, and Anthrax, Koch inspired and instructed an entire generation of German microbiologists who valued camaraderie. Together, his pupils identify the causative organism and in many cases effective treatments for typhoid, diptheria, gonorrhea, leprosy, syphilis and many others. Koch made sure to give credit where credit was due. Every hospital has a little room full of Petri dishes, named for Koch s assistant Petri, full of agar medium which was an idea of Koch's wife, upon which sputum and blood are streaked in pursuit of marauding animalicules. Koch's postulates discredited the Miasma theory of Disease, and the Germans could once again gallivant confidently through the night air.
Paul Ehrlich
In spite of Koch's non-competitive spirit, one can imagine the trepidation of Paul Ehrlich when he found that both he was and Koch were scheduled to speak on the same topic, methods of staining the tuberculosis pathogen, at the Imperial Public Health Office. This wariness likely grew to terror after Koch spoke, and Ehrlich realized he, a mere student at the time, was going to stand before Koch and his colleagues and explain the superiority of his method to Kochs. Rather than chiding the upstart, Koch applauded Ehrlich and the two began a life long friendship.
P. Falciparem
While Koch had effectively discredited the age old Miasma Theory of Disease, those living near the equator still had reason to fear the night air. Rather than Mal Aria or “bad air,” the disease Malaria is spread by the bite of mosquitoes which take flight at dusk. Unlike the majority of diseases being studied during this seminal period of microbiology, Malaria isn't caused by bacteria. The causative agent, Plasmodium Falciparem, is a protist which is a type of single celled eukaryote. Eukaryotes are millennia of milleninia ahead of bacteria in terms of complexity and even today there are no effective vaccines against protists such as those that cause Malaria, African Sleeping Sickness, and Giardia. Ehrlich concluded that if a vaccine could not be produced to prevent Malaria, then perhaps the agent itself could be attacked. He theorized that because his favorite stain, Methylene Blue, binded so readily to the Malaria pathogen, in higher doses it might even kill it without harming the human host. He trialed methylene blue in two Malaria patients; lo and behold, their fevers subsided and the nasty little protists no longer showed in blood samples. This was the first synthetic antibiotic agent, and it's discovery the herald of a whole new means by which to treat the microscopic beasts within. 
Bioenergetics, Natto, and Whisky
Methylene Blue (MB) has a rather unique means by which it kills organisms. Understanding this mechanism requires a cursory understanding of bioenergetics: the study of how energy flows through living systems. It's important to become familiar with the compounds involved and a great place to start is at the desired end product: ATP.

Adenosine Triphosphate (ATP)

All known organisms use ATP to power protein machines within cells. It consists of one Adenosine with three phosphate groups bound in a sequence. When the high energy bond which links the third phosphate is broken, energy is released in a way proteins can use, leaving behind an ADP and a phosphate.

 There are many ways that ATP can be synthesized and bacteria are particularly diverse in these pathways. Geobacter Metallireducens for example uses uranium instead of oxygen as an electron receptor. Archae breathe in carbon dioxide and breathe out methane gas. Amongst Eukaryotes, yeasts are of particular note due to the inebriating effects of the ethanol they produce. Ethanol does not result from the ATP producing steps per se, but rather during fermentation which is a way that the fungi can “reset” a very important intermediary energy molecule called NAD.

Nicotinamide adenine dinucleotide (NAD)

 NAD is a coenzyme found in all living cells. It facilitates Redox reactions in glycolysis and is reduced to NADH. NADH is then transported to the mitochondria where it is oxidized back to NAD. This allows the NAD to once again facilitate redox reactions in glycolysis and provides the hydrogen and electrons which the Electron Transport Chain uses to synthesize ATP. 

Production of ATP starts outside of the body with a saccharide fuel source. Regardless of whether this is healthy plump little grape or the last extant twinkie, digestion begins in the mouth with salivary enzymes that break down complex carbohydrates into simpler monomer forms such as glucose. This happens two more times, once via enzymes from the pancreas, and again from the brush borders of the small intestines.
Then, absorption occurs. The glucose first enters the cells of the small intestines. Then it enter the blood and travels around a bit before finally being absorbed into the cell which will break it down for energy. This is when “Cellular Respiration” begins, with the process called Glycolysis. The purpose of Glycolysis is to synthesize ATP the energy of life, but the process isn't free.Glycolysis requires NAD. 

NAD facilitates Redox reactions during glycolysis and are converted to the reduced form NADH. This means that NAD accepts electrons in order to bond with a hydrogen ion. This is essential for the payoff steps of glycolysis to occur. Glycolysis is occurring pretty much continuously in cells and organisms and must have a way to “reset” the NADH back into it's oxidized form NAD. As discussed above, booze producing yeasts such as Saccharomyces cerevisiae use a non-energy producing process called fermentation to reset NADH to NAD. Fermentation is also used to make vinegar, pickles, and yogurt. More exotic fermented foods include Surströmming, a swedish dish made of fermented Baltic Herring, and Natto, a fermented soy bean dish from Japan with a texture often compared to snot.The human body also performs fermentation if cells aren't receiving an adequate supply of oxygen resulting in lactic acid. Accumulated lactic acid is responsible for much of the muscle pain associated with intense exercise. When sufficient amounts of oxygen are available, the NADH is re-oxidzed instead by a series of enzymes within the mitochondria, which make up the Electron Transport Chain.

Redox Reactions within The Electron Transport Chain
Mitochondria have an outer membrane and an inner membrane. The ETC is a set of proteins complexes on the inner membrane which oxidizes NADH and pumps the hydrogen ion (H+) into the space between the two membrane. There, the H+ accumulates like air in a balloon, awaiting the opportunity to escape. This concentration gradient of H+ is harnessed by an enzyme called ATP-Synthase to make ATP. This is something like a water-wheel in that H+ is allowed to flow, and it's movement powers the ATP assembly mechanisms within the ATP-synthase enzyme.
The terminology of redox reactions is somewhat counter-intuitive. The term Redox is a portmanteau of “reduction” and “oxidation.” Reduction and oxidation occur at the same time during a redox reaction. Historically, reduction referred to a loss of weight observed in heated metal ores. Heating an ore releases oxygen gas, and thus the metal is “reduced.” When a metal rusts, it's oxidized because oxygen bonds to the metal and it gains weight. The terminology became confusing when researchers discovered that when a metal is heated and releases oxygen, it also gains electrons. Thus reduction is now used to indicate the gain of electrons. When oxygen binds to a substance, such as when metal rusts, the metal loses electrons and thus oxidation has come to mean the loss of electrons.
The confounding nature of this terminology is evident when speaking about compounds like NAD. NADH, albeit larger, is the reduced form of NAD because NAD gains electrons in order to bind to the H+ and form NADH. NAD is the oxidized form, in spite of the fact that the two compounds have the exact same amount of oxygen. NAD is the oxidized form because it lost electrons in order to kick out the H+. Complex I of the Electron Transport Chain is responsible for this reaction. Complex I strips away the electrons from NADH and pumps away the hydrogen, leaving behind an NAD which can then be used again in glycolysis. 
Methylene Blue, like NAD, can pick up H+ and assume a reduced form. It's so efficient at this process, that it can pull electrons out of the ETC itself and stop the process. This is how Methylene Blue attacks malaria. High doses of Methylene Blue inhibit the malarial mitochondria from producing adequate ATP to continue living. Methylene Blue essentially starves the malaria to death. The reason it can be used safely in humans is that the ETC in malaria lacks a few structures, such as complex I, which makes the malarial ETC more susceptible to the theft of electrons by methylene blue. 
Oxidative Stress and The Palace of the Mad
In 1899, Paul Ehrlich assumed directorship of the Institute of Experimental Therapy in Frankfurt near an insane asylum called, “Das Irrenschloss...” The Palace of the Mad. It was here that Dr. Alzheimer first described his eponymous disease which is first expressed as a difficulty thinking abstractly and progresses to a nearly complete loss of memory. Alzheimer Disease (AD) progresses until sufferers can no longer perform even the most basic activities to sustain life. During the 1980s, the Cholinergic Hypothesis emerged, claiming AD results from a reduction in the ability to make the neurotransmitter Acetylcholine. The majority of medications prescribed for AD works to increase Acetylcholine. Unfortunately, they've shown no impact on the progression of AD, and only moderately decrease its symptoms. In the 1990s, research was focused on the Amyloid Hypothesis. The brains of AD patients were found to contain extensive deposits of Beta-Amyloid Plaques. These plaques were though to be the underlying cause and an experimental vaccine was developed to prevent plaque formation. This vaccine was successful, but in spite of the absence of plaques, patient continued to grow progressively worse and die. Further research on these beta-amyloids revealed they have a non-pathological role and are found to a lesser extent in all healthy adults.
Beta-amyloids function to protect us against oxidative stress and their overabundance in those suffering from AD seems to indicate that its cause is associated with the most notorious electron leaking, super-oxide producer in the cell: the Electron Transport Chain. Research in the first decade of the twenty first century has shown that those with AD exhibit a depression of all electron transport chain complexes possibly due to oxidative damage. As these complexes become less numerous and less efficient, more leakage of electrons occur, and the ETC is further damaged. If one could find a substance capable of picking up excess electrons which leak out of the ETC and prevent the formation of superoxides, it would be possible to slow the progression of AD; furthermore, if that same substance could then return those electrons to the ETC, cells would have an increase in available energy. 

Methylene Blue is this substance. The proposed trade name is “Rember.” A 2008 clinical trial of low dose Methylene Blue found an 81% reduction in the speed of disease progression and trials are currently underway to see if Methylene Blue is capable of preventing formation of AD before symptoms arise. MB not only slows the disease, patients demonstrate a significant improvement in memory function. It's not a cure, but all research so far indicates that MB is the most promising treatment for AD found so far. Research has also indicated that low dose MB may be efficacious in the treatment of anxiety, depression, and Parkinsons disease.  
While these finding are certainly exciting for those suffering from such diseases, I most likely don't have Alzheimer's and I certainly don't have Malaria. The paramount data yielded from research on methylene blue is that in normal healthy adults it can improve memory, decrease anxiety, and perhaps even slow the aging process. Rather than interpreting the research for you, you'll find eight articles addressing these three effects of interest.

1. Behavioral, Physiological and Biochemical Hormetic Responses to the Autoxidizable Dye Methylene Blue



Dosage Calculations

Methylene Blue exhibits what's referred to as a hormetic dose response, where a small dose has the opposite effect of a large dose. For example, when 200mg Malaria treatment dosages are administered, the ETC within cells of the brain are actually less active and memory functions are inhibited. Methylene Blue is excreted rather rapidly, it's taken as often as 3 times a day in 5 hour intervals. Dosages vary between studies and nearly all the studies use animal models rather than humans so what we're trying for here is more of an appropriate range than a specific dose. The dose has to be high enough to have an effect, but low enough that the effect is the one we want. I'm going to start by calculating out doses equivalent to those used in the studies I've read, and then we'll discuss the dosages that have been found most effective by those who have experimented on themselves. I think you'll be surprised at the discrepancy between the two.

Take a look at these values taken from the articles listed above:

50mg kg-1
Study 1
Amnesic Effects
5mg kg-1
Study 1
No effect noted
4mg kg-1
Study 2
Improved object memory recognition
1mg kg-1
Study 1
Enhanced memory retention
Study 3
Enhanced discriminative learning
0.05mg kg-1
Study 1
Enhanced memory retention, less than 1mg kg-1

It would seem from these values that we want a dose higher than 0.05mg kg-1, but less than 4mg kg-1; however, don't forget that these values are for rats! First we have to do Allometric Scaling, which is a kind of animal-to-animal dose conversion. The underlying concepts are pretty interesting, but for brevity sake, I'll spare you the details. After scaling, our values are as follows:

2.77mg kg-1
This dose is the human equivalent of 4mg kg-1, and represents our upper limit
0.069mg kg-1
This dose is the human equivalent of 1mg kg-1 and represents our goal dose
0.035mg kg-1
This dose is the human equivalent of .05mg kg-1 and represents our lower limit

Now that we have our human equivalents, we can move onto the next problem... these rats were receiving MB intravenously. According to the article, Pharmacokinetics and organ distribution of intravenous and oral methylene blue, only 60% of methylene Blue is absorbed through the GI tract. So we have to correct for this:

4.61mg kg-1
2.77mg kg-1/ 0.6 = 4.61mg kg-1 is the oral upper limit
0.115mg kg-1
0.069mg kg-1/0.6 = 0.115mg kg-1 is the oral goal dose
.058mg kg-1
0.035mg kg-1/0.6 = .058mg kg-1 is the oral lower limit

According to these values, dosage should fall between 58 ug and 4.6mg per kg. If ones isn't familiar with the metric system, you'd first divide your weight in lbs by 2.2 and then multiply by 0.115mg. If I weigh 180 lbs, then my MB dose would be 9.4mg. This might seem a rather reasonable number when one compares it to the 60mg proposed dose for “Rember.” 
Interestingly, the majority of experimental users of MB report the greatest results from a dose three orders of magnitude lower. Many people report the greatest benefits from a dose of 60 micrograms. That's a mere 0.006 mg, yet many swear by it. Before trying MB for yourself, it would behoove to read some of the experiences reported by self-experimenter on forums such as Longecity or Mind and Muscle.
Because of these vastly different quantities, I'm going to provide two different dilutions for Methylene Blue. The first method will provide a dilution where each drop contains 60 mcg. I'd advise anyone using this augmentation to start with the lowest dose and working up toward the larger doses until one finds their own optimal amount. The second method will provide 1mg per drop to assist those who choose to trial the higher doses as used in the studies above. The nice thing about MB is it's safety. According to NIH Toxicological studies, the LD50 for rats is 1250mg kg-1. The average human doesn't show any indication of toxicity under 600mg, so playing around with doses between 60mcg and 10mg is pretty harmless as long as your not on an SSRI and don't suffer from Favism. 
Methylene Blue is widely available, perfectly legal to own, and cheap as dirt. It generally comes in one of two different concentrations.
The 1% concentration is available from chemical and science supply houses and is used as a biological stain and as an indicator in redox reactions. A 1 ounce bottle goes for as little as $3.50 plus shipping. 
I have purchased this type from The Science Company

A 2.303% solutions can be bought from pet stores and aquarium supply shops. It's an effective treatment for fungal infections and protozoa such as Ich which afflicts fish. A 4 ounce bottle retails for around 8 dollars, so this is the most economic way to purchase MB.
A third source is from suppliers such as Provepharm, which claim to produce a better product with less heavy metals. The “cheap” aquarium MB doesn't harm fish though... and fish are far more sensitive to heavy metals than people. I'd advise you to save your money and buy the cheaper product unless you have significant amounts of disposable income. 

To accurately dilute your methylene blue, you'll need:

There are guides which explain how to do these dilutions with an eye dropper, but the volume from droppers varies by as much as 20%. It's worth dropping 40 dollars to do it right, particularly since a single bottle of methylene blue contains far more than a years worth of doses. It isn't unusual to spend 50$ a month on a single Nootropic such as Oxiracetam, so this is good investment. 
The dilution formula is:
V1 x C1 = V2 x C2
V1 is the starting volume needed to make the solution
C1 is the concentration of starting solution
V2 is the volume of the final solution
C2 is the concentration of the final solution

It's a simple formula to use and if one was feeling lazy they could instead choose to use one of the many Online Dilution Calculators. To be nice though, I'm going to walk you through each math problem. (Thanks for the format Elus. It was more visually appealing than anything else I could come up with.) All that's left is for you to choose the dosage you want and use the formula for the percent concentration of MB that you bought.

Diluting a 2.303% solution to 60mcg/ml
This is the calculation to dilute 2.303% MB solution down to 60 micrograms/ml with a final volume of 100ml. Each ml of the final solution will provide a 60mcg dose and there will be 100 doses available.
V1 x (0.023 grams/mL) = (100mL) x (60 x 10-6 grams/mL) ...Solving for V1 we get
V1 = 0.26 mL
Therefore, we mix 0.26 mL of 2.303% MB solution and 99.74 mL water to obtain a 60 micrograms per mL solution of MB with a total volume of 100 mL. This is how it's done:
  1. Using a 1mL pipette, draw up 0.26 mL of 2.303% MB solution
  2. Place MB solution in a 100ml graduated cylinder
  3. Fill the graduated cylinder to the 100ml mark with purified water
One mL of this solution is a 60 micrograms dose.

Diluting a 1% solution to 60mcg/ml
This is the calculation to dilute 1% MB solution down to 60 micrograms/ml with a final volume of 100ml. Each ml of the final solution will provide a 60mcg dose and there will be 100 doses available.
V1 x (0.01 grams/mL) = (100mL) x (60 x 10-6 grams/mL) ...Solving for V1 we get
V1 = 0.6 mL
Therefore, we mix 0.6 mL of 1% MB solution and 99.4ml water to obtain a 60 micrograms per mL solution of MB with a total volume of 100 mL. This is how it's done:
  1. Using a 1mL pipette, draw up 0.6 mL of 1% MB solution
  2. Place MB solution in a 100ml graduated cylinder
  3. Fill the graduated cylinder to the 100ml mark with purified water
One mL of this solution is a 60 micrograms dose.

Diluting a 2.303% solution to 1mg/ml
This is the calculation to dilute 2.303% MB solution down to 1 milligram/ml with a final volume of 100ml. Each ml of the final solution will provide a 1 mg dose and there will be 100 doses available.
V1 x (0.023 grams/mL) = (100mL) x (1 x 10-3 grams/mL) ...Solving for V1 we get
V1 = 4.35 mL
Therefore, we mix 4.35 mL of 2.303% MB solution and 95.65 mL water to obtain a 1 milligram per mL solution of MB with a total volume of 100 mL. This is how it's done:
  1. Using a 5mL pipette, draw up 4.35 mL of 2.303% MB solution
  2. Place MB solution in a 100ml graduated cylinder
  3. Fill the graduated cylinder to the 100ml mark with purified water
One mL of this solution is a 1 milligram dose.

Diluting a 1% solution to 1mg /ml
This is the calculation to dilute 1% MB solution down to 1 milligram/ml with a final volume of 100ml. Each ml of the final solution will provide a 1 mg dose and there will be 100 doses available.
V1 x (0.01 grams/mL) = (100mL) x (1 x 10-3 grams/mL) ...Solving for V1 we get
V1 = 10 mL
Therefore, we mix 10 mL of 1% MB solution and 90.00 mL water to obtain a 1milligram per mL solution of MB with a total volume of 100 mL. This is how it's done:
  1. Using a 10mL pipette, draw up 10 mL of 1% MB solution
  2. Place MB solution in a 100ml graduated cylinder
  3. Fill the graduated cylinder to the 100ml mark with purified water
One mL of this solution is a 1 milligram dose.

Tips and Technique
Sometimes people unfamiliar with lab equipment get confused by the numbering on pipettes. They are labeled so that one measures as they eject the fluid, so if you want 0.26 mL, you draw up an entire mL of solution and then eject it out until you get to the 0.26 line. This wastes a negligible 0.73 mL of MB, so if you want to be thrifty you can subtract the amount you want (0.25 mL) from 1 mL. If you draw up to the mark on the pipette showing the difference, in this case the 0.73 mark, you'll have exactly 0.26ml.
Reading the amount in a graduated cylinder is also a bit tricky if you have no experience. You'll find that you don't get a nice flat line at the top of the fluid. Instead, the fluid forms a meniscus with the sides being higher and the center of the surface being lower. The volume is always read from the lowest point of the meniscus.
As I stated before, eye droppers are notoriously inaccurate but they are more convenient then carrying around a 1ml syringe. If you choose to use an eye dropper, 20 drops usually equals around 1ml. One last tip: Methylene Blue is a stain. Whatever it touches, be it your counter, your carpet, your clothes or your skin... it is now blue. Skin is constantly being shed, so stains on your hands will eventually fade. Stains to dental work such as crowns and dentures will not. While the low concentrations of MB aren't an issue, larger concentrations such as the 1mg/ml might if held in the mouth for long. I've also read about people using alternative means of administration such as through a mucosal administration device, or via sublingual route. I simply haven't seen research indicating these routes to be any better than by mouth.
If you read through forums, you'll also find reports of people combining MB with 1-2 grams of vitamin C. This is rather cool to do because the MB bonds to the excess hydrogen ions yielded by ascorbic acid, and the solution turns clear. Some claim this somehow makes the MB work more effectively, although I have my doubts and there isn't any supporting research. Because the doses of MB we're discussing maintain serum levels near homeostasis because the reduced and oxidized form, the “starting state” of the MB should be irrelevant. Vitamin C is good for you though! The color change is a neat trick, and it makes it into a kind of sour-orange cocktail.
Precautions and Contraindications
Of course, prior to using any substance, you should check with your medical doctor. I'm not advising you take anything and you should do your own due diligence. I'm simply reporting on something I find interesting. Methylene blue has a number of other medical uses we haven't discussed in the treatment of cyanide poisoning, an interesting condition called Methemoglobinemia. A famous example of people with methemoglobinemia are the Blue Fugates of Troublesome Creek, who were known for their vivid blue skin coloration. The condition is a hereditary disorder of the blood which requires treatment with oxygen and methylene blue. MB is administered at a dose of 2mg Kg-1 IV over as little as 5 minutes. Because we corrected for GI absorption rates, this value is nearly the same dose as the animal studies were using. Although adverse reactions aren't common, they do occur. Side effects can include hypertension and chest pain. Sometimes people experience confusion, dizziness, and headache. GI irritation can occur and both the feces and urine can be stained blue but this isn't really harmful. MB is damn safe under most circumstances but there are two contraindication that if not heeded can be downright lethal: Antidepressants and “favism.”
In high doses, MB has been found to exhibit MAO-I properties. When an MAO-I is combined with a serotonergic drug such as Prozac, Paxil, or Zoloft, an excess of the neurotransmitter serotonin can cause a number of serious psychiatric symptoms as well as CNS toxicity. Now, I've also read a considerable number of subjective accounts claiming that low dose MB, specifically the 60mcg dose, actually augments the action of these antidepressants; there are obviously no studies that support this and a serotonin storm isn't a good thing.
Favism is an antiquated term for a hereditary disorder that results in a haemolytic response to the consumption of broad beans, which are called “fava” beans in Italy. The condition is now called a G6PD deficiency. Methylene Blue is amongst a long list of drugs which can cause hemolytic anemia in sufferers.
As with any substance to which your body is naïve, start small. Some users have even reported effects from doses as low as 5mcg. This rings of crockery like homeopathy to me but if you find it works best for you, my opinion is irrelevant. I haven't personally gone above a 2mg total dose because it was less effective for me. I felt “cloudy” and tired, thus I haven't even tried the dosing used in the animal studies. Of course, curiosity will get the better of me sooner or later... The vast majority of MB proponents really are using a mere 60mcg dose and so this is a great place to start.

There is but one area in which Methylene Blue disappoints me: It's affect upon the eyes. You see, throughout WWII, high dose Methylene Blue was a rather unpopular treatment and preventative agent for Malaria amongst US soldiers. There are many reports of soldiers being non-compliant as they didn't like two side effects of MB: it turns the urine and the schlera of the eyes blue. As a die hard Frank Herbert fan... I aspire to blue schlera. My disappointment stems from the fact that the doses required to turn ones eye blue is significantly higher than the optimal doses for memory enhancement. Because of the hormetic dose response, such large doses would actually result in decreased mental faculties. Alas... MB is inexpensive, easy to acquire, and safe as long as one follows certain precautions and takes a reasonable dose. It shows considerable promise as a brain function enhancing agent, an anxiolytic, and may even prevent a person from developing a neurodegenerative disease. As such, I find Methylene Blue to be an augmentation worthy of consideration.

Holmes C. Long-term effects of Abeta42 immunisation in Alzheimer's disease: follow-up of a randomised, placebo-controlled phase I trial. Lancet. 2008;372(9634):216–23. doi:10.1016/S0140-6736(08)61075-2. PMID 18640458.


  1. Thank you so much for going in details and explaining it so the average person can grasp the content. You actually made me really interested in biology and chemistry again =)

  2. Hi, this is a very informative article, but something troubles me about the calculations. You have considered total intestinal absorption of MB. However, GI doesn't absorb it all at once, but rather slowly releases it in the bloodstream, and it has been said that peak blood concentration of MB after an iv dose is 10 times greater than after an oral dose. So, do you think it should be accounted for and values for iv administration multiplied by 10 to give appropriate dose values for oral ingestion?

    1. Have you found something regarding that?

  3. Hi, I really appreciated your dosage analysis and how to prepare the solution. I have been scouring the internet for pharmaceutical grade methylene blue, because I would feel more confident in the product if it wasn't intended for staining microscope slides or as a fish disinfectant. The chemical synthesis of methylene blue involves heavy metals and solvents like dioxane/carbon tetrachloride, so I feel that concerns about purity/contaminants are legitimate. The only sources of pharma grade methylene blue I could locate are a seller on ebay and this website: Do you have any idea whether these sources are legit?

  4. Hey anon, I have no idea; however, there's a great post here:

    The advantage of this method is that the author is describing the use of a pure reagent being diluted rather than serial dilutions of an inferior source like I've described above. Good stuff! Check it out.

  5. Another benefit of MB:
    Methylene Blue Reverses Human Cell Aging, Treats Progeria Symptoms in New Study:

  6. My uncle once suffered from Alzheimer's disease,i never thought there is a cure to it, we have used different medication but no solution,
    I got a herbal product from Dr Benard for the first two month my Uncle took the medication, he was about to remember past,he was also getting back his memory. now my uncle is living a normal life,no more sign of Alzheimer. he is completely cured. any one with such case can contact Dr Phillip on

  7. If your goal is blue schlera then why not put a very small dose in your eyes? It stains your tongue so I'm sure it would stain your eyes. I'm a dune fan too so this is a tempting experiment. I wonder what dilution would be required to not cause damage. Looks like someone else had it in their eyes and it wasn't good. Maybe if you used a very very very small amount and then slowly increased the dose until you reach the desired eye color.

  8. What does the negative exponent mean in the dosage ranges "less than 1mg kg-1" ? I haven't seen anything expressed this way before. Thanks.

    1. That's a standard scientific usage denoting "per." "1mg kg-1" is the same as saying "1mg per kg."