Methylene Blue, Mitochondrial Bioenergetics, and Disease Metabolism

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First, this post will briefly explain the process of cellular respiration. Then it will highlight the importance of proper mitochondrial function (bioenergetics) for maintaining the health of the organism, and will further show how poor mitochondrial function/energy production is an essential feature of nearly every known illness. When you understand the basics, then you can truly appreciate how Methylene Blue fits into the picture. So even though it will be long and tedious for some, it is highly recommended that you read all of the information before skipping to the studies at the bottom of the post.

How the cell produces energy: The basics of oxidative metabolism

According the current framework, the source of cellular energy is Adenosine Tri-Phosphate (ATP), which supposedly contains usable energy in its third phosphate bond. To make ATP, the body derives electrons and protons from food via digestion and subsequently breaks each molecule down into its constituent parts. The electrons and protons are passed along a series of molecules called redox (oxidation/reduction) molecules. Reduction refers to accepting electrons, and oxidation means to lose electrons. Redox molecules exist in two states, one being oxidised and the other being reduced.

In normal conditions, the (simplified) process is as follows:

Extracting energy from carbohydrate: 1. Glycolysis ===> 2. Acetyl CoA ===> 3. Krebs Cycle (TCA/Citric acid cycle) ===> 4. Electron transport chains/Oxidative Phosphorylation = ATP

Extracting energy from fat is slightly different: 1. Beta-Oxidation ===> 2. Acetyl CoA + Glycerol (which enters back into glycolysis) ===> 3. Krebs cycle (TCA/Citric acid cycle) ===> 4. Electron transport chain/Oxidative Phosphorylation = ATP

Below is a diagram of the first stage of glucose metabolism (glycolysis):

aqa_a2_biology_unit_4_complete_51_638.jpg


Notice the end product of this stage is Pyruvate (aka pyruvic acid) + 2 ATP + 2 NADH. This is a a redox reaction. NAD (Nicotinamide adenine dinucleotide) is a molecule derived from niacinamide (vitamin B3) and is a typical redox agent which participates in energy transfer. NAD+ is it's oxidised state, and NADH is it's reduced state. So in the above process, NAD+ is reduced (accepts electrons) so that it now becomes NADH. All that needs to be understood is that some of the electrons from food have been passed onto this carrier molecule (NAD), and that they will later to used to drive ATP synthesis in the final stage. However, to derive more of the energy from food, the next phase must proceed.

Under normal, healthy conditions in the presence of oxygen, most of the Pyruvate will be oxidised and will donate electrons to another molecule to produce Acetyl CoA. important note: pyruvate dehydrogenase is the enzyme which coverts pyruvate to Acetyl CoA.. This point is essentially the beginning of mitochondrial energy production, and aside from Acetyl CoA it also yields CO2 and more NADH.

PYRUVATE_TO_ACETYLCOA.png


The next stage involves Acetyl CoA entering the Krebs (citric acid) cycle whereby lots of redox reactions take place. This process yield more NADH, CO2, and FADH2. FAD (Flavin adenine dinucleotide) is derived from riboflavin (vitamin B2) and is similar to NAD - being a key redox component in ATP production. All that is important to know in this context is that more electrons are being derived from the original food product and are attached to carrier molecules to be used later. The Krebs cycle can be disrupted due to several factors, including if there is a deficiency of any of the intermediates involved, or a lack of oxygen.

For those who are wondering, this is now the point where fatty acids enter. Beta oxidation breaks down fatty acids into 2-carbon molecules and directly produces Acetyl CoA. So fatty acids basically bypass the whole process involving glycolysis and pyruvate oxidation.

Krebs.png


Finally, the electron carriers NADH and FADH2 enter the "electron transport chain", which is made up of five protein complexes. The details on this can be found in biology text books, online, or on youtube for those who are interested.

c8_9x16_chemiosmosis.jpg


Basically, NADH and FADH2 from each of the previous stages donate electrons the complex 1 and 2, which are passed to complex 3, and then finally through complex 4 to reduce oxygen and make water. This is the only stage in energy production where oxygen plays a role. But it is the final (and most important) stage, because when oxygen accepts those electrons it drives complex 5 (ATP Synthase) to produce 34 ATP. What is essential to understand here is that when there is no oxygen present, ATP cannot be synthesised in the mitochondria. .

RECAP

- Carbohydrate is broken down into glucose, which is converted into pyruvate via glycolysis. This yields 2 ATP.
- Pyruvate is converted into Acetyl CoA by the enzyme Pyruvate dehydrogenase when oxygen is present . In a similar way, fats are broken down into fatty acids, which also get converted into Acetyl CoA, and glycerol, which enters glycolysis.
-Acetyl CoA goes through the Krebs cycle to make NADH and FADH2
- Electrons from NADH and FADH2 run through the electron transport chain to reduce oxygen and this results in a massive production of ATP.

What happens when there is no oxygen?

When no oxygen in present, or when pyruvate dehydrogenase (the enzyme responsible for oxidising pyruvate into Acetyl CoA) is not functioning properly, the cell is left with only one option: glycolysis. Look back at the diagram of glycolysis. See that it only produces a measly 2 ATP, yet the only way it can do so is by having sufficient levels of NAD+ so that it can reduce them to NADH. In a situation where no oxygen is present, there are significant amounts of Pyruvate and NADH, and not enough NAD+ to keep the glycolytic cycle running.

B9780323071550000216_f21_07_9780323071550.jpg


In basic terms, the cell has a choice of 2 units of energy (ATP), or no energy at all (because there is a lack of oxygen). So in order to survive, it NEEDS to provide an avenue for NADH's electrons to be donated, otherwise energy production completely stops. Hence, it uses Pyruvate as the "electron sink", where NADH recycles back to NAD+ by donating it's electrons to pyruvate, which goes on to form lactic acid. The reaction is catalized by the enzyme lactate dehydrogenase. This is called anaerobic respiration, and is essentially a temporary measure made by the cell the survive in oxygen deprived conditions.

Main point: Energy is needed by every cell to function properly, to repair, and to differentiate into what is required at any given moment. Lactate and lactate dehydrogenase are markers of glycolysis - a highly inefficient energy source. This means that the level of lactate or lactate dehydrogenase enzymes can indicate whether the mitochondria are working properly so that there is enough energy to do what the cell needs to do, or whether it is resorting the the backup mechanism for whatever reason.

How does this look in real life?

Cancer
Serum lactate dehydrogenase and survival following cancer diagnosis.
RESULTS:
At the end of follow-up, 5799 participants were deceased. Hazard ratios (HRs) and 95% confidence intervals (CIs) for overall and cancer-specific death in the multivariable model were 1.43 (1.31-1.56) and 1.46 (1.32-1.61), respectively, for high compared with low prediagnostic LDH. Site-specific analysis showed high LDH to correlate with an increased risk of death from prostate, pulmonary, colorectal, gastro-oesophageal, gynaecological and haematological cancers. Serum LDH assessed within intervals closer to diagnosis was more strongly associated with overall and cancer-specific death.
CONCLUSIONS:
Our findings demonstrated an inverse association of baseline serum LDH with cancer-specific survival, corroborating its role in cancer progression.
https://www.ncbi.nlm.nih.gov/pubmed/26469834

Type 1 and 2 Diabetes
Lactate, a Neglected Factor for Diabetes and Cancer Interaction

Fasting plasma lactate level is increased in patients with DM including T1DM and T2DM versus nondiabetic persons [28–36]. Diabetic patients with obesity exhibit higher fasting plasma lactate levels than nondiabetic individuals with obesity [37, 38]. Barnett et al. proposed that diabetes-associated hyperlactatemia might be an early change in the time course of the disease [39]. Recently, Berhane et al. [40] demonstrated that lactate production progressively rises during hyperinsulinemic euglycemic clamp study, a condition of hyperinsulinemia similar to the early stages in the development of T2DM. Intriguingly, similar previous studies also report elevated lactate concentrations during the early stages of diabetes, prediabetes, and the hyperinsulinemia condition. In addition, Brouwers et al. [41] reported increased lactate levels in patients with poorly controlled T1DM and glycogenic hepatopathy, implying that enhanced plasma lactate concentrations are part of the clinical spectrum of these diseases. Furthermore, lactate has also been revealed to predict diabetes occurrence in the future [42, 43].

The mechanisms underlying diabetes-associated hyperlactatemia include serious changes in the intracellular glucose metabolism in insulin-sensitive tissues, for example, diminished glycogen synthesis, compromised glucose oxidative metabolism, and increased whole-body rate of nonoxidative glycolysis [28, 31, 44]. Importantly, when compared with controls, nonoxidative glycolysis rate retains higher in T2DM patients during hyperglycemic [31, 44, 45] and hyperinsulinemic [31, 44] status. In addition, the postprandially nonoxidative glycolysis is elevated in these patients relative to healthy controls and blood lactate level rises under this condition [36]. Insulin resistance plays a vital role in the pathogenesis of T2DM [46] and can be used as an early marker for the disease [40]. Under the insulin resistant condition, high levels of insulin promote glycolysis through activating two rate limiting enzymes, namely, phosphofructokinase and pyruvate dehydrogenase [47]. Thus, patients with insulin resistance/diabetes exhibit augmented activity of glycolysis [31, 48]. The elevated glycolysis results in enhanced formation of NADH and pyruvate and reduced NAD+ levels. Pyruvate is converted into lactate by LDH accompanied by NAD+ generation from NADH in a redox reaction. This reaction may be accentuated in insulin resistance since hyperinsulinemia induces enhanced glycolysis.

https://www.hindawi.com/journals/mi/2016/6456018/

Arthritis - RH and OA

Lactate dehydrogenase activity and its isoenzymes in serum and synovial fluid of patients with rheumatoid arthritis and osteoarthritis.

By determining the total activity of total lactate dehydrogenase (LDH-T) and its isoenzymes in serum and synovial fluid (SF) of patients with rheumatoid arthritis (RA) and osteo-arthritis (OA) we demonstrated in RA serum increased (p less than 0.02) activity of hepatic LDH (LDH-H) and a shift of the LDH isoenzymatic profile towards the M forms; in rheumatoid SF increased (p less than 0.001) activity of the total LDH-T and LDH-H which makes possible the use of these markers of inflammation in assessing RA activity. Values for LDH-T and LDH-H of 400-700 U/l and 300-500 U/l, respectively, correspond to moderate disease activity, while values exceeding 750 U/l and 550 U/l, respectively, correspond to high RA activity. The anaerobic isoenzymatic distribution of LDH in rheumatoid SF results in a significant (p less than 0.001) decrease in LDH1 and LDH2 and an increase (p less than 0.001) in LDH4 and LDH5
https://www.ncbi.nlm.nih.gov/pubmed/1593572

Alzheimer's
Activities of key glycolytic enzymes in the brains of patients with Alzheimer's disease.

The activities of hexokinase, aldolase, pyruvate kinase, lactate dehydrogenase and glucose 6-phosphate dehydrogenase were determined in brains of patients with Alzheimer's disease (AD) and in age matched controls. For pyruvate kinase and lactate dehydrogenase a significant increase in specific activity was found in frontal and temporal cortex of AD brains, while the activities of aldolase and hexokinase are not changed. Glucose 6-phosphate dehydrogenase activity was significantly reduced in hippocampus. The increase of some glycolytic enzyme activities is correlated with increased contents of lactate dehydrogenase and glial fibrillary acidic protein (GFAP) in homogenates of frontal and temporal cortex and elevated phosphofructokinase (PFK) and GFAP in astrocytes from the same brain areas. The data extend previous findings on an increase in brain PFK specific activity in AD and suggest that the increased activity of some glycolytic enzymes may be, at least in part, the result of the reactive astrocytosis developing in the course of AD.
https://www.ncbi.nlm.nih.gov/pubmed/10443553

Multiple Sclerosis

Lactic dehydrogenase isoenzymes in adolescents with multiple sclerosis.

Multiple sclerosis is an immune-mediated demyelinating disease with high morbidity and major mortality. To determine the potential use of lactic dehydrogenase activity and lactic dehydrogenase isoenzyme concentrations in cerebrospinal fluid as biomarkers of multiple sclerosis, we reviewed the files of all patients with multiple sclerosis who attended our tertiary pediatric medical facility from 1999-2005. The study group included three adolescent patients with multiple sclerosis (cerebrospinal fluid analysis at diagnosis) and one patient with recurrent optic neuritis (cerebrospinal fluid analysis during a disease episode). The isoenzyme pattern was abnormal in all patients with multiple sclerosis, with higher-than-normal levels of lactic dehydrogenase-2, lactic dehydrogenase-3, and lactic dehydrogenase-5 in two patients, and lower-than-normal levels of lactic dehydrogenase-4 in one patient. It was not necessarily, however, the same two patients who had the abnormally high levels of lactic dehydrogenase-2, -3, and -5. The patient with optic neuritis also exhibited an abnormal lactic dehydrogenase isoenzyme pattern that shared common features with the others. Multiple sclerosis appears to be characterized by an abnormal lactic dehydrogenase isoenzyme pattern in cerebrospinal fluid. The importance of this finding and its diagnostic potential use warrant further investigation.
https://www.ncbi.nlm.nih.gov/pubmed/19748045

Consider that lactate dehydrogenase is just one biomarker that can be tested for. Looking through the research, it seems that mitochondrial inefficiency is a common factor in almost every pathology. Dr Doug Wallace, pioneering researcher on mitochondria, has essentially shown that mitochondrial heteroplasmy (mtDNA mutation rate) is associated with around 80% of diseases we see today. The following article briefly outlines some of his work:

New work by a pioneering scientist details how subtle changes in mitochondrial function may cause a broad range of common metabolic and degenerative diseases. Mitochondria are tiny energy-producing structures within our cells that contain their own DNA.

The new research shows that small changes in the ratio of mutant to normal mitochondrial DNA within the thousands of mitochondrial DNAs inside each cell can cause abrupt changes in the expression of numerous genes within the nuclear DNA. Furthermore, the different proportions of mutant mitochondrial DNA that result in altered nuclear gene expression correspond to the same proportions of mutations in mitochondrial DNA that are associated with diabetes and autism; brain, heart and muscle disease; or lethal infantile disease.

By showing that subtle changes in the cellular proportion of the same mitochondrial DNA mutation can result in a wide range of different clinical manifestations, these findings challenge the traditional model that a single mutation causes a single disease,” said study leader Douglas C. Wallace, Ph.D., director of the Center for Mitochondrial and Epigenomic Medicine at The Children’s Hospital of Philadelphia. He added, “The research offers key insights into understanding the underlying cause of metabolic and neurodegenerative disorders such as diabetes, Alzheimer, Parkinson and Huntington disease, as well as human aging.”

“The discrete changes in nuclear gene expression in response to small increases in mitochondrial DNA mutant level are analogous to the phase changes that result from adding heat to ice,” said Wallace. “As heat is added, the ice abruptly turns to water and with more heat, the water turns abruptly to steam.” Here a quantitative change (an increasing proportion of mitochondrial DNA mutation) results in a qualitative change (coordinate changes in nuclear gene expression together with discrete changes in clinical symptoms).

The study by Wallace and colleagues appeared online Sept. 3 in the Proceedings of the National Academy of Sciences.

Existing in hundreds or thousands of copies outside the nucleus of every cell, mitochondria have their own DNA, distinct from the well-known DNA inside the cell nucleus. Although mitochondrial DNA (mtDNA) holds far fewer genes than nuclear DNA, mtDNA exchanges signals with nuclear DNA and participates in complicated networks of biochemical reactions essential to life.

Wallace’s current study rests on his investigations into the mysteries of mitochondria for over 40 years. In 1988, he was the first to demonstrate that mitochondrial DNA mutations can cause human disease. He has continued to build a body of research into mechanisms by which mutations in mtDNA contribute to both rare and common diseases by disrupting the body’s energy production. In the current study, Wallace’s team investigated the impacts of steadily increasing levels of a pathogenic mutation in one particular base of mitochondrial DNA.

Researchers already knew that if 10 to 30 percent of a person’s mitochondrial DNA has this mutation, a person has diabetes, and sometimes autism. Individuals with an mtDNA mutation level of 50 to 90 percent have other multisystem diseases, particularly MELAS syndrome, a severe condition which involves brain and muscle impairments. Above the 90 percent level, patients die in infancy.

In the current study, conducted in cultured human cells, Wallace and colleagues analyzed cells with different levels of this pathogenic mtDNA mutation to determine the effects on the gene expression of the cell. The researchers measured variations in cellular structure and function, nuclear gene expression, and production of different proteins.

The mutations in mitochondria impair their ability to produce energy, and mitochondria transmit distress signals to the cell nucleus,” said Wallace. “But the nucleus can respond in only a limited number of ways.” Those responses may manifest themselves in discrete, profound consequences for patients.

Findings May Pertain to Common Conditions

Wallace argues that the medical significance of this research extends beyond the province of the relatively rare disorders typically classified as mitochondrial diseases. The gene expression profile—the pattern of gene activity seen at the level at which mtDNA mutations trigger brain disorders—parallels the profiles found in Alzheimer, Parkinson and Huntington diseases. “The findings in this study provide strong support for the concept that common metabolic diseases such as diabetes and obesity, heart and muscle diseases, and neurodegenerative diseases have underpinnings in energy deficiencies from malfunctioning mitochondria,” said Wallace. “Thus this concept brings together a cluster of diseases previously considered to be separate from one another.”

Significantly, Wallace added that the research also pertains to aging. Because mitochondrial mutations accumulate as people age, mitochondrial energy production declines, with deleterious effects on the heart, the brain and on interrelated biological systems that sustain health and life.

Next steps in research, says Wallace, include investigations of how different diseases are associated with the sorts of abrupt phase changes his group found in the current cellular study. Some of the cellular changes, signaling patterns and protein activity levels found in the current research might become useful biomarkers in disease studies and drug development. “For instance, a preclinical screen for potential drugs that could reverse gene expression profile changes of the mitochondrial DNA mutant cells could reveal new therapies,” he added.

Wallace’s current study reinforces arguments he has presented over the course of his career, that mitochondria play a central, largely under-recognized role in all common human diseases. He has long argued that a traditional biomedical approach focusing on anatomy and individual organs does not provide the insights generated from a systems biology, bioenergetics-focused approach.

Wallace’s paradigm-shifting hypotheses remain controversial in biomedicine. This latest study, he says, implies that the complexity of common diseases is rooted in the disconnect between continuous, linear changes in mtDNA mutations and the discontinuous, sudden phase changes in nuclear gene expression that result. Even as his overall arguments about the role of mitochondria contend for broader acceptance, the current findings may provide useful, versatile tools for understanding and treating disease.

Overwhelming evidence supports this notion, showing that defects in mitochondrial respiration underlie almost all illness.

Neurodegenerative diseases

Mitochondrial defects are found in pathological studies of all major neurodegenerative diseases, said Vamsi Mootha of Harvard Medical School. The range of mitochondrial defects includes fragmentation and other morphological changes, increased mutation rates in mtDNA, changes in permeability of mitochondrial membranes, changes in redox potential, accumulation of mutant proteins, and impaired oxidative phosphorylation (Reddy and Reddy, 2011).
https://www.ncbi.nlm.nih.gov/books/NBK208519/

Cancer

As an alternative to the somatic mutation theory, emerging evidence suggests that cancer is primarily a mitochondrial metabolic disease (Seyfried and Shelton, 2010; Hu et al., 2012; Verschoor et al., 2013; Seyfried et al., 2014). The view of cancer as a metabolic disease originated with the experiments of Otto Warburg (Warburg, 1956a,b; Burk et al., 1967). Respiratory insufficiency is the origin of cancer according to Warburg's theory. All other phenotypes of the disease, including the somatic mutations, arise either directly or indirectly from insufficient respiration (Warburg, 1956a; Seyfried, 2012a; Seyfried et al., 2014). Warburg's metabolic theory was also in line with the concepts of C. D. Darlington and others showing that cancer is largely a cytoplasmic mitochondrial disease (Woods and Du Buy, 1945; Darlington, 1948).
[..]
In summary, the information presented here supports the notion that cancer originates from damage to the mitochondria in the cytoplasm rather than from damage to the genome in the nucleus. The genomic damage in tumor cells follows, rather than precedes, the disturbances in cellular respiration. This view is also consistent with the previous findings of Roskelley et al. (1943), Hu et al. (2012).
http://journal.frontiersin.org/article/10.3389/fcell.2015.00043/full

Skin diseases

Aberrant mitochondrial structure and function influence tissue homeostasis and thereby contribute to multiple human disorders and ageing. Ten per cent of patients with primary mitochondrial disorders present skin manifestations that can be categorized into hair abnormalities, rashes, pigmentation abnormalities and acrocyanosis. Less attention has been paid to the fact that several disorders of the skin are linked to alterations of mitochondrial energy metabolism. This review article summarizes the contribution of mitochondrial pathology to both common and rare skin diseases. We explore the intriguing observation that a wide array of skin disorders presents with primary or secondary mitochondrial pathology and that a variety of molecular defects can cause dysfunctional mitochondria. Among them are mutations in mitochondrial- and nuclear DNA-encoded subunits and assembly factors of oxidative phosphorylation (OXPHOS) complexes; mutations in intermediate filament proteins involved in linking, moving and shaping of mitochondria; and disorders of mitochondrial DNA metabolism, fatty acid metabolism and heme synthesis. Thus, we assume that mitochondrial involvement is the rule rather than the exception in skin diseases. We conclude the article by discussing how improving mitochondrial function can be beneficial for aged skin and can be used as an adjunct therapy for certain skin disorders. Consideration of mitochondrial energy metabolism in the skin creates a new perspective for both dermatologists and experts in metabolic disease.
https://www.ncbi.nlm.nih.gov/pubmed/24980550

Cardiovascular disease

Whereas the pathogenesis of atherosclerosis has been intensively studied and described, the underlying events that initiate cardiovascular disease are not yet fully understood. A substantial number of studies suggest that altered levels of oxidative and nitrosoxidative stress within the cardiovascular environment are essential in the development of cardiovascular disease; however, the impact of such changes on the subcellular or organellar components and their functions that are relevant to cardiovascular disease inception are less understood. In this regard, studies are beginning to show that mitochondria not only appear susceptible to damage mediated by increased oxidative and nitrosoxidative stress, but also play significant roles in the regulation of cardiovascular cell function. In addition, accumulating evidence suggests that a common theme among cardiovascular disease development and cardiovascular disease risk factors is increased mitochondrial damage and dysfunction.
https://www.ncbi.nlm.nih.gov/pubmed/15855047

The list is exhaustive, and anyone who is interested can do a search on PubMed and see that every non-genetic disease is essentially linked with mitochondrial respiratory defects. Ultimately, any factor which hampers the mitochondria's ability to produce enough energy is a potential disease-causing agent. A cell needs sufficient energy to be able to deal with life's stressors, and when that energy source declines, pathology kicks in. What this means is that energy metabolism is fundamentally coupled with a healthy living state. Nobel prize winner Albert Szent-Gyorgi emphasised in his book The Living State: With Observations on Cancer that "a living cell requires energy not only for all its functions, but also for the maintenance of its structure. He could see that structure and function were interdependent on every level, and he believed that in order for a system to increase its level of order and coherence, it must have sufficient energy to do so. Likewise, a lack of energy would decrease order, organisation, and would promote entropy in that system. This can be applied to the human body, and we see that the research supports this. The disease-state seems to be chaotic, where information transfer is poor and the system is incoherent as a whole, probably lacking in information. This is not limited to biological processes however, ala Gabor Mate and such individuals who show that the emotional/psychological state has the ability to promote or to cure disease. So in this sense, looking at the body almost like an information system which can be affected by all factors, external and internal, is probably the most constructive way to look at it. However, this thread is aimed at fixing some biological processes, so on this topic, I think we should attempt to increase the energy available to cell. We can do this by targeting the mitochondria, and by examining factors which are associated with defective mitochondrial metabolism.

Enter stress-metabolism

Any stressful stimuli (both external and physiological) requires increased metabolic efficiency to provide extra energy so that certain requirements can be met. If the stress persists, glycogen stores are eventually depleted (which happens fairly quickly), so adrenaline and cortisol are released via the HPA axis. Cortisol begins to break down muscle tissue, the liver performs gluconeogenesis and the proteins are converted to glucose. Adrenaline simultaneously activates lipolysis, breaking down adipose tissue and cell membranes fats to be used for energy by the mitochondria. However, due to the Randle Effect, fatty acids and glucose cannot undergo oxidative phosphorylation at the same time. Fatty acid oxidation inhibits pyruvate dehydrogenase (remember, the enzyme which allows pyruvate to enter the mitochondria), therefore glucose must undergo glycolysis. The simultaneous breakdown of fat via oxidative phosphorylation and glucose via glycolysis provides the body with maximum energy. However, the activation of HPA-axis hormones suppress the thyroid, and this process is only designed to be a temporary measure. The over production of lactate has several negative consequences, including further inhibition of mitochondrial respiration, and overall damage to cellular structures. A reliance on cortisol means that cholesterol's conversion to protective hormones pregnenelone, DHEA, and progesterone is stopped.

Ray Peat explains some of the processes involved in cancer "stress-metabolism" in the following:
Lactate formation from glucose is increased when anything interferes with respiratory energy production, but lactate, through a variety of mechanisms, can itself suppress cellular respiration. (This has been called the Crabtree effect.) Lactate can also inhibit its own formation, slowing glycolysis. In the healthy cell, the mitochondrion keeps glycolysis working by consuming pyruvate and electrons (or "hydrogens") from NADH, keeping the cell highly oxidized, with a ratio of NAD+/NADH of about 200. When the mitochondrion's ability to consume pyruvate and NADH is limited, the pyruvate itself accepts the hydrogen from NADH, forming lactic acid and NAD+ in the process. As long as lactate leaves the cell as fast as it forms, glycolysis will provide ATP to allow the cell to survive. Oxygen and pyruvate are normally "electron sinks," regenerating the NAD+ needed to produce energy from glucose.

But if too much lactate is present, slowing glycolytic production of ATP, the cell with defective respiration will die unless an alternative electron sink is available. The synthesis of fatty acids is such a sink, if electrons (hydrogens) can be transferred from NADH to NADP+, forming NADPH, which is the reducing substance required for turning carbohydrates and pyruvate and amino acids into fats.

This transfer can be activated by the transhydrogenase enzymes in the mitochondria, and also by interactions of some dehydrogenase enzymes.

The enzyme, fatty acid synthase (FAS), normally active in the liver and fat cells and in the estrogen-stimulated uterus, is highly active in cancers, and its activity is an inverse indicator of prognosis. Inhibiting it can cause cancer cells to die, so the pharmaceutical industry is looking for drugs that can safely inhibit it. This enzyme is closely associated with the rate of cell proliferation, and its activity is increased by both cortisol and estrogen.

The first biochemical event when a cell responds to estrogen is the synthesis of fat. Estrogen can activate transhydrogenases, and early studies of estrogen's biological effects provided considerable evidence that its actions were the result of the steroid molecule's direct participation in hydrogen transfers, oxidations and reductions. E.V. Jensen's claim that estrogen acts only through a "receptor protein" which activated gene transcription was based on his experimental evidence indicating that estrogen doesn't participate in oxidation and reduction processes in the uterus, but subsequently his claim has turned out to be false.

Glycolysis is very inefficient for producing usable energy compared to the respiratory metabolism of the mitochondria, and when lactate is carried to the liver, its conversion to glucose adds to the energy drain on the organism.

The hypoglycemia and related events resulting from accelerated glycolysis provide a stimulus for increased activity of the adaptive hormones, including cortisol. Cortisol helps to maintain blood sugar by increasing the conversion of protein to amino acids, and mobilizing free fatty acids from fat stores. The free fatty acids inhibit the use of glucose, so the stress metabolism relies largely on the consumption of amino acids. This increases the formation of ammonia, yet the combination of glycolysis and fat oxidation provides less carbon dioxide, which is needed for the conversion of ammonia to urea. Ammonia stimulates the formation of lactate, while carbon dioxide inhibits it.

Starving an animal with a tumor increases the stress hormones, providing free fatty acids and amino acids, and accelerates the tumor's growth (Sauer and Dauchy, 1987); it's impossible to "starve a tumor," by the methods often used. Preventing the excessive breakdown of protein and reducing the release of fatty acids from fat cells would probably cause many cancer cells to die, despite the availability of glucose, because of lactate's toxic effects, combined with the energy deficit caused by the respiratory defect that causes their aerobic glycolysis. Recently, the intrinsically high rate of cell death in tumors has been recognized. The tumor is maintained and enlarged by the recruitment of "stem cells." These cells normally would repair or regenerate the tissue, but under the existing metabolic conditions, they fail to differentiate properly.

The extracellular matrix in the tumor is abnormal, as well as the metabolites and signal substances being produced there, and the new cells fail to receive the instructions needed to restore the normal functions to the damaged tissue. These abnormal conditions can cause abnormal differentiation, and this cellular state is likely to involve chemical modification of proteins, including remodeling of the chromosomes through acetylation of the histones (Alam, et al., 2008; Suuronen, et al., 2006). The protein-protective effects of carbon dioxide are replaced by the protein-damaging effects of lactate and its metabolites.

The ability of lactic acid to displace carbon dioxide is probably involved in its effects on the blood clotting system. It contributes to disseminated intravascular coagulation and consumption coagulopathy, and increases the tendency of red cells to aggregate, forming "blood sludge," and makes red cells more rigid, increasing the viscosity of blood and impairing circulation in the small vessels. (Schmid-Schönbein, 1981; Kobayashi, et al., 2001; Martin, et al., 2002; Yamazaki, et al., 2006.)

The features of the stress metabolism include increases of stress hormones, lactate, ammonia, free fatty acids, and fat synthesis, and a decrease in carbon dioxide. Factors that lower the stress hormones, increase carbon dioxide, and help to lower the circulating free fatty acids, lactate, and ammonia, include vitamin B1 (to increase CO2 and reduce lactate), niacinamide (to reduce free fatty acids), sugar (to reduce cortisol, adrenaline, and free fatty acids), salt (to lower adrenaline), thyroid hormone (to increase CO2). Vitamins D, K, B6 and biotin are also closely involved with carbon dioxide metabolism. Biotin deficiency can cause aerobic glycolysis with increased fat synthesis (Marshall, et al., 1976).

A protein deficiency, possibly by increasing cortisol, is likely to contribute to increased FAS and fat synthesis (Bannister, et al., 1983), but the dietary protein shouldn't provide an excess of tryptophan, because of tryptophan's role as serotonin precursor--serotonin increases inflammation and glycolysis (Koren-Schwartzer, et al., 1994).

Incidental stresses, such as strenuous exercise combined with fasting (e.g., running or working before eating breakfast) not only directly trigger the production of lactate and ammonia, they also are likely to increase the absorption of bacterial endotoxin from the intestine. Endotoxin is a ubiquitous and chronic stressor. It increases lactate and nitric oxide, poisoning mitochondrial respiration, precipitating the secretion of the adaptive stress hormones, which don't always fully repair the cellular damage.

Aspirin protects cells in many ways, interrupting excitotoxic processes by blocking nitric oxide and prostaglandins, and consequently it inhibits cell proliferation, and in some cases inhibits glycolysis, but the fact that it can inhibit FAS (Beynen, et al., 1982) is very important in understanding its role in cancer.

There are several specific signals produced by lactate that can promote growth and other features of cancer, and it happens that aspirin antagonizes those: HIF, NF-kappaB, the kinase cascades, cyclin D1, and heme oxygenase.

Lactate and inflammation promote each other in a vicious cycle (Kawauchi, et al., 2008).

The toxic mechanism of bacterial endotoxin (lipopolysaccharide) involves inappropriate stimulation (Wang and White, 1999) of cells, followed by inflammation and mitochondrial inhibition. The stimulation seems to be a direct "biophysical" action on cells, causing them to take up water (Minutoli, et al., 2008), which is especially interesting, since estrogen's immediate excitatory effect causes cells to take up water.

The whole story is really complex, and there are so many avenues that link in with one another. I am only beginning to get my head around some of Peat's work, but I digress. A couple of things are for certain: Hypoxia (lack of oxygen) is present in almost every pathology, and in those that are not hypoxic, a defect in respiratory enzymes or complexes can be identified.

A small part of the picture: Nitric Oxide

Inefficient mitochondrial metabolism results in less production of carbon dioxide. Carbon dioxide is required by cells for several functional reasons, including the vasodilation of blood vessels, and maybe most importantly to allow the body to absorb oxygen via the respiratory tract. The amount of oxygen which can be absorbed from air is dependent on how much carbon dioxide is present in the blood, and this is due to the "Bohr Effect". Basically, if the mitochondria are not working properly, there will be low carbon dioxide (CO2), which also means that less oxygen diffuse into the blood stream. This is an initial cause of hypoxia. Remember that mitochondria need oxygen to produce ATP, so any oxygen deficiency is going to force many mitochondria to revert back to primitive glycolytic metabolism.

Additionally, since CO2 is the main vasodilator of blood vessels, in conditions of low CO2 the body is required upregulate the synthesis of Nitric Oxide (another potent vasodilator) via Nitric Oxide Synthase enzymes so that oxygen can reach the tissues. Nitric oxide elevations are meant to be a temporary measure against hypoxia.

However, during its own detoxification Nitric Oxide can bind "irreversibly" with Cytochrome C Oxidase, a key component of the mitochondrial electron transport chain. This means that for the cell to continue producing ATP via oxidative phosphorylation, it must produce a new cytochrome c oxidase, although this can be a lengthy and energy hungry process.

Regulation of mitochondrial respiration by nitric oxide inhibition of cytochrome c oxidase.

Nitric oxide (NO) and its derivatives inhibit mitochondrial respiration by a variety of means. Nanomolar concentrations of NO immediately, specifically and reversibly inhibit cytochrome oxidase in competition with oxygen, in isolated cytochrome oxidase, mitochondria, nerve terminals, cultured cells and tissues. Higher concentrations of NO and its derivatives (peroxynitrite, nitrogen dioxide or nitrosothiols) can cause irreversible inhibition of the respiratory chain, uncoupling, permeability transition, and/or cell death. Isolated mitochondria, cultured cells, isolated tissues and animals in vivo display respiratory inhibition by endogenously produced NO from constitutive isoforms of NO synthase (NOS), which may be largely mediated by NO inhibition of cytochrome oxidase. Cultured cells expressing the inducible isoform of NOS (iNOS) can acutely and reversibly inhibit their own cellular respiration and that of co-incubated cells due to NO inhibition of cytochrome oxidase, but after longer-term incubation result in irreversible inhibition of cellular respiration due to NO or its derivatives. Thus the NO inhibition of cytochrome oxidase may be involved in the physiological and/or pathological regulation of respiration rate, and its affinity for oxygen.
https://www.ncbi.nlm.nih.gov/pubmed/11239484

More on the destructive effects of excess Nitric Oxide:

The nitric oxide theory of aging revisited.

Bacterial and viral products, such as bacterial lipopolysaccharide (LPS), cause inducible (i) NO synthase (NOS) synthesis, which in turn produces massive amounts of nitric oxide (NO). NO, by inactivating enzymes and leading to cell death, is toxic not only to invading viruses and bacteria, but also to host cells. Injection of LPS induces interleukin (IL)-1beta, IL-1alpha, and iNOS synthesis in the anterior pituitary and pineal glands, meninges, and choroid plexus, regions outside the blood-brain barrier. Thereafter, this induction occurs in the hypothalamic regions (such as the temperature-regulating centers), paraventricular nucleus (releasing and inhibiting hormone neurons), and the arcuate nucleus (a region containing these neurons and axons bound for the median eminence). Aging of the anterior pituitary and pineal with resultant decreased secretion of pituitary hormones and the pineal hormone melatonin, respectively, may be caused by NO. The induction of iNOS in the temperature-regulating centers by infections may cause the decreased febrile response in the aged by loss of thermosensitive neurons. NO may play a role in the progression of Alzheimer's disease and parkinsonism. LPS similarly activates cytokine and iNOS production in the cardiovascular system leading to coronary heart disease. Fat is a major source of NO stimulated by leptin. As fat stores increase, leptin and NO release increases in parallel in a circadian rhythm with maxima at night. NO could be responsible for increased coronary heart disease as obesity supervenes. Antioxidants, such as melatonin, vitamin C, and vitamin E, probably play important roles in reducing or eliminating the oxidant damage produced by NO.
https://www.ncbi.nlm.nih.gov/pubmed/16399888

This suggests to me that Nitric Oxide in any excess is likely to have a destructive effect on health, especially if the mitochondria are already working suboptimally.

Methylene Blue: A potential solution for restoring mitochondrial efficiency

Heres a chemical which has some pretty amazing properties, and can be used in many cases to increase mitochondrial function, and in some cases actually reverse pathology.

Methylene blue (or MB) is a basic aniline dye with the molecular formula C16H18N3SCl. At room temperature, it appears as a solid, odorless, dark green powder that yields a blue solution when dissolved in water. It has many uses in a number of different fields. For instance, chemists use it to detect oxidizing agents and biologists use it to stain tissue samples and detect nucleic acids. In medicine, it is used as a treatment for various illnesses and disorders, including methemoglobinemia, schizophrenia, kidney stones, and herpes infections. In aquaculture, it is used to prevent freshwater fish eggs from being infected by bacteria and fungi.

Methylene blue has been described as "the first fully synthetic drug used in medicine." Methylene blue was first prepared in 1876 by German chemist Heinrich Caro.[40]

Its use in the treatment of malaria was pioneered by Paul Guttmann and Paul Ehrlich in 1891. During this period before the first World War, researchers like Ehrlich believed that drugs and dyes worked in the same way, by preferentially staining pathogens and possibly harming them. Methylene blue continued to be used in the second World War, where it was not well liked by soldiers, who observed, "Even at the loo, we see, we pee, navy blue." Antimalarial use of the drug has recently been revived.[41] It was discovered to be an antidote to carbon monoxide poisoning and cyanide poisoning in 1933 by Matilda Brooks.[42]

Among its many other benefits... I think some of the most interesting ones relate the it's effects on mitochondrial respiration:

1. Methylene Blue can artificially replace oxygen in the electron chain transport, meaning that hypoxia can essentially be mitigated and ATP synthesis can restored.
2. Methylene Blue can dissassociate Nitric Oxide from cytochrome C oxidase. From what I understand, the only other thing capable of doing this is red light.
3. Methylene Blue also inhibits Nitric Oxide Synthase

NOTE: It is best used in conjunction with red light, due to complementary physiological effects.

Here is an article from SelfHacked outlining some of the research on Methylene Blue:

1) Methylene Blue is an Anti-Depressant

Methylene Blue (MB) is a monoamine oxidase inhibitor (MAOI) (R).

MB inhibits MAO-A more than MAO-B, but it inhibits both at large doses (R).

At doses exceeding 5 mg/kg, it may cause serious serotonin toxicity/serotonin syndrome, if combined with any SSRIs or other serotonin reuptake inhibitor (R).

2)Methylene Blue is Anti-Cancer

Methylene blue appears to induce selective cancer cell apoptosis by the NQO1-dependent generation of cellular oxidative stress (R).

3) Methylene Can Help Alzheimer’s, Dementia and Parkinson’s and Huntington’s

Methylene blue has been investigated for the treatment of Alzheimer’s dementia (R, R2).

Methylene blue is proposed to affect neurodegeneration in Alzheimer’s disease via inhibition of tau protein aggregation and amyloids (R, R2).

It also may help Alzheimer’s by increasing acetylcholine (via acetylcholinesterase inhibition) (R).

It also partially repairs impairments in mitochondrial function and cellular metabolism (R).

By acting as an electron carrier and mitochondrial enhancer, it has promise in treating Parkinson’s disease (R).

Methylene blue helps Huntington’s by increasing autophagy and activating AMPK (R).

Methylene blue can alleviate mitochondrial abnormalities in a cellular model of progeria (R).

4) Methylene Blue Increases Blood Pressure

In diseased states, blood pressure often drops too low.

Methylene blue, an inhibitor of nitric oxide synthase and guanylate cyclase has been found to improve the hypotension associated with various clinical states (R).

5) Methylene Blue Improves Cognitive Performance in Health People

http://journal.frontiersin.org/article/10.3389/fphar.2015.00206/full
A randomized, double-blinded, placebo-controlled clinical trial of twenty-six subjects (age range, 22–62 years) was conducted on low dose methylene blue (~280mg, which is actually a high dose in my book) (R).

In this randomized study, low-dose methylene blue increased functional MR imaging activity during sustained attention and short-term memory tasks and potentiated memory retrieval (R).

Compared with control subjects, oral administration of low-dose methylene blue increased functional MR imaging response during the encoding, maintenance, and retrieval components of a short-term memory task in multiple clusters in the prefrontal, parietal, and occipital cortex (R).

Administration of methylene blue increased response (more brain activity) in the bilateral insular cortex during a psychomotor vigilance task (Z = 2.9–3.4, P= .01–.008) and functional MR imaging response during a short-term memory task involving the prefrontal, parietal, and occipital cortex (Z = 2.9–4.2, P = .03–.0003). Methylene blue was also associated with a 7% increase in correct responses during memory retrieval (P = .01) (R).

The insular cortex is important for sustained attention (R).

The study concluded: “Low-dose methylene blue can increase functional MR imaging activity during sustained attention and short-term memory tasks and enhance memory retrieval” (R).

Methylene blue has been shown to support memory consolidation and is neuroprotective as well (R).

In rat hippocampal slices, glutamate-mediated synaptic transmission is abolished by relatively high concentrations (5–50 mM) of MB (R).

On the other hand, MB is known to enhance memory retention and other brain functions in which ionotropic glutamate receptors are involved. It is possible that MB benefits cognitive function by modulating AMPA/kainate and NMDA-type ionotropic glutamate receptors (R).

Part of the cognitive enhancing effects are mediated by improvements in mitochondrial function.

6) Methylene Blue Helps Mitochondrial Function

It also has been shown that in low doses methylene blue protects the brain from disease by acting as an antioxidant in the mitochondria. It also acts as an artificial electron donor to complex I-IV of the mitochondria.

MB increases heme synthesis, cytochrome c oxidase (complex IV), and mitochondrial respiration (R), all of which help cognitive function.

This means that it can increase ATP production. ATP is the currency of life and the energy that powers humans. If our production of ATP declines, our physical and mental performance declines. Even healthy individuals can benefit from a boost in ATP production.

High concentrations of MB promote oxidative stress. Therefore, it is expected that low MB doses or concentrations will be, in general, more effective than large ones at facilitating physiological effects within mitochondria.

In fact, at high local concentrations, MB can potentially “steal” electrons away from the electron transport chain complexes, disrupting the redox balance and acting as a pro-oxidant (R).

It is well established that reduced MB can donate electrons to coenzyme Q and possibly to cytochrome c, thus increasing cytochrome oxidase (complex IV) activity and oxygen consumption (R).

At low concentrations, MB can interact with oxygen to form water, which would decrease the superoxide radicals produced during the process of oxidative phosphorylation. MB can also trap leaking electrons produced by mitochondrial inhibitors and preserve the metabolic rate by bypassing blocked points of electron flow, thus improving mitochondrial respiration (R, R2, R3).

In a rat model of cerebral ischemia, MB was able to speed up the removal of damaged mitochondria from a cell prior to cell death (mitophagy) (R).

MB is capable of reducing the mitochondrial damaging effects of amyloid beta in animal models (R).

Thus, MB is a potential target for mitochondrial dysfunction (R).

MB is able to stimulate glucose metabolism in conditions without oxygen (R), and increase NAD+ by mitochondria (R).

Some of the outcomes of improved mitochondrial function include increased fat burning (βeta-oxidation) (R), glucose utilization, ATP synthesis and extracellular matrix production (R).

In rat models of pancreatitis, Methylene blue reduces mitochondrial dysfunction (R).

7) Methylene Blue is Antimicrobial

Methylene blue was first used in 1891 to treat malaria.

Photodynamic therapy using the light-activated antimicrobial agent, MB kills methicillin-resistant staphylococcus aureus (MRSA) in superficial and deep excisional wounds (R).

MB in combination with light also inactivates viral nucleic acid of hepatitis-C and human immunodeficiency virus (HIV-1) and treats cases of resistant plaque psoriasis (R).

MB is an antifungal agent and can inhibit candida by causing mitochondrial dysfunction in this species (R).

8) Methylene Blue Can Extinguish Fear

Preclinical studies have shown that low-dose methylene blue increases memory retention after learning tasks, including fear extinction (R).

Adult participants displaying marked claustrophobic fear were randomly assigned to double-blind administration of 260 mg of methylene blue (N=23) or administration of placebo (N=19) immediately following six 5-minute extinction trials in an enclosed chamber (R).

The study concluded that Methylene blue enhances memory and the retention of fear extinction when administered after a successful exposure session but may have a deleterious effect on extinction when administered after an unsuccessful exposure session (R).


From: Methylene Blue: Revisited

METHYLENE BLUE IN SEPTIC SHOCK
A release of nitric oxide has been incriminated in the cardiovascular alterations of septic shock. Since guanylate cyclase is the target enzyme in the endothelium dependent relaxation mediated by nitric oxide, Methylene blue- a potent inhibitor of guanylate cyclase has been found very effective in improving the arterial pressure and cardiac function in septic shock.3

Studies have found improvement in mean arterial pressure (MAP) and systemic vascular resistance (SVR) while decreasing vasopressor requirements in septic shock.14

METHYLENE BLUE AND HEPATOPULMONARY SYNDROME
The hypoxemia in hepatopulmonary syndrome results from widespread pulmonary vasodilatation due to increased C-GMP. Methylene blue is found to ↑PaO2 and ↓alveolar-arterial difference for partial pressure of oxygen in all pts with hepatopulmonary syndrome. This was due to ↓C-GMP levels by Methylene Blue-a potent inhibitor of guanylate cyclase.2


METHYLENE BLUE AS ANTIMALARIAL
Methylene Blue has already been used some 100 yr ago against malaria, but it disappeared when chloroquine (CQ) and other drugs entered the market. However recent studies has shown the efficacy of Methylene Blue as an effective and cheap antimalarial agent especially in countries with increasing resistance of P. falciparum to existing 1st line antimalarial agents-CQ and pyrimethamine-sulfadoxine.

Methylene Blue, a specific inhibitor of P.falciparum glutathione reductase has the potential to reverse CQ resistance and it prevents the polymerization of haem into haemozoin similar to 4-amino-quinoline antimalarials.

A dose of 36-72mg/kg over 3 days is the most effective schedule.15

Apart from the intrinsic antimalarial activity and CQ sensitizing action it was also considered to prevent methemoglobinemia- a serious complication of malarial anemia.16

METHYLENE BLUE IN METHEMOGLOBINEMIA
Methemoglobinemia is a life threatening condition that can be congenital or acquired. It is characterized by the inability of hemoglobin to carry oxygen because the ferrous part of the heme molecule has been oxidized to a ferric state.

Methylene Blue acts by reacting within RBC to form leukomethylene blue, which is a reducing agent of oxidized hemoglobin converting the ferric ion (fe+++) back to its oxygen carrying ferrous state(fe++).17

Dose commonly used is 1-2mg/kg of 1% Methylene Blue solution.17,18

METHYLENE BLUE AND CANCER
Recent research suggests that Methylene Blue and other redox cyclers induce selective cancer cell apoptosis by NAD (P) H: quinine oxidoreductase (NQO1)-dependent bioreductive generation of cellular oxidative stress. Hence Methylene Blue is being investigated for the photodynamic treatment of cancer.19

IFOSFAMIDE NEUROTOXICITY
Another, less well known use of Methylene Blue is its utility for treating ifosfamide neurotoxicity. A toxic metabolite of ifosfamide, chloroacetaldehyde, disrupts the mitochondrial respiratory chain, leading to accumulation of nicotinamide adenine dinucleotide hydrogen (NADH).

Methylene blue acts as an alternative electron acceptor, and reverses the NADH inhibition of hepatic gluconeogenesis while also inhibiting the transformation of chloroethylamine into Chloroacetaldehyde, and also inhibits multiple amine oxidase activities, preventing the formation of Chloroacetaldehyde.20

Hence it has prophylactic and therapeutic role in ifosfamide - induced encephalopathy.21

NEUTRALIZATION OF HEPARIN
Methylene blue effectively neutralizes heparin especially in pts with protamine allergy. However work still needs to be done to determine the safety of the drug at the higher doses necessary to neutralize heparin levels achieved in bypass patients.26


METHYLENE BLUE AND PRIAPISM

Methylene blue has been used to treat high flow priapism by intra-cavernous injection which is known to antagonize endothelial derived relaxation factor.27


METHYLENE BLUE AND ALZHEIMER'S DISEASE

The relationship between Methylene blue and Alzheimer's disease has recently attracted increasing scientific attention. It has been shown to attenuate the formations of amyloid plaques and neurofibrillary tangles and partial repair of impairments in mitochondrial function and cellular metabolism.28


METHYLENE BLUE COMBINED WITH LIGHT
Photodynamic therapy using the light activated anti-microbial agent, Methylene blue kills methicillin resistant staphylococcus aureus (MRSA) in superficial and deep excisional wounds.29 Methylene blue in combination with light also inactivates viral nucleic acid of hepatitis-C and human immunodeficiency virus (HIV-1) and treats cases of resistant plaque psoriasis.30,31
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3087269/

Methylene blue-mediated photodynamic therapy enhances apoptosis in lung cancer cells

Combined treatment with a photosensitizer and iodide laser [photodynamic therapy (PDT)] has improved the outcome of various cancers. In this study, we investigated the effects of using the photosensitizer methylene blue (MB) in PDT in human lung adenocarcinoma cells. We found that MB enhances PDT-induced apoptosis in association with downregulation of anti-apoptotic proteins, reduced mitochondrial membrane potential (MMP), increased phosphorylation of the mitogen-activated protein kinase (MAPK) and the generation of reactive oxygen species (ROS). In MB-PDT-treated A549 cells, we observed PARP cleavage, procaspase-3 activation, downregulation of the anti-apoptotic proteins Bcl-2 and Mcl-1, and the reduction of mitochondrial membrane potential (MMP). Western blot data showed that phosphorylation of p38 was increased in MB-PDT-treated A549 cells, indicating that several signaling molecules participate in the apoptotic cascade. Our data also showed that apoptotic cell death in MB-PDT-treated cells occurred through a series of steps beginning with the photochemical generation of ROS. Demonstrating the role of ROS, pretreatment of A549 cells with the antioxidant N-acetylcysteine (NAC) followed by MB-PDT resulted in increased cell viability and reduced proteolytic cleavage of PARP.
https://www.spandidos-publications.com/10.3892/or.2013.2494

Photodynamic therapy with intralesional methylene blue and a 635 nm light-emitting diode lamp in hidradenitis suppurativa: a retrospective follow-up study in 7 patients and a review of the literature
Hidradenitis suppurativa is a chronic inflammatory skin disease which has an estimated prevalence of 1%. It is characterized by the formation of recurrent painful suppurative nodules and abscesses in the flexural areas of the body. It is believed that its pathogenesis involves an aberrant, genetically-determined activation of innate immunity against the bacterial commensal flora of intertriginous areas. It has been found that the formation of antibiotic-resistant bacterial biofilms is a common finding in hidradenitis lesions. Photodynamic therapy with different compounds and light sources has demonstrated its efficacy in a number of infectious diseases such as nail mycosis and chronic periodontitis. We retrospectively report our experience in the treatment of hidradenitis with photodynamic therapy using intralesional methylene blue and a 635 nm light-emitting diode lamp in 7 patients. Two patients received one session whereas 5 patients received two sessions. At one month follow-up good response was achieved in 6 patients. After 6 months, 5 patients (71%) maintained remission of the disease in the treated area. In view of the results and literature review, we regard methylene blue as an ideal photosensitizer for photodynamic therapy in this disease.
http://pubs.rsc.org/en/Content/ArticleLanding/2016/PP/c6pp00082g#!divAbstract


Methylene blue alleviates experimental autoimmune encephalomyelitis by modulating AMPK/SIRT1 signaling pathway and Th17/Treg immune response.
Methylene blue (MB) is an effective neuroprotectant in many neurological disorders. AMP-activated protein kinase (AMPK)/silent mating-type information regulation 2 homolog 1 (SIRT1) plays a crucial role in maintaining inflammatory responses and shows a synergistic effect on cell homeostasis. We investigated the effect of MB on experimental autoimmune encephalomyelitis (EAE), a classical animal model of multiple sclerosis (MS). MB treatment reduced the clinical scores of EAE significantly and attenuated pathological injuries in spinal cords. Furthermore, the protective effects of MB were related to the activation of AMPK/SIRT1 signaling pathway. In addition, MB treatment alleviated T helper type17 (Th17) responses and increased regulatory T cell (Treg) responses. Our findings suggest that MB could be a promising reagent to treat autoimmune diseases and MS
 

Keyhole

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SIRT1 activation by methylene blue, a repurposed drug, leads to AMPK-mediated inhibition of steatosis and steatohepatitis.

"...In mice fed on a high-fat diet for 8 weeks, MB treatment inhibited excessive hepatic fat accumulation and steatohepatitis. The ability of MB to activate SIRT1 promotes mitochondrial biogenesis and oxygen consumption and activates AMPK, contributing to anti-lipogenesis in the liver. Our results provide new information on the potential use of MB for the treatment of steatosis and steatohepatitis."
https://www.ncbi.nlm.nih.gov/pubmed/24486702

I have written elsewhere that nicotine also activates SIRT1, a NAD+ dependent gene associated with health and longevity. So it seems that nicotine and MB may work in conjunction to increase mitochondrial function.

The role of dopamine in methylene blue-mediated inhibition of estradiol benzoate-induced anterior pituitary hyperplasia in rats.

Recently, we demonstrated that methylene blue partially inhibited estradiol-benzoate-induced anterior pituitary hyperplasia in rats. Since central dopaminergic systems participate in the regulation of estrogen-induced anterior pituitary growth and tumor transformation, this study examined whether a 3-week treatment with methylene blue could affect anterior pituitary levels of dopamine (DA), dihydroxyphenylalanine (DOPA), and dihydroxyphenylacetic acid and dopamine (D-2) receptors in male rats. Compared to controls, methylene blue significantly decreased anterior pituitary weight, increased basal anterior pituitary DA levels, and inhibited estradiol benzoate-induced decreases in anterior pituitary DA concentrations. Furthermore, we found that methylene blue alone decreased anterior pituitary D-2 receptor number. Methylene blue given in combination with estradiol benzoate partially inhibited estradiol benzoate-induced anterior pituitary growth and estradiol benzoate-induced increases in D-2 receptor number. Estradiol benzoate-treated rats had significantly lower anterior pituitary DOPA accumulation after intraperitoneal administration of 3,4-hydroxybenzyl-hydrazine dihydrochloride (NSD-1015), an irreversible inhibitor of L-aromatic amino acid decarboxylase whereas methylene blue did not affect anterior pituitary DOPA accumulation when compared to controls. Methylene blue decreased anterior pituitary prolactin levels and inhibited increases in anterior pituitary prolactin after estradiol benzoate administration. The present results suggest that anterior pituitary DA may play an important role in estrogen-induced anterior pituitary hyperplasia and tumor formation and that antioxidant drugs such as methylene blue may attenuate estrogen-induced pituitary growth. This may occur via increases in anterior pituitary DA levels associated with down-regulation of anterior pituitary D-2 receptors.
https://www.ncbi.nlm.nih.gov/pubmed/11343835

The regulation of adenohypophyseal prolactin secretion: effect of triiodothyronine and methylene blue on estrogenized rat adenohypophysis.

The purpose of the present article is to provide a review about neurotransmitters and their receptors involved in estrogen-induced anterior pituitary growth and in the antagonistic effects of triiodothyronine (T3) and methylene blue (MB). Central dopaminergic and noradrenergic systems are the most important factors regulating pituitary growth and function. Recently nitric oxide (NO) was added to the list of the neurotransmitters and neuropeptides involved in the control of the anterior pituitary secretion. Our data suggest that estrogen-induced anterior pituitary growth is associated with decreased synthesis and metabolism of central catecholamines, reduction of adenohypophyseal beta-adrenergic receptors and increase of dopamine DA-2 receptors. We found that the treatment with T3 or MB prevented both estrogen-induced catecholaminergic inhibition and dopamine DA-2 receptor increment in the anterior pituitary. In contrast to T3, MB given alone also slightly decreased the anterior pituitary weight. Serum levels and anterior pituitary content of prolactin were increased after treatment with estradiol benzoate (EB), whereas T3 or MB partially attenuated prolactin hypersecretion after estrogen administration. This is in accord with the attenuation of EB-induced inhibition of dopaminergic system by T3 and MB. MB given in combination with EB also partially attenuated EB-promoted rise of adenohypohyseal NO synthase activity which plays an important role in the regulation of prolactin secretion. Further studies on central catecholaminergic systems, pituitary receptors, the nitrergic system and mechanisms of intracellular signal transduction are necessary for better understanding of pituitary tumor transformation and possibly for the discovery of new approaches towards treating patients with these diseases.
https://www.ncbi.nlm.nih.gov/pubmed/10984069
 

Gaby

SuperModerator
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FOTCM Member
Very interesting!

It has been used in urinary tract infections and to prevent formation of kidney stones. As a treatment of vasoplegic syndrome and its placebo effect was also very curious:

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

Medical uses

Methemoglobinemia

While many texts indicate that methylene blue has oxidizing agent properties, its effects as an oxidizing agent occur only at very high doses.[citation needed] At pharmacologic doses it has reducing agent properties. It is owing to this reason that methylene blue is employed as a medication for the treatment of methemoglobinemia. This can arise from ingestion of certain pharmaceuticals, toxins, or broad beans.[7] Normally, through the NADH or NADPH dependent methemoglobin reductase enzymes, methemoglobin is reduced back to hemoglobin. When large amounts of methemoglobin occur secondary to toxins, methemoglobin reductases are overwhelmed. Methylene blue, when injected intravenously as an antidote, is itself first reduced to leucomethylene blue, which then reduces the heme group from methemoglobin to hemoglobin. Methylene blue can reduce the half life of methemoglobin from hours to minutes.[8] At high doses, however, methylene blue actually induces methemoglobinemia, reversing this pathway.[8]

Combined with light

Methylene blue combined with light has been used to treat resistant plaque psoriasis,[9]

Urinary tract infection

Methylene blue is a component of a frequently prescribed urinary analgesic/anti-infective/anti-spasmodic known as "Prosed", a combination of drugs which also contains phenyl salicylate, benzoic acid, hyoscyamine sulfate, and methenamine (aka hexamethylenetetramine and not to be confused with 'methanamine').[10]

Cyanide poisoning

Since its reduction potential is similar to that of oxygen and can be reduced by components of the electron transport chain, large doses of methylene blue are sometimes used as an antidote to potassium cyanide poisoning, a method first successfully tested in 1933 by Dr. Matilda Moldenhauer Brooks in San Francisco,[11] although first demonstrated by Bo Sahlin of Lund University, in 1926.[11][12]

Dye or stain

Human cheek cells stained with methylene blue

Methylene blue is used in endoscopic polypectomy as an adjunct to saline or epinephrine, and is used for injection into the submucosa around the polyp to be removed. This allows the submucosal tissue plane to be identified after the polyp is removed, which is useful in determining if more tissue needs to be removed, or if there has been a high risk for perforation. Methylene blue is also used as a dye in chromoendoscopy, and is sprayed onto the mucosa of the gastrointestinal tract in order to identify dysplasia, or pre-cancerous lesions. Intravenously injected methylene blue is readily released into the urine and thus can be used to test the urinary tract for leaks or fistulas.

In surgeries such as sentinel lymph node dissections, methylene blue can be used to visually trace the lymphatic drainage of pertinent tissues. Similarly, methylene blue is added to bone cement in orthopedic operations to provide easy discrimination between native bone and cement. Additionally, methylene blue accelerates the hardening of bone cement, increasing the speed at which bone cement can be effectively applied. Methylene blue is used as an aid to visualisation/orientation in a number of medical devices, including a Surgical sealant film, TissuePatch.

When methylene blue is "polychromed" (oxidized in solution or "ripened" by fungal metabolism,[13] as originally noted in the thesis of Dr D L Romanowsky in 1890s), it gets serially demethylated and forms all the tri, di, mono and non methyl intermediates - which are Azure B, Azure A, Azure C and thionine respectively.[14] This is the basis of the basophilic part of the spectrum of Romanowski-Giemsa effect. If only synthetic Azure B and Eosin Y is used, it may serve as a standardized Giemsa stain; but, without methylene blue, the normal neutrophilic granules tend to overstain and look like toxic granules. On the other hand, if methylene blue is used it might help to give the normal look of neutrophil granules and may additionally also enhances the staining of nucleoli and polychromatophilic RBCs (reticulocytes).[15]

A traditional application of methylene blue is the intravital or supravital staining of nerve fibers, an effect first described by Paul Ehrlich in 1887.[16] A dilute solution of the dye is either injected into tissue or applied to small freshly removed pieces. The selective blue coloration develops with exposure to air (oxygen) and can be fixed by immersion of the stained specimen in an aqueous solution of ammonium molybdate. Vital methylene blue was formerly much used for examining the innervation of muscle, skin and internal organs.[17][18][19] The mechanism of selective dye uptake is incompletely understood; vital staining of nerve fibers in skin is prevented by ouabain, a drug that inhibits the Na/K-ATPase of cell membranes.[20]

Placebo

Methylene blue has been used as a placebo; physicians would tell their patients to expect their urine to change color and view this as a sign that their condition had improved.[21] This same side effect makes methylene blue difficult to test in traditional placebo-controlled clinical studies.[22]

Ifosfamide toxicity

Another use of methylene blue is to treat ifosfamide neurotoxicity. Methylene blue was first reported for treatment and prophylaxis of ifosfamide neuropsychiatric toxicity in 1994. A toxic metabolite of ifosfamide, chloroacetaldehyde (CAA), disrupts the mitochondrial respiratory chain, leading to an accumulation of nicotinamide adenine dinucleotide hydrogen (NADH). Methylene blue acts as an alternative electron acceptor, and reverses the NADH inhibition of hepatic gluconeogenesis while also inhibiting the transformation of chloroethylamine into chloroacetaldehyde, and inhibits multiple amine oxidase activities, preventing the formation of CAA.[23] The dosing of methylene blue for treatment of ifosfamide neurotoxicity varies, depending upon its use simultaneously as an adjuvant in ifosfamide infusion, versus its use to reverse psychiatric symptoms that manifest after completion of an ifosfamide infusion. Reports suggest that methylene blue up to six doses a day have resulted in improvement of symptoms within 10 minutes to several days.[24] Alternatively, it has been suggested that intravenous methylene blue every six hours for prophylaxis during ifosfamide treatment in patients with history of ifosfamide neuropsychiatric toxicity.[25] Prophylactic administration of methylene blue the day before initiation of ifosfamide, and three times daily during ifosfamide chemotherapy has been recommended to lower the occurrence of ifosfamide neurotoxicity.[26]

Vasoplegic syndrome

Some literature has reported the use of methylene blue as an adjunct in the management of people experiencing vasoplegic syndrome after cardiac surgery

Methylene blue as an inhibitor of stone formation.
https://www.ncbi.nlm.nih.gov/pubmed/351913

Kinetics of growth and dissolution of calcium oxalate monohydrate were examined in the presence of small concentrations of methylene blue. The data presented show moderate retardation in growth and dissolution rates. It was also found that methylene blue decreased the decalcification rate of calcium oxalate renal calculi.

Methylene blue in renal calculi. Results of five-year study.
https://www.ncbi.nlm.nih.gov/pubmed/1202715

Methylene blue in a dose of 65 mg. three times a day has been reported to be useful in the management of chronic renal calculous disease. Sixty-eight patients with renal calculi, in whom there was no biochemical abnormality, were started on this drug five or more years ago. Forty-six per cent of formers of calcium oxalate stone have passed no further stones, and 20 per cent have been improved. Initial studies had reported success in the management of infected stones. However, only 27 per cent obtained any benefit in this study. Methylene blue is useful in the management of patients who form multiple small calculi that contain calcium oxalate dihydrate and may be useful in the prevention of new stone formation.
 

RedFox

The Living Force
FOTCM Member
After the last C's session, I had a look to see if Methylene Blue could help with stems cells:

_https://www.ncbi.nlm.nih.gov/pubmed/28269766
Methylene Blue (Tetramethylthionine Chloride) Influences the Mobility of Adult Neural Stem Cells: A Potentially Novel Therapeutic Mechanism of a Therapeutic Approach in the Treatment of Alzheimer's Disease.

An interest in neurogenesis in the adult human brain as a relevant and targetable process has emerged as a potential treatment option for Alzheimer's disease and other neurodegenerative conditions. The aim of this study was to investigate the effects of tetramethylthionine chloride (methylene blue, MB) on properties of adult murine neural stem cells. Based on recent clinical studies, MB has increasingly been discussed as a potential treatment for Alzheimer's disease. While no differences in the proliferative capacity were identified, a general potential of MB in modulating the migratory capacity of adult neural stem cells was indicated in a cell mobility assay. To our knowledge, this is the first time that MB could be associated with neural mobility. The results of this study add insight to the spectrum of features of MB within the central nervous system and may be helpful for understanding the molecular mechanisms underlying a potential therapeutic effect of MB.

_https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5217567/
Oxidative status predicts quality in human mesenchymal stem cells

Abstract
Background

Human bone marrow-derived mesenchymal stem cells (MSC) are adult progenitor cells with great potential for application in cell-based therapies. From a cell-based therapy perspective, there are two limitations to MSC use: (1) these therapies require large numbers of cells, and long-term expansion of MSC in vitro promotes replicative senescence; and (2) patient variability is a challenge for defining MSC quality standards for transplantation. This study aimed to determine whether low or high oxidative status of MSC correlate with changes in cell expansion and differentiation potentials.

Methods

We investigated functional aspects of mitochondria, such as cell metabolic activity indicators and expression of antioxidant enzymes. Furthermore, we tested if senescence-induced changes in oxidative status of MSC could be counteracted by methylene blue (MB), an alternative mitochondrial electron transfer known to enhance cell bioenergetics.

Results

MSC isolated from donors of the same age showed distinctive behavior in culture and were grouped as weak (low colony-forming units (CFU) and a short life in vitro) and vigorous MSC (high CFU and a long life in vitro). In comparison to weak MSC, vigorous MSC had oxidative status characterized by lower mitochondrial membrane potential, lower mitochondrial activity, and fewer reactive oxygen species production, as well as reduced mitochondrial biogenesis. Vigorous MSC had a significantly higher expansion potential compared to weak MSC, while no differences were observed during differentiation. MB treatment significantly improved expansion and differentiation potential, however only in vigorous MSC.

Conclusions

Together, these results demonstrate the importance of mitochondrial function in MSC in vitro, and that cells with low oxidative status levels are better candidates for cell-based therapies.

_https://cmns.umd.edu/news-events/features/3352
Safe, Inexpensive Chemical Found to Reverse Symptoms of Progeria in Human Cells

Finding could lead to treatments for rare genetic illness as well as normal aging

Progeria is a rare genetic disease that mimics the normal aging process at an accelerated rate. Symptoms typically appear within the first year of life, and individuals with the disease develop thin, wrinkled skin, fragile bones and joints, full-body hair loss and organ failure, among other complications. Most do not survive past their teen years.

New work from the University of Maryland suggests that a common, inexpensive and safe chemical called methylene blue could be used to treat progeria—and possibly the symptoms of normal aging as well. A new study shows for the first time that small doses of methylene blue can almost completely repair defects in cells afflicted with progeria, and can also repair age-related damage to healthy cells. The study was published online in the journal Aging Cell on December 10, 2015.

“We tried very hard to examine the effect of methylene blue on all known progeria symptoms within the cell,” said Kan Cao, senior author on the study and an associate professor of cell biology and molecular genetics at UMD. “It seems that methylene blue rescues every affected structure within the cell. When we looked at the treated cells, it was hard to tell that they were progeria cells at all. It’s like magic.

Progeria results from a defect in a single gene. This gene produces a protein called lamin A, which sits just inside the cell’s nucleus, under the nuclear membrane. Healthy cells snip off a small piece of each new lamin A molecule—a small edit that is necessary for lamin A to work properly. Cells with progeria, however, skip this important editing step. The defective lamin A interferes with the nuclear membrane, causing the nucleus to form bulges and deformations that make normal functioning impossible.

Cells with progeria also have misshapen and defective mitochondria, which are the small organelles that produce energy for the cell. Although previous studies suggested damage to mitochondria in progeria cells, the current study is the first to document the nature and extent of this damage in detail. Cao and her colleagues found that a majority of the mitochondria in progeria cells become swollen and fragmented, making it impossible for the defective mitochondria to function.

The team found that methylene blue reverses the damages to both the nucleus and mitochondria in progeria cells remarkably well. The precise mechanism is still unclear, but treating the cells with the chemical effectively improved every defect, causing progeria cells to be almost indistinguishable from normal cells.

Cao and her colleagues also tested methylene blue in healthy cells allowed to age normally. The normal aging process degrades mitochondria over time, causing these older mitochondria to resemble the mitochondria seen in progeria cells. Once again, methylene blue repaired these damages.

“We have repeated these experiments many times and have not seen a single one fail,” said Zheng-Mei Xiong, lead author on the study and a postdoctoral associate in the UMD Department of Cell Biology and Molecular Genetics. “This is such an exciting result with so much potential, both for progeria and normal aging. Methylene blue is common and inexpensive. It is fully water soluble and non-toxic. People use it to clean fish tanks because it is so safe for the fish eggs.”

Because methylene blue can repair the cell defects that ultimately lead to whole-body symptoms in progeria patients, Cao, Xiong and their colleagues believe methylene blue could be used as a treatment for the disease in the future. Similarly, methylene blue could show promise as an over-the-counter treatment for the symptoms of normal aging, perhaps as an additive to cosmetic products or nutritional supplements.

Cao and her team are moving quickly to complete the next crucial step: testing in animal models.

“So far, we have done all of our work in stem cell lines. It is critical to see whether the effect extends to whole animals,” Cao explained. “We also want to see if methylene blue can repair specific effects of progeria in various cell types, such as bone, skin, cardiovascular cells and others. Further down the line, other groups might begin human clinical trials. It’s very exciting.”
 

Nico

Jedi Council Member
Thank you Keyhole,
There a site selling methylene blue, I have not enough knowledge nor interest (apart from testing) to use it, but it's maybe useful.

_https://mitoblue.com/
 

Ina

Dagobah Resident
As a child the cure for sore throats was two three drops of Methylene Blue on a sugar cube. I also used it during my first pregnancy because I did not want to take any medicine. It was used in solution form as an antiseptic. Recently I have been asking the pharmacists for it only to find out from the older ones that it hasn't been in use for a long time.
 

Mikey

The Living Force
Keyhole said:
Methylene blue (or MB) is a basic aniline dye with the molecular formula C16H18N3SCl. At room temperature, it appears as a solid, odorless, dark green powder that yields a blue solution when dissolved in water.

Note that there are at least two types of Methylene Blue - which are structurally similar, but not identical. They may have different properties. Vendors rarely specify the chemical formula and rarely deliver a quality certificate with the product.

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

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

New Methylene Blue seems to be sold as a Zinc Chloride (ZnCl2) salt, and appears as a dark brown/reddish powder rather than a greenish powder.

As always, do lots of research before considering utilization of any substance!
 

dj

Padawan Learner
Thank you Keyhole for the first rate presentation of this info. I wish I'd had a teacher like you for my chem classes.

This is very useful and timely info for me - I'm looking at cancer treatments and this opens up a whole new window to explore.

Jack Kruse seemed to drop hints in some of his stuff that I was able to access (I have no membership as it's kind of expensive) but your info, for my purposes, is way better, cleaner, and more concise.

Again, Thanks
 

fabric

SuperModerator
Moderator
FOTCM Member
This is very very interesting stuff, methylene blue (MB). It seems like it has been around for quite a long time, and is also known as a nootropic (brain enhancing drug). There are also tons of research papers (over 11,000) and a lot of them promising. I’ll post a few here that I found to be worth reading to get a better idea of how MB works and its benefits. I think this is something worth experimenting with and there seems to be a rather wide safety margin. Interestingly effects of dosing seem to vary quite greatly (microgram range up to milligram range) but all of the studies I read used dosages in the milligram range. However some people reported effects in the microgram range so similar to Iodine, it could be different depending on the person. More on that later. Here are the links:

Cellular and Molecular Actions of Methylene Blue in the Nervous System

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3005530/ (full article)

Abstract

Methylene Blue (MB), following its introduction to biology in the 19th century by Ehrlich, has found uses in various areas of medicine and biology. At present, MB is the first line of treatment in methemoglobinemias, is used frequently in the treatment of ifosfamide-induced encephalopathy, and is routinely employed as a diagnostic tool in surgical procedures. Furthermore, recent studies suggest that MB has beneficial effects in Alzheimer's disease and memory improvement. Although the modulation of the cGMP pathway is considered the most significant effect of MB, mediating its pharmacological actions, recent studies indicate that it has multiple cellular and molecular targets. In the majority of cases, biological effects and clinical applications of MB are dictated by its unique physicochemical properties including its planar structure, redox chemistry, ionic charges, and light spectrum characteristics. In this review article, these physicochemical features and the actions of MB on multiple cellular and molecular targets are discussed with regard to their relevance to the nervous system.

Neurometabolic mechanisms for memory enhancement and neuroprotection of methylene blue

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3265679/ (full article)

Abstract

This paper provides the first review of the memory-enhancing and neuroprotective metabolic mechanisms of action of methylene blue in vivo. These mechanisms have important implications as a new neurobiological approach to improve normal memory and to treat memory impairment and neurodegeneration associated with mitochondrial dysfunction. Methylene blue’s action is unique because its neurobiological effects are not determined by regular drug-receptor interactions or drug-response paradigms. Methylene blue shows a hormetic dose-response, with opposite effects at low and high doses. At low doses, methylene blue is an electron cycler in the mitochondrial electron transport chain, with unparalleled antioxidant and cell respiration-enhancing properties that affect the function of the nervous system in a versatile manner. A major role of the respiratory enzyme cytochrome oxidase on the memory-enhancing effects of methylene blue is supported by available data. The memory-enhancing effects have been associated with improvement of memory consolidation in a network-specific and use-dependent fashion. In addition, low doses of methylene blue have also been used for neuroprotection against mitochondrial dysfunction in humans and experimental models of disease. The unique auto-oxidizing property of methylene blue and its pleiotropic effects on a number of tissue oxidases explain its potent neuroprotective effects at low doses. The evidence reviewed supports a mechanistic role of low-dose methylene blue as a promising and safe intervention for improving memory and for the treatment of acute and chronic conditions characterized by increased oxidative stress, neurodegeneration and memory impairment.

Another good one with human studies:

Multimodal Randomized Functional MR Imaging of the Effects of Methylene Blue in the Human Brain.

https://www.ncbi.nlm.nih.gov/pubmed/27351678 (sci-hub is your friend)

Abstract

Purpose: To investigate the sustained-attention and memory-enhancing neural correlates of the oral administration of methylene blue in the healthy human brain.

Materials and Methods: The institutional review board approved this prospective, HIPAA-compliant, randomized, double-blinded, placebo-controlled clinical trial, and all patients provided informed consent. Twenty-six subjects (age range, 22-62 years) were enrolled. Functional magnetic resonance (MR) imaging was performed with a psychomotor vigilance task (sustained attention) and delayed match-to-sample tasks (short-term memory) before and 1 hour after administration of low-dose methylene blue or a placebo. Cerebrovascular reactivity effects were also measured with the carbon dioxide challenge, in which a 2 × 2 repeated-measures analysis of variance was performed with a drug (methylene blue vs placebo) and time (before vs after administration of the drug) as factors to assess drug × time between group interactions. Multiple comparison correction was applied, with cluster-corrected P < .05 indicating a significant difference.

Results: Administration of methylene blue increased response in the bilateral insular cortex during a psychomotor vigilance task (Z = 2.9-3.4, P = .01-.008) and functional MR imaging response during a short-term memory task involving the prefrontal, parietal, and occipital cortex (Z = 2.9-4.2, P = .03-.0003). Methylene blue was also associated with a 7% increase in correct responses during memory retrieval (P = .01).

Conclusion: Low-dose methylene blue can increase functional MR imaging activity during sustained attention and short-term memory tasks and enhance memory retrieval.


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

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2867617/ (full article)

Abstract

The goals of this review were to identify methylene blue (MB) as a compound that follows hormetic behavior for a wide range of effects and to address the question of what is unique about MB that could account for its wide applicability and hormetic behavior as a drug. The MB hormetic dose-response relationship is exemplified by an increase in various behavioral, physiological and biochemical responses with increasing dose, followed by a decrease in the same responses with an even higher dose, until the responses are equal to control responses. With MB doses increasing beyond the hormetic zone, the responses decrease even further, until they are below the control responses. At doses spanning its hormetic zone, MB can increase select responses until they are 130–160% of control. For example, low doses of MB produce maximum behavioral and biochemical responses with averages of approximately 140% of control. As MB dose is raised outside the hormetic zone the response decreases below the control response, as exemplified by MB’s ability to increase cytochrome oxidase activity at intermediate doses, while decreasing cytochrome oxidase activity at higher doses. It is proposed that MB’s autoxidizable chemical property may be responsible for its unique biological action as both a metabolic energy enhancer and antioxidant that is frequently characterized by hormetic dose-response relationships.

There’s also this rather interesting snippet from Jack Kruse:

TIME #14: BIOHACKING “TIME” WITH METHYLENE BLUE

_https://www.jackkruse.com/biohacking-time-with-methylene-blue/

[...]
Methylene blue is a monoamine oxidase inhibitor. MAOI’s act by inhibiting the activity of monoamine oxidase, thus preventing the breakdown of monoamine neurotransmitters and thereby increasing their availability. Thusly, methylene blue increases dopamine, melatonin, serotonin, and melanin levels on our surfaces to increase our ability to deal with sunlight properly. This makes MB a classic silent vagal stimulant. It means that vagal tone and UV light assimilation are fundamentally linked in the autonomic nervous system. It will also increase epinephrine and nor-epinephrine levels to increase BP and muscle power on a short term basis. This explains why cold thermogenesis increases beta three sympathetic receptors to liberate protons in brown fat for beta oxidation on mitochondria.

During my searches for more info, I also came across this patent which mentions MB as one of the compounds used for their testing methods. Reduced MB is mentioned on which I will expand on.

https://www.google.com/patents/US7335505

[...]

Stabilisation of Reduced Form

Some of these compounds of interest are known to circulate in the body predominantly in the reduced form. For example, for a discussion of the pharmacokinetics of MB, see e.g. DiSanto, A. et al. (1972) Journal Pharm. Sci. 61(7):1086 and DiSanto, A. et al. (1972) Journal Pharm. Sci 61(7):1090. Thirdly, only the reduced form of compounds such as MB is found to cross the blood-brain barrier (Chapman, D. M. (1982) Tissue and Cell 14(3):475; Müller, T. (1992) Acta Anat. 144:39; Müller, T. (1994) J. Anat. 184:419; Becker, H. et al. (1952) Zeitschrift für Naturforschung 7:493; Müller, T. (1995) It. J. Anat. Embryol. 100(3):179; Müller, T. (1998) Histol. Histopathol. 13:1019).

Such references as these illustrate that the reduced form of compounds such as MB represents a feasible and pharmaceutically-acceptable formulation for administration to subjects. MB has previously been used clinically in an oral preparation. Further toxicological tests are, however, required before its clinical acceptability is achieved. The half live of MB and related compounds (e.g. tolonium chloride) in blood is approximately 100 minutes. It is evident that slow release formulations of compounds with such, relatively short, half lives can substantially improve compound availability and hence therapeutic efficacy.

[...]

Dosage of Therapeutics

[...]

The pharmacokinetics of methylene blue have been studied in humans, dogs and rats by DiSanto and Wagner, J Pharm Sci 1972, 61:1086-1090 and 1972, 61:1090-1094. Further data on urinary excretion in humans is also available from Moody et al., Biol Psych 1989, 26: 847-858. Combining data on urinary excretion of MB in humans, it is possible to derive an overall model for distribution of MB following single 100 mg dose in a 70 kg subject, assuming instantaneous absorbtion (FIG. 19B). Urinary excretion accounts for 54-98% of the ingested dose. This variability is most likely due to variability in absorbtion, although variability in metabolism cannot be excluded. From urinary excretion data, it is possible to calculate that whole body clearance is 56 mg/kg/hr. Therefore, the dosage required to achieve an effective target tissue concentration of 4 μM is 1.73 mg/kg/day (0.58 mg/kg tds) if there were complete absorbtion. However, from Moody et al., it is clear that total urinary excretion, and hence effective bioavailability, is itself a function of dose. The oral dose required to deliver 1.73 mg/kg/day is approximately 2× the dosage calculated on the basis of whole-body clearance. Therefore the actual required dosage is on the order of 3.2 mg/kg/day. This is close to the minimum routine oral dosage used clinically in humans, eg in the treatment of chronic urinary tract infection (390 mg/day). The maintenance oral dosage in humans is therefore approximately 225 mg/day, or 75 mg tds. Peak tissue levels are reached at approximately 1 hr and the tissue half-life is about 12 hours.

Methylene blue exists in the charged blue oxidised form, and the uncharged colourless reduced leukomethylene blue form. We have shown experimentally in cells that the target tissue concentration in cells required to prevent tau aggregation by 50% (ie the EC50) is 4 μM for reduced methylene blue, and that it is the leuko-form which is preferentially active. It is shown by DiSanto and Wagner (1972) that approximately 78% of the methylene blue recovered in urine is in the reduced form, and from anatomical studies following iv administration, the only form which is bound to tissues is the colourless reduced form, which becomes oxidised to the blue colour on exposure to air after post-mortem dissection. The only form of methylene blue which crosses the blood-brain barrier after iv administration is the reduced form (Muller, Acta Anat 1992, 144:39-44 and Becker and Quadbeck, 1952). Therefore, orally absorbed methylene blue is very rapidly reduced in the body, and remains so until excretion, possibly undergoing further chemical modification which stabilises it in a reduced form.

It is highly likely that variability in oral absorbtion is determined largely by the efficiency of initial reduction in the GI tract. One way to achieve more reliable absorbtion is therefore be to pre-reduce methylene blue with ascorbic acid. We have shown from in vitro studies that this conversion is rather slow, so that it takes 3 hours to achieve 90% reduction of methylene blue in water in the presence of 2× mg ratio of ascorbic acid. Therefore, the dosage of methylene blue which is most likely to ensure reliable absorbtion will be 3.5 mg/kg/day of methylene blue pre-reduced for at least 3 hours in the presence of 7 mg/kg/day of ascorbic acid.

It is also possible that MB may be active at lower concentrations in man, and that a range of clinically feasible doses would be therefore 20 mg tds, 50 mg tds or 100 mg tds, combined with 2× mg ratio of ascorbic acid in such a manner as to achieve more than 90% reduction prior to ingestion.


One of the questions I was trying to figure out was, how much are you supposed to take to reach a therapeutic dose? And so far it seems to be a rather wide range. In mouse studies and rat studies, they use amounts of 0.5mg/kg to 4mg/kg but there are also some human studies where they used a similar amount (for example, here and here ). I’d like to find more human studies but what I’d like to know is how they arrived at 4mg/kg for humans when that is the upper limit used for rats (in other words, it seems it should be much lower when converting to human doses). For example, a method that is commonly used to convert dosages used in animal studies to human dosages is called BSA (body surface area) scaling . If you use this method to scale the dose you end up with much lower ranges (about 0.162mg/kg to 0.649mg/kg) for a 60kg human. If you go the other way, and convert a human 4mg/kg to a rat dosage, it brings it to 24.8mg/kg which is outside the dose that is mentioned in the rat studies where they saw improvement. I think there’s something to the pharmacokinetics of MB that I’m missing, and perhaps that is the reason for not scaling. Or perhaps they aren’t using the BSA method and some other version for scaling dose which returns equivalent values for both rat and human studies. Note that range is IV so oral will be higher due to absorption rates which can vary. For a starting point, I'm going with the dosages used in human studies.

Here in this article (gives a good explanation of the cellular process, then about MB and dosing), the author shows how he arrived at dosing for humans but I can’t verify his calculations though they seem to line up better with the ones in the human studies.

That said, both studies did show improvement in the areas they were studying and there is also this upcoming study that uses around 4mg/kg (280mg dose). It will be interesting to see the results.

Another interesting read was user experiences at _http://www.longecity.org/forum/topic/51983-methylene-blue-experiences. Though they were dosing in the microgram range, which is not even close to the doses used in the studies (but is closer if rat dosages are scaled using BSA but still a lot more than those users were taking). There is also a thread on MB research _http://www.longecity.org/forum/topic/23947-methylene-blue-research/ that has good info.

Back to reduced MB. One of the things that came up which I found also interesting was whether to take the MB in its reduced form or just as is (the reduced form in colourless and doesn’t stain your mouth and also said to cross the blood/brain barrier). There doesn’t seem to be any clear indication of which is better but what I’ve been able to find does seem to point towards reduced MB as preferred.

I think it is mentioned in the above research thread that one company that makes a drug for treatment of Alzheimer's is switching to a reduced form for better permeability to the blood brain barrier and lower side effects.

http://www.alzforum.org/news/research-news/will-tau-drug-show-its-true-colors-phase-3-trials

The company has since developed a modified version of the drug, LMTX™, which it will use in the upcoming Phase 3 trials. The new compound is a stabilized, reduced form of methylthionine, and is more readily absorbed and tolerated at a 10-fold higher dose than Rember®, Wischik said. So far, TauRx has secured LMTX patents in Europe, the U.S., Canada, South Korea, Hong Kong, and Singapore.


http://www.alzforum.org/news/conference-coverage/vienna-and-burkina-faso-whats-new-methylene-blue

Q: You also reported at ICAD that you have developed a new, colorless formulation of MTC?

A: Yes. This follows directly from the mathematical analysis I described above. The formulation is critical as the absorption of MT is a complex process, something we had to learn the hard way from the RemberTM Phase 2 trial. This is fully explained in our published patent WO07/110627. When administered as MTC, the MT moiety is presented as the chloride salt of the oxidized form of MT. In order for MT to be absorbed as the beneficial monomer, it has first to be reduced to the uncharged form (i.e., leuco-MT, or LMT), which is able to cross the gut lining and enter the blood. We believe this reduction process is probably enzyme-mediated, and occurs most favorably at the low pH of the stomach. We believe from our experimental work and analysis of our Phase 2 data that it is the LMT form which carries the activity required to retard the progression of AD. The oxidized form is the predominant form absorbed if the formulation is not right (as, e.g., with our 100 mg capsule), and it has the potential to give low efficacy coupled with increased side effects.

We have discovered a process to produce a pure LMT form that can be administered as a tablet. Yes, it is substantially colorless (actually, a light yellowy-green). We have found that when administered orally to animals, this LMT form enhances availability of the active moiety in the brain while reducing toxicity.

Here is the patent they mention:

https://patentscope.wipo.int/search/en/detail.jsf;jsessionid=2901A12ECA704123B984D8F4779A4795.wapp1nC?docId=WO2007110627&recNum=102&maxRec=2695&office=&prevFilter=&sortOption=&queryString=%28Alzheimer*%29%2520AND%2520%28tau%29%2520AND%2520%28%28IC%2FA61*%29%2520OR%2520%28IC%2FC07*%29%29&tab=PCTDescription


The "reduced form" (the "leuko form") is known to be unstable, and is readily and rapidly oxidized to give the corresponding "oxidized" form.

May et al. (Am J Physiol Cell Physiol, 2004, Vol. 286, pp. C1390-C1398) have shown that human erythrocytes sequentially reduce and take up MTC; that MTC itself is not taken up by the cells; that it is the reduced from of MTC that crosses the cell membrane; that the rate of uptake is enzyme dependent; and that both MTC and reduced MTC are concentrated in cells (reduced MTC re-equilibrates once inside the cell to form MTC).

MTC and similar drugs are taken up in the gut and enter the bloodstream. Unabsorbed drug percolates down the alimentary canal, to the distal gut. One important undesired side-effect is the effect of the unabsorbed drug in the distal gut, for example, sensitisation of the distal gut and/or antimicrobial effects of the unabsorbed drug on flora in the distal gut, both leading to diarrhoea. Therefore, it is desirable to minimize the amount of drug that percolates to the distal gut. By increasing the drug's update in the gut (i.e., by increasing the drug's bioavailability), dosage may be reduced, and the undesired side-effects, such as diarrhoea, may be ameliorated.

Since it is the reduced form of MTC that is taken up by cells, it would be desirable to administer the reduced form. This would also reduce reliance on the rate limiting step of enzymatic reduction.


It turns out that vitamin C (ascorbic acid) when added to MB reduces it. To make reduced methylene blue, you add around 2 to 2.5 grams of vit C to every gram of MB. So if you take a 50mg dose, then add around 100 - 150mg of vit C and leave it for a few minutes (studies say it can take up to 3 hours but when I’ve done it it took only about 10 minutes). It will turn clear, but will still make you pee blue. I’m still not sure if one form is better than the other and I think I will have to try both forms over a period of time to see until more research is available for the leuco (reduced) form. Currently I'm using the reduced form and I haven’t experienced any negative side effects but can’t say that I noticed anything beneficial that stood out (this was actually with both forms). If anything the effect is subtle. Two things that I think may be an effect is that I seem to be able to get by on less sleep. I usually need about 7.5 to 8 hrs. If I get less than that it catches up to me and I’m really tired by the end of the week. The last few weeks I was getting around 6.5 to 7hrs but I don’t feel extremely tired. It could be that I’m getting better quality of sleep but not really sure if that’s also an effect of the MB. One thing that I have noticed more recently is that after taking MB in the morning, I feel quite alert and focused. I’m currently taking about 50mg (so just under 1mg/kg as I weigh about 56kg) but will do some experimenting at the 4mg/kg as well as that was the amount used in the human studies as well as more with the oxidized form. Although I don’t really expect any miracles from this, what I’m more interested in is its neuroprotective benefits, which thus far seems well documented.


Nico said:
Thank you Keyhole,
There a site selling methylene blue, I have not enough knowledge nor interest (apart from testing) to use it, but it's maybe useful.

_https://mitoblue.com/

One thing to keep in mind is that some people say you can use fish tank cleaner (sold as methylene blue, very cheap) but that’s not what they were using for these studies so you’ll want to stay away from that. Also, the premade liquid forms of MB are not really practical in terms of reaching the doses used in the studies as the concentration is too diluted (one drop has 0.5mg of MB - so you will need 100 drops to get a 50mg dose) and it will get expensive really fast. What you want to do is make your own using >99% pure USP grade as it contains very low amounts of contaminants. In the EU you can order from APC (they also ship internationally but there may be sources in the US that USP grade can be sourced from). This is the stuff you want:

https://apcpure.com/product/methylene_blue_powder_99_5_bp73_usp

So for £8.50 you get 25grams vs £39.99 for 1 gram (10mg/ml in 100ml) of methylene blue.

Easiest way to make the solution:

http://www.brainprotips.com/methylene-blue-nootropic/ (rest of article also a good read)

Methylene Blue Dosing Protocol

For convenience, I prepared a solution of methylene blue so that ten drops of solution would contain 13 mg of methylene blue. Since 10 drop is roughly equivalent to 0.5 mL, and the total volume of my empty dropper bottle is 30 mL, we obtain the following:

Desired dose: 13 mg
Volume of ten drops: 0.5mL
Dropper bottle volume: 30 mL
(13 mc / 0.5mL) = 26 mg/ml (desired final concentration of MB)
(26 mg/ml) * 30 mL = 780 mg = 0.78 g of methylene blue

Hence, I will need to dissolve 780 mg of methylene blue in a volume of 30 mL distilled water (the volume of my dropper bottle) to attain a final concentration of 26 mg/mL, so that ten drops of solution will contain about 13 mg +/- 5 mg.

I had a 50mL bottle that I used to make the solution and was aiming for 1 drop to give about 2.5mg of MB. So for 10 drops that would be 25mg.

Volume of ten drops: 0.5ml
Dropper bottle: 50ml
(25mg/0.5ml) = 50mg/ml
50mg/ml * 50ml = 2500mg of methylene blue

So I weighed out 2.5g for powder, put that into the bottle, then added distilled water. Don’t fill it too much as you’ll need to leave room for the dropper to displace the liquid. You can suck some into the dropper so it doesn’t displace as much or if you’ve overfilled a bit.

Another thing is to find a prep area or use a mat or something you don’t mind getting stained. MB is a very strong dye and will stain everything blue even if you get the tiniest bit of powder on the table or clothes so be careful. If you do happen to stain something inadvertently, you can use a combination of vitamin C with vinegar to clean the stain.

When methylene blue is chemically reduced it becomes decolorised. This decolorisation is also acid-catalysed. Make up a solution containing 1 part vinegar (acid) and 1 part saturated Vitamin C (reducing agent), then rub that into the stain. We teach a 2nd-year kinetics experiment using this exact reaction at our university. If it works, rinse it with water. If it comes back, repeat experiment. [...]

I also read that MB has an effect on gut bacteria but I was not able to find much detail on whether it was detrimental or not. In either case, it doesn’t hurt to supplement with probiotics. Taking them at least a couple of hours apart from MB is suggested. Also, since one of the benefits is increased energy, better to take it in the morning or during the day rather than right before bed. Another thing is to keep in mind that besides making your urine or stools blue, some other side effects could include:

mild bladder irritation
dizziness
headache
increased sweating
nausea
abdominal pain
diarrhea
upset stomach

I have also seen this mentioned in various places:
https://en.wikipedia.org/wiki/Methylene_blue

Methylene blue is a monoamine oxidase inhibitor (MAOI),[32] and if infused intravenously at doses exceeding 5 mg/kg, may precipitate serious serotonin toxicity, serotonin syndrome, if combined with any selective serotonin reuptake inhibitors (SSRIs) or other serotonin reuptake inhibitor (e.g., duloxetine, sibutramine, venlafaxine, clomipramine, imipramine).[33]

It causes hemolytic anemia in carriers of the G6PD (favism) enzymatic deficiency.

Although at doses < 4mg/kg, it seems to be generally quite safe and I did read a study where at low doses (< 2mg/kg) those with favism weren’t affected. Though in both situations I’d err on the side of caution and avoid experimenting with methylene blue. And stop taking MB or reduce the dose if you are experiencing uncomfortable side effects.


Data said:
As always, do lots of research before considering utilization of any substance!

Exactly! Make sure you understand the implications of what you are doing and risks involved so you can stay safe.
 

Gaby

SuperModerator
Moderator
FOTCM Member
fabric said:
Another thing is to keep in mind that besides making your urine or stools blue, some other side effects could include:

mild bladder irritation
dizziness
headache
increased sweating
nausea
abdominal pain
diarrhea
upset stomach

Bolded those that I had with only 4 mg of pharmaceutical grade methylene blue. I read that 10mg twice per week was a good and low mitochondrial booster dose. I figured I would start with 4mg and see how it goes.

It also makes me sleepy. But I think I had more mental clarity the next day I took the MB.

Then, there is this paper:

Adverse Effects of Methylene Blue on the Central Nervous System
http://anesthesiology.pubs.asahq.org/article.aspx?articleid=1932089

It seems that the range of dose which had neurotoxic effects was from 5mg/kilo to 50mg/kilo in IV. The closer to 50mg per kilo, the worse. This was done in rats, but it is a concern because doctors might use 5mg/kg in parathyroid surgery and sometimes patients wake up confused and disoriented from surgery...

Perhaps the maximum safe dose is 1mg/kilo.

Then,

Severe anaphylactic shock due to methylene blue dye
http://www.sciencedirect.com/science/article/pii/S2213576614000256

But that applies to everything, i.e. foods, medication, etc. Starting with a very small dose will make any reaction more manageable though.
 

Odyssey

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I've been experimenting with MB (Kordon) for about 4 days. The first time I took roughly 2mg (at once)and felt a boost in both energy and mood. The next day I split it to 1mg in am and pm and found that my sleep was poor. I tried a night dose again and sleep was poor so I won't take any at night anymore.

Most of the people on the forums I've visited (Ray Peat, Longecity) have been using MB in the microgram doses. Higher doses in the milligram range (15mg+) are usually reserved for severe depression, cancer, alzheimer's and other serious disorders.

As with anything, it requires research and experimentation to find the 'sweet spot' that works for you and your current state of mitochondrial health.

_http://www.longecity.org/forum/topic/51983-methylene-blue-experiences/
_https://raypeatforum.com/community/threads/methylene-blue.5344/page-2
 

Ant22

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Wow, this is a really interesting thread! Methylene blue is one of those substances that were around when I was little, like iodine, but I never looked into them. MB was the last resort substance for recurring cystitis back then.

Keyhole's initial post contains information on beneficial impact of Methylene Blue on issues that run in my family a lot (arthritis, cancer, cardiovascular diseases) so I've decided to give it a try. I find the topics of stress metabolism and cellular level energy production to be quite fascinating too. I myself have been feeling very well recently but I'm always interested in ways to protect myself if issues that have been plaguing my family members start to rear their head in my case. Although I've been very careful with my diet over the last year (paleo, gluten free and nearly grain free, 60-70% meat and lots of animal fat) whilst my family loooove all the stuff they should not be eating (like sugar, gluten and dairy). So I am in fact taking steps to protect myself better than they do.

As they say: the genes load the gun but it's the environment that pulls the trigger.

Data said:
Keyhole said:
Methylene blue (or MB) is a basic aniline dye with the molecular formula C16H18N3SCl. At room temperature, it appears as a solid, odorless, dark green powder that yields a blue solution when dissolved in water.

Note that there are at least two types of Methylene Blue - which are structurally similar, but not identical. They may have different properties. Vendors rarely specify the chemical formula and rarely deliver a quality certificate with the product.

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

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

New Methylene Blue seems to be sold as a Zinc Chloride (ZnCl2) salt, and appears as a dark brown/reddish powder rather than a greenish powder.

As always, do lots of research before considering utilization of any substance!

This is handy to know. I've been looking for a seller online and like you said, most of them don't provide the chemical formula. And the only purity information provided is "purest quality", whatever that means.

fabric said:
(...) One thing to keep in mind is that some people say you can use fish tank cleaner (sold as methylene blue, very cheap) but that’s not what they were using for these studies so you’ll want to stay away from that. Also, the premade liquid forms of MB are not really practical in terms of reaching the doses used in the studies as the concentration is too diluted (one drop has 0.5mg of MB - so you will need 100 drops to get a 50mg dose) and it will get expensive really fast. What you want to do is make your own using >99% pure USP grade as it contains very low amounts of contaminants. In the EU you can order from APC (they also ship internationally but there may be sources in the US that USP grade can be sourced from). This is the stuff you want:

https://apcpure.com/product/methylene_blue_powder_99_5_bp73_usp (...)

Thank you for recommending the supplier fabric, I've just ordered it and it should be with me mid next week. The seller's product has the chemical formula of C16H18ClN3S, which is what Methylene Blue (and not New Methylene Blue) should be according to the links provided by Data.

What caught my attention is that the 'hazards' section on the seller's website says: "Do no eat, drink or smoke when using this product. IF SWALLOWED: Call a POISON CENTER or doctor/physician if you feel unwell. Rinse mouth."

I'd assume this is not supposed to be ingested if it wasn't for the fact that my bottle of Lugol says "for external use only" too.

Other sources, such as this paper for example, are less dramatic in their description of ingestion hazards: "Ingestion: May cause discomfort if swallowed".


Gaby said:
fabric said:
Another thing is to keep in mind that besides making your urine or stools blue, some other side effects could include:

mild bladder irritation
dizziness
headache
increased sweating
nausea
abdominal pain
diarrhea
upset stomach

Bolded those that I had with only 4 mg of pharmaceutical grade methylene blue. I read that 10mg twice per week was a good and low mitochondrial booster dose. I figured I would start with 4mg and see how it goes.

It also makes me sleepy. But I think I had more mental clarity the next day I took the MB.

Thank you for your testimonial and the summary of symptoms you experienced Gaby! I must say the one I'm scared of most is being sleepy. I had issues with chronique fatigue for like a decade and I've had the most 'awake' and productive couple of months recently. I'll ditch MB at the faintest sign these issues start to come back. But the 'mental clarity' part you mentioned is a good enough reason for me to give it a try.

Odyssey said:
I've been experimenting with MB (Kordon) for about 4 days. The first time I took roughly 2mg (at once)and felt a boost in both energy and mood. The next day I split it to 1mg in am and pm and found that my sleep was poor. I tried a night dose again and sleep was poor so I won't take any at night anymore.

Most of the people on the forums I've visited (Ray Peat, Longecity) have been using MB in the microgram doses. Higher doses in the milligram range (15mg+) are usually reserved for severe depression, cancer, alzheimer's and other serious disorders. (...)

Thanks for the testimonial Odyssey. I guess I'll apply the same approach as was advised with Lugol: start with low doses and build it up.

Since it affected your sleep, I guess it would be advisable to take it in the morning. That's when I take my Lugol so I was trying to find information on any possible reasons to avoid combining them.

Well, it looks like Lugol and MB are in fact used together in staining in gastrointestinal endoscopy or
double staining for detection of GST-Pi and telomerase in the early diagnosis of esophageal carcinoma
.

I will take them 2-3 hours apart though.
 

Ant22

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My methylene blue arrived two days after I posted in this thread and I thought I'd share my experience with it. I must say it was a bit interesting.

Let me start with my main reason for wanting to give it a try: I used to struggle with fatigue and tiredness and although the problem is mostly gone now, I still occasionally get very sleepy around 6-7pm. The sleepiness comes back every couple of weeks and stays for a couple of days at a time. This is usually combined with a quite intense brain fog that puts an end to any productivity that evening. On those occasions I am simply too tired to focus even on reading.

I was hoping methylene blue would address this issue - and it actually did it much better than I expected. I had really high levels of energy, alertness and mental clarity and I really felt great overall! Despite increased energy levels during the day I didn't have any sleeping problems and I was able to fall asleep without any issues within my regular 10.30-11pm window.

In fact, I didn't experience any negative symptoms apart from very faint headache.

Another surprising effect was something that I didn't see discussed here - my period. Ever since I started taking iodine in December 2015 it has been ridiculously irregular and painful. I usually also get massive stomach aches that last for about a week after my period is over. (This problem started before my iodine adventure so it's not related to taking it though.) I sometimes have to resort to prescription strength painkillers in order to be able to function at work.

I started taking methylene blue a week before my period and to my surprise it was MUCH better! I wouldn't say it was perfect but the stomach ache was minimal and simple ibuprofen itself did the job.

Also, I have been diagnosed with hemochromatosis and one of the symptoms is scarce period bleeding. Which is not exactly desirable since period bleeding helps remove excess iron. That issue was solved too so I was quite excited about the results.

But after around 10 days my great results vanished and all the symptoms I used to suffer from returned, skin rashes, light sensitivity, nausea, and especially fatigue and brain fogs. I can't say I missed struggling to keep my eyes open and making enormous effort to keep track of conversations.

Needless to say I was devastated! I discontinued methylene blue immediately but the symptoms persisted and I even started to worry that I ruined nearly two years of hard work to regain my health! I wasn't sure what to do so I resorted to my go-to trick that helps me whenever I start getting sluggish and sleepy, namely DMSA. Other people here have reported really bad symptoms while taking it while I feel OK on it. The two days after I finish a round are quite challenging but I make sure to remineralise and I'm back to feeling great quickly. DMSA actually helped this time too but surprisingly enough, despite taking a lower dose I felt really bad on it. It also took me longer to recover during the remineralisation phase.

I still can't say I'm back to where I was before I started taking methylene blue though. I do wonder whether my MB was contaminated or something? Or maybe I took too much? It's hard to say how much I took as it was in a powder form. I put a wee little bit (like a square millimetre) of it into an empty capsule and topped it up with water. I think it's about time I bought a milligram sensitive scale really.

Despite the scary part of my experience I must say the initial days of taking MB were really good. Gosh, this was probably the best I've ever felt! I managed to do more within that short amount of time than I normally do in a month and I really wish I could keep it for longer.

I had every intention of starting with a low dose but I guess I didn't realise just how low it actually has to be. I do wonder whether lowering the dose to a tiny fraction of what I was taking would help? Maybe if I diluted that square millimetre in a litre of water and just took a couple of drops would help?

Well, unless I have allergy to it. :/

Either way, I have no intention of having another go until at least a couple of months down the line - and only if I get back to where I was before I took it.

On a different note, MB has extremely potent colour! A grain of invisible size would probably be enough o paint the whole room blue. I made the mistake of opening the tub with my teeth, my face and the bathroom looked like I murdered a smurf!

In case someone has a similar accident, vitamin C as ascorbic acid removes methylene blue. It's not as quick as with iodine and it has to be left on it for a couple of minutes up to half an hour, but it does work.


Edit = spelling
 

fabric

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Ant22 said:
[...]

I still can't say I'm back to where I was before I started taking methylene blue though. I do wonder whether my MB was contaminated or something? Or maybe I took too much? It's hard to say how much I took as it was in a powder form. I put a wee little bit (like a square millimetre) of it into an empty capsule and topped it up with water. I think it's about time I bought a milligram sensitive scale really.

Despite the scary part of my experience I must say the initial days of taking MB were really good. Gosh, this was probably the best I've ever felt! I managed to do more within that short amount of time than I normally do in a month and I really wish I could keep it for longer.

I had every intention of starting with a low dose but I guess I didn't realise just how low it actually has to be. I do wonder whether lowering the dose to a tiny fraction of what I was taking would help? Maybe if I diluted that square millimetre in a litre of water and just took a couple of drops would help?

[...]

I'm sorry to hear about the setback Ant22! Yea, MB is definitely something you should not be guessing at when it comes to dosing. One thing you should have noticed if you were taking too much was if the colour of your urine was blue. A small amount will slightly discolour it (so like a greenish yellow) to a deeper blue for larger amounts. In my case at around 10mg I would have green pee but when I take around 50mg (which is just under 1mg/kg for me) it is very blue. There is a pretty narrow range in which benefits were noticed and outside of that either no effect or undesirable effects. Not sure if you had a look through some of the experiences reported here: _http://www.longecity.org/forum/topic/51983-methylene-blue-experiences/ but most were dosing in the microgram range (which was a lot less from the studies) and had varying reports on whether it worked for them or not. I'm still not sure exactly what the right dose would be and this has to be adjusted since everyone responds differently (similar to iodine). If you are going try a lower dose, make sure you get a scale (one that can read 0.001g) and dropper bottle and make it as per the example earlier.

Methylene Blue Dosing Protocol

For convenience, I prepared a solution of methylene blue so that ten drops of solution would contain 13 mg of methylene blue. Since 10 drop is roughly equivalent to 0.5 mL, and the total volume of my empty dropper bottle is 30 mL, we obtain the following:

Desired dose: 13 mg
Volume of ten drops: 0.5mL
Dropper bottle volume: 30 mL
(13 mc / 0.5mL) = 26 mg/ml (desired final concentration of MB)
(26 mg/ml) * 30 mL = 780 mg = 0.78 g of methylene blue

Hence, I will need to dissolve 780 mg of methylene blue in a volume of 30 mL distilled water (the volume of my dropper bottle) to attain a final concentration of 26 mg/mL, so that ten drops of solution will contain about 13 mg +/- 5 mg.

So another example: You have a 50mL bottle to make the solution with and are aiming for 1 drop to give about 0.06mg of MB. So for 10 drops that would be 0.6mg.

Volume of ten drops: 0.5ml
Dropper bottle: 50ml
(0.6mg/0.5ml) = 1.2mg/ml
1.2mg/ml * 50ml = 60mg of methylene blue

So weigh out 60mg powder, put that in the bottle then fill it with distilled water. Give a good shake and one drop from the dropper should give you 60ug, similar

One thing I noticed was that at first, when I would take 50mg, my urine would be dark blue in the evenings (I take my MB in the mornings) but the next day it would be back to normal. However after several days, I would still pee blue even in the morning. So I would skip that day but my urine would continue to be blue for another day. I'm thinking there was some kind of accumulation going on or my body is processing it differently so I backed down the dose and started taking it every other day instead. I haven't noticed much in the way of extra high energy levels or alertness but on days I do take it it seems that I have better recall and do better when studying my flashcards. Other than that, nothing really noticeable. However, I had the same when I took iodine. At first I was feeling very alert and energized but after awhile nothing. Or just a headache if I took too much.
 
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