Methylene Blue Today: Mitochondria, Brain Research and Safety Findings

Table of Contents

1. A Dye That Kept Finding New Uses
2. Return to Malaria: Trials in Burkina Faso
3. The Mitochondrial Electron Shuttle
4. Hormesis: Opposite Effects at Low and High Doses
5. Human Brain Imaging Studies
6. The Alzheimer’s Disease Trials and Their Negative Result
7. Mood Disorders: From Naylor to Alda
8. Serotonin Toxicity and Monoamine Oxidase A
9. G6PD Deficiency and Red-Cell Findings
10. Legacy of Caro and Guttmann
11. Key Research Papers
12. Connections
13. Featured Videos

1. A Dye That Kept Finding New Uses

Few substances in medicine have had as many careers as methylene blue. It began as a textile colour. Within ten years of its synthesis Paul Ehrlich was using it to stain living nerve tissue, and by 1891 it had been given to malaria patients in Berlin. A 2011 review by Schirmer and colleagues, written to mark roughly 120 years of the dye in medicine, called it “the first synthetic drug” — a historians’ shorthand, since a few synthetic substances such as chloral hydrate were in medical use earlier, but one that captures how early methylene blue came to be used against a specific disease.

The dye’s greater influence may have been as a model. The chemists Ohlow and Moosmann, in a 2011 review titled “the seven lives of pharmacology’s first lead structure”, traced how the phenothiazine ring at the core of methylene blue became the starting point for whole families of later drugs: anti-worm and antibacterial agents in the 1930s and 1940s, then antihistamines, sedatives and, in the 1950s, the antipsychotic chlorpromazine. Wainwright and Crossley, reviewing the dye in 2002, described it as the basis both of antimicrobial chemotherapy and of the antimalarial line that followed, and as a forerunner of the neuroleptic drugs.

Ehrlich’s work with dyes also fed his later idea of the “magic bullet” — a chemical that seeks out and harms a disease-causing target while sparing the body’s own cells. Strebhardt and Ullrich, looking back over a century of that concept in 2008, followed it all the way to the targeted cancer drugs and antibodies of the modern era. Methylene blue, in other words, did not only find new uses for itself; it gave later chemists a template to build on.

From the late twentieth century, researchers returned to the original molecule. Three threads stand out: a revived interest in malaria, especially in stopping transmission; research on the dye’s chemistry inside mitochondria and the brain; and a growing body of safety findings. The sections below follow each in turn.

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2. Return to Malaria: Trials in Burkina Faso

Methylene blue was set aside as an antimalarial in the twentieth century as other drugs, above all the synthetic chloroquine, took over, and later as artemisinin — the compound extracted from the sweet wormwood plant, Artemisia annua — became the backbone of treatment. But resistance to one drug after another kept the search for partner medicines alive, and in the early 2000s a German–Burkinabé research group began testing the old dye again in children in Burkina Faso, West Africa.

The gametocyte trial

The most cited of these studies, published by Coulibaly and colleagues in 2009, was an open-label randomised controlled trial in 180 children aged 6 to 10 with uncomplicated falciparum malaria in Nouna, north-western Burkina Faso. The children were assigned to one of three combinations: methylene blue with artesunate (an artemisinin derivative), methylene blue with amodiaquine, or artesunate with amodiaquine, which was the local standard of care. Compared with the standard combination, both methylene blue regimens were associated with significantly fewer children carrying gametocytes on days 3, 7 and 14 of follow-up.

Gametocytes are the sexual stage of the malaria parasite — the form a mosquito picks up when it bites an infected person and then carries to the next. A drug that clears them does not necessarily make a patient feel better faster, but it can cut the chain of transmission. The authors concluded that methylene blue showed pronounced activity against both existing and developing gametocytes and that methylene-blue-based combinations had the potential to reduce the spread of falciparum malaria where it is common.

A partner for artemisinin-based therapy

By 2013, according to Müller and colleagues, methylene blue was being investigated for its benefit when added to artemisinin-based combination therapy (ACT), the current first-line treatment for falciparum malaria. A 2018 systematic review by Lu and colleagues pulled together 21 studies with 1,504 patients across the dye’s history as an antimalarial. They described methylene blue as the first synthetic antimalarial ever discovered, found it highly effective against malaria, noted its strong effect on gametocytes, and recorded the main side effects as blue-coloured urine together with mild urinary and digestive complaints.

The story had come full circle: the dye Guttmann and Ehrlich gave to two Berlin patients in 1891 was being weighed again, more than a century later, against the same disease — this time as a companion to a plant-derived medicine. The site’s page on antimalarial drugs and ACT covers today’s treatments in more detail.

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3. The Mitochondrial Electron Shuttle

Methylene blue is a redox dye: it switches easily between a blue, oxidised form and a colourless, reduced form called leucomethylene blue, taking up and giving away electrons as it does so. That property, explained on the wing’s chemistry page, is what lets it reverse methaemoglobinaemia. In the last few decades it has also drawn researchers to the mitochondria, the small structures inside cells that turn food into usable energy.

Mitochondria make energy by passing electrons along a chain of protein complexes, the electron transport chain, ending with the enzyme cytochrome c oxidase, which hands the electrons to oxygen. Reviews by Rojas, Bruchey and Gonzalez-Lima (2012) and by Tucker, Lu and Zhang (2018) describe how methylene blue, at low concentrations, can accept electrons from NADH and pass them on toward cytochrome c — in effect acting as an alternative electron carrier that can route around parts of the chain. Rojas and colleagues called it an “electron cycler” and reported that the available data support a major role for the respiratory enzyme cytochrome oxidase in its memory effects; Tucker and colleagues described the rerouting as increasing the activity of complex IV, the cytochrome oxidase step.

The appeal of this idea is that several brain diseases involve faltering mitochondria. In laboratory work, the reviews report, methylene blue was associated with better memory consolidation and with protection of nerve cells in animal models of stroke, global cerebral ischaemia, Alzheimer’s disease, Parkinson’s disease and traumatic brain injury. Both reviews are built largely on animal and cell experiments; they set out a mechanism and a research direction rather than proof of benefit in people. The site’s page on methylene blue’s mitochondrial mechanism and the animated mitochondria page explain the electron chain step by step.

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4. Hormesis: Opposite Effects at Low and High Doses

One of the most important findings in the modern research is that methylene blue does not simply do “more” as the amount rises. Rojas and colleagues described its dose response as hormetic: low doses and high doses can have opposite effects. The word hormesis comes from toxicology and describes a substance that is stimulating or protective at low exposure but harmful at high exposure.

The reason lies in the same redox chemistry. Rojas and colleagues tied the dye’s memory and nerve-protecting effects specifically to low doses, at which it behaves as an electron cycler with antioxidant and respiration-enhancing properties; they stressed that its effects are not governed by the usual drug–receptor rules, in which a bigger dose simply produces a bigger effect. A molecule that can both give and take electrons can support the respiratory chain at one concentration and work against it at another.

The same two-sidedness appears in the clinic. Methylene blue is the standard antidote for acquired methaemoglobinaemia, yet the 2016 Alzheimer’s trial described below excluded patients taking other medicines that carry warnings of methaemoglobinaemia, because the oxidised form of the dye at high doses can itself induce the condition. A substance that corrects a disorder at one level and can provoke it at another is a clear example of why the dose-response curve sits at the centre of this research.

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5. Human Brain Imaging Studies

Animal experiments raised the question of whether low-dose methylene blue changes brain activity in people. In 2016 a team at the University of Texas, including Gonzalez-Lima, published one of the first attempts to look directly. Rodriguez and colleagues ran a randomised, double-blind, placebo-controlled trial in 26 healthy adults aged 22 to 62, published in the journal Radiology.

The volunteers had functional magnetic resonance imaging (fMRI) — brain scans that track changes in blood flow as areas become active — while doing a sustained-attention task and a short-term-memory task, before and one hour after taking either a low oral dose of methylene blue or a placebo. The researchers reported increased fMRI responses in brain regions involved in these tasks in the methylene blue group, and a 7% increase in correct responses during memory retrieval compared with placebo.

The study was small, involved healthy people rather than patients, and measured effects over a single hour, so it shows that a single low dose can alter task-related brain activity under scan conditions; it does not show lasting benefit or treatment of any disease. It is frequently cited in discussions of methylene blue as a “nootropic”, which makes its limits worth stating plainly. The site’s main methylene blue page reviews the wider literature on brain effects.

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6. The Alzheimer’s Disease Trials and Their Negative Result

In Alzheimer’s disease, a protein called tau clumps together inside nerve cells into tangles. Laboratory work suggested that methylthioninium — the chemical core of methylene blue — can block tau from aggregating, and Schirmer’s 2011 history review discussed this tau research as one of the dye’s newest chapters. That led to a large drug-development programme using a stable, reduced form of the molecule called leuco-methylthioninium bis(hydromethanesulfonate), or LMTM.

The 2016 phase 3 trial

The results of the first big phase 3 trial were published in The Lancet in 2016 by Gauthier and colleagues. It was a 15-month randomised, double-blind trial at 115 centres in 16 countries, which enrolled 891 people with mild to moderate Alzheimer’s disease. Participants received one of two higher doses of LMTM or a very low “control” dose of LMTM, chosen to keep the trial blinded, since the drug turns urine and stools blue. Many participants were also taking standard Alzheimer’s medicines.

The result was negative. In the authors’ own words, the prespecified primary analyses “did not show any treatment benefit at either of the doses tested” on the two main measures of thinking and daily function, and the results did not suggest benefit of LMTM as an add-on treatment for mild to moderate Alzheimer’s disease. Digestive and urinary effects were the most common adverse events at the higher doses and the most common reasons for stopping, and small dose-related drops in haemoglobin were the most common laboratory change.

Discussions of methylene blue and memory sometimes leave this trial out. Its published primary result is negative, and it is the largest controlled test of the methylthioninium molecule in a brain disease described on this page. The site’s Alzheimer’s disease page covers the condition and its research more broadly.

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7. Mood Disorders: From Naylor to Alda

Methylene blue has a long, scattered history in psychiatry. A 2019 review by the Canadian psychiatrist Martin Alda noted that it has been used in psychiatry for over a century, and its phenothiazine descendant chlorpromazine arrived in the 1950s. Two controlled trials in bipolar disorder stand out.

Naylor and colleagues, 1986

Naylor, Martin, Hopwood and Watson ran a two-year double-blind crossover trial in 31 people with manic-depressive illness (now called bipolar disorder), all also maintained on lithium. Each person spent one year on a higher daily dose of methylene blue and one year on a very low dose. Seventeen completed the full two years. During the higher-dose year, participants were significantly less depressed; there was no significant difference in manic symptoms. The authors themselves listed the trial’s obvious limitations: a small number of subjects, many dropouts, simple rating scales, doubts about whether blinding held (blue urine is hard to hide), and uncertainty over whether the low dose could be treated as a placebo.

Alda and colleagues, 2017

Three decades later, Alda and colleagues in Canada ran a six-month double-blind crossover study in 37 people with bipolar disorder who were already taking the mood stabiliser lamotrigine and still had lingering symptoms. They compared an active dose of methylene blue with a very low dose used as a “placebo”. The active dose significantly improved symptoms of depression on two standard rating scales and reduced anxiety; manic symptoms stayed low and stable; and there was no significant effect on cognitive (thinking) symptoms. Side effects were described as mild and short-lived.

Taken together, the two trials are small and both used methylene blue as an add-on to another mood medicine, so they describe a research signal rather than an established treatment. They also connect directly to the safety question in the next section, because many people with mood disorders take antidepressants that act on serotonin. The site’s bipolar disorder page covers the condition itself.

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8. Serotonin Toxicity and Monoamine Oxidase A

The most serious safety finding of the modern era came not from a trial but from case reports. Patients taking selective serotonin reuptake inhibitor (SSRI) antidepressants were reported to develop serotonin toxicity (also called serotonin syndrome) after being given methylene blue, a dye still in clinical use, including in hospital and surgical settings.

The mechanism: MAO-A inhibition

In 2007 Ramsay, Dunford and Gillman tested a theoretical prediction: that methylene blue was blocking monoamine oxidase A (MAO-A), the enzyme that breaks down serotonin. In measurements on purified human enzyme published in the British Journal of Pharmacology, they found that methylene blue is a potent, reversible inhibitor of MAO-A. When MAO-A is blocked in someone already taking a drug that raises serotonin, serotonin can build up to toxic levels. Monoamine oxidase inhibitors were already known to cause serotonin toxicity when combined with SSRIs; the finding placed methylene blue in that same group. The authors calculated that at concentrations reported after intravenous use, MAO-A would be completely inhibited.

A fatal case

In 2014 Top, Gillman, de Langen and Kooy, writing in the Netherlands Journal of Medicine, reported what they described as the first published fatal case of serotonin toxicity associated with methylene blue, in a patient who was taking the antidepressant venlafaxine. The authors noted that methylene-blue-associated serotonin toxicity had been described before, usually as mild, and that its presentation after general anaesthesia may be atypical and harder to recognise.

The site’s page on methylene blue drug interactions and serotonin syndrome lists the classes of medicine involved.

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9. G6PD Deficiency and Red-Cell Findings

Glucose-6-phosphate dehydrogenase (G6PD) is an enzyme that helps red blood cells protect themselves from oxidative damage. Inherited G6PD deficiency is one of the most common enzyme deficiencies in the world, and it is especially frequent in regions where malaria has long been common, because the trait offers partial protection against the parasite. People with the deficiency can have haemolysis — the breaking-up of red blood cells — after exposure to certain drugs, foods (such as fava beans) or infections.

The link to methylene blue is chemical. Inside red cells, the dye has to be reduced to its colourless form before it can reverse methaemoglobinaemia, and that reduction depends on NADPH, which G6PD supplies. With too little G6PD the dye works less well and can add oxidative stress of its own. Methylene blue is therefore on the list of drugs that can potentially trigger haemolysis in G6PD deficiency, alongside several other antimalarials.

The West African data

Because the Burkina Faso trials treated children in a region where G6PD deficiency is common, the research group pooled the data to look at this risk directly. Müller and colleagues (2013) analysed four clinical studies conducted between 2003 and 2007 in 1,005 West African children with falciparum malaria: 199 were G6PD-deficient and 806 were not; 844 received methylene-blue-containing regimens and 161 did not. In children with a full G6PD defect, methylene blue treatment was associated with a significant fall in haemoglobin, and two episodes of haemolysis occurred among the 1,005 children — one in a girl carrying one copy of the deficiency and one in a boy with the full defect, both of whom had received methylene blue.

The authors concluded that methylene blue treatment of malaria in Africa was associated with slightly reduced haemoglobin in children with a full G6PD defect, an effect they judged to be of limited clinical relevance but one that needs to be monitored. The site’s pages on G6PD deficiency and on methaemoglobinaemia and G6PD deficiency explain the enzyme and its testing.

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10. Legacy of Caro and Guttmann

Heinrich Caro died in Dresden in 1910; Paul Guttmann died in Berlin in 1893. Neither lived to see the antidote years of the 1930s, the phenothiazine drugs of the 1950s or the trials of the twenty-first century. Yet each left something that outlasted him.

Caro: the industrial research laboratory

Caro’s most lasting contribution may be less a single dye than a way of working. As head of research at the Ludwigshafen dye works from 1868, he ran what the Dictionary of Scientific Biography calls probably the first true industrial research organisation — a place where trained chemists worked systematically to create new substances. The historian Anthony Travis, Caro’s biographer, has traced how the chemistry of synthetic dyes, beginning with the synthesis of alizarin, fed directly into Ehrlich’s idea of chemical “side chains” and receptors, and from there into the way modern drugs are designed. The coal-tar dye industry that Caro helped build was, in part, an attempt to replace natural colours from plants such as madder and indigo; the drug industry that grew out of it would eventually make synthetic versions and successors of plant medicines such as quinine as well.

Guttmann: the clinical test

Guttmann’s legacy is the clinical step. Ehrlich’s staining had shown that methylene blue reached malaria parasites; it was the hospital physician who put the idea to a test in sick patients, and their 1891 report, reprinted in Ehrlich’s collected papers, stands at the start of the line of synthetic antimalarials that runs through pamaquine and chloroquine. Guttmann’s long-used textbook of clinical examination reflects the same habit of careful observation at the bedside.

A molecule that is still being tested

The modern record is mixed, and that is part of the legacy too. In malaria, methylene blue showed strong effects on gametocytes and is still studied as a partner for artemisinin-based drugs. As an antidote for methaemoglobinaemia it remains in standard use. In mitochondrial and brain research it has produced a coherent mechanism and some intriguing small human studies. In the large Alzheimer’s trial the primary result was negative. And its safety profile now includes two well-documented findings — MAO-A inhibition with serotonin toxicity, and haemolysis risk in G6PD deficiency — that no one in 1891 could have known about. A blue dye for cotton, in short, became one of the longest-running experiments in the history of pharmacology.

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Key Research Papers

1. Schirmer RH, Adler H, Pickhardt M, Mandelkow E. “Lest we forget you--methylene blue...”. Neurobiol Aging. 2011;32(12):2325.e7-16. PubMed PMID: 21316815
2. Ohlow MJ, Moosmann B. Phenothiazine: the seven lives of pharmacology’s first lead structure. Drug Discov Today. 2011;16(3-4):119-31. PubMed PMID: 21237283
3. Wainwright M, Crossley KB. Methylene Blue--a therapeutic dye for all seasons? J Chemother. 2002;14(5):431-43. PubMed PMID: 12462423
4. Strebhardt K, Ullrich A. Paul Ehrlich’s magic bullet concept: 100 years of progress. Nat Rev Cancer. 2008;8(6):473-80. PubMed PMID: 18469827
5. Coulibaly B, Zoungrana A, Mockenhaupt FP, Schirmer RH, Klose C, Mansmann U, Meissner PE, Müller O. Strong gametocytocidal effect of methylene blue-based combination therapy against falciparum malaria: a randomised controlled trial. PLoS One. 2009;4(5):e5318. PubMed PMID: 19415120
6. Lu G, Nagbanshi M, Goldau N, Mendes Jorge M, Meissner P, Jahn A, Mockenhaupt FP, Müller O. Efficacy and safety of methylene blue in the treatment of malaria: a systematic review. BMC Med. 2018;16(1):59. PubMed PMID: 29690878
7. Müller O, Mockenhaupt FP, Marks B, Meissner P, Coulibaly B, Kuhnert R, Buchner H, Schirmer RH, Walter-Sack I, Sié A, Mansmann U. Haemolysis risk in methylene blue treatment of G6PD-sufficient and G6PD-deficient West-African children with uncomplicated falciparum malaria: a synopsis of four RCTs. Pharmacoepidemiol Drug Saf. 2013;22(4):376-85. PubMed PMID: 23135803
8. Rojas JC, Bruchey AK, Gonzalez-Lima F. Neurometabolic mechanisms for memory enhancement and neuroprotection of methylene blue. Prog Neurobiol. 2012;96(1):32-45. PubMed PMID: 22067440
9. Tucker D, Lu Y, Zhang Q. From Mitochondrial Function to Neuroprotection-an Emerging Role for Methylene Blue. Mol Neurobiol. 2018;55(6):5137-5153. PubMed PMID: 28840449
10. Rodriguez P, Zhou W, Barrett DW, Altmeyer W, Gutierrez JE, Li J, Lancaster JL, Gonzalez-Lima F, Duong TQ. Multimodal Randomized Functional MR Imaging of the Effects of Methylene Blue in the Human Brain. Radiology. 2016;281(2):516-526. PubMed PMID: 27351678
11. Gauthier S, Feldman HH, Schneider LS, Wilcock GK, Frisoni GB, Hardlund JH, Moebius HJ, Bentham P, Kook KA, Wischik DJ, Schelter BO, Davis CS, Staff RT, Bracoud L, Shamsi K, Storey JM, Harrington CR, Wischik CM. Efficacy and safety of tau-aggregation inhibitor therapy in patients with mild or moderate Alzheimer’s disease: a randomised, controlled, double-blind, parallel-arm, phase 3 trial. Lancet. 2016;388(10062):2873-2884. PubMed PMID: 27863809
12. Naylor GJ, Martin B, Hopwood SE, Watson Y. A two-year double-blind crossover trial of the prophylactic effect of methylene blue in manic-depressive psychosis. Biol Psychiatry. 1986;21(10):915-20. PubMed PMID: 3091097
13. Alda M, McKinnon M, Blagdon R, Garnham J, MacLellan S, O’Donovan C, Hajek T, Nair C, Dursun S, MacQueen G. Methylene blue treatment for residual symptoms of bipolar disorder: randomised crossover study. Br J Psychiatry. 2017;210(1):54-60. PubMed PMID: 27284082
14. Alda M. Methylene Blue in the Treatment of Neuropsychiatric Disorders. CNS Drugs. 2019;33(8):719-725. PubMed PMID: 31144270
15. Ramsay RR, Dunford C, Gillman PK. Methylene blue and serotonin toxicity: inhibition of monoamine oxidase A (MAO A) confirms a theoretical prediction. Br J Pharmacol. 2007;152(6):946-51. PubMed PMID: 17721552
16. Top WM, Gillman PK, de Langen CJ, Kooy A. Fatal methylene blue associated serotonin toxicity. Neth J Med. 2014;72(3):179-81. PubMed PMID: 24846936
17. Travis AS. Chemical Modeling: From Paul Ehrlich’s Dyes to β-Blockers-A Brief History. J Comput Biol. 2019;26(7):726-734. PubMed PMID: 31066580
18. Guttmann P, Ehrlich P. Über die Wirkung des Methylenblau bei Malaria. In: The Collected Papers of Paul Ehrlich. Elsevier; 1960:9-14 (reprint of the 1891 Berliner klinische Wochenschrift paper). DOI: 10.1016/b978-0-08-009056-6.50006-3

PubMed Topic Searches

1. PubMed: methylene blue and malaria
2. PubMed: methylene blue and mitochondria
3. PubMed: methylene blue and serotonin toxicity
4. PubMed: methylene blue and G6PD deficiency
5. PubMed: history of methylene blue

Further Reading

• Reinhardt C, Travis AS. Heinrich Caro and the Creation of Modern Chemical Industry. Dordrecht: Springer Netherlands; 2000. DOI: 10.1007/978-94-015-9353-3

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Connections

• Heinrich Caro and Paul Guttmann: Methylene Blue, from Textile Dye to the First Synthetic Antimalarial
• Heinrich Caro and Paul Guttmann: Two Lives Behind Methylene Blue
• From Dye to Drug: Methylene Blue, Ehrlich’s Staining and the 1891 Malaria Cures
• The Science of Methylene Blue: Coal Tar, Redox Chemistry and the Antidote Years
• Pharmacology
• Paul Ehrlich
• Methylene Blue
• Methylene Blue: Mitochondrial Mechanism
• Methylene Blue: Drug Interactions and Serotonin Syndrome
• Malaria
• G6PD Deficiency
• Alzheimer’s Disease

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