Methylene blue

Methylene blue is used to treat methemoglobinemia by chemically reducing the ferric iron in hemoglobin to ferrous iron.{{cite book |title=British national formulary: BNF 69 |date=2015 |publisher=British Medical Association |isbn=978-0-85711-156-2 |edition=69 |page=34}} Methemoglobinemia can arise from ingestion of certain pharmaceuticals, toxins, or broad beans in those susceptible.{{cite book | vauthors = Thomé SD, Petz LD | chapter = Hemolytic Anemia: Hereditary and Acquired | veditors = Mazza J |title=Manual of Clinical Hematology |date=2002 |publisher=Lippincott Williams & Wilkins |location=Philadelphia |isbn=978-0-7817-2980-2 |page=102 |edition=3rd | chapter-url = https://books.google.com/books?id=NzMKMPzbxZwC&pg=PA102 }} Specifically, it is used to treat methemoglobin levels that are greater than 30% or in which there are symptoms despite oxygen therapy. 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.

{{Cite book| vauthors = Brent J | title =Critical care toxicology: diagnosis and management of the critically poisoned patient.| publisher = Elsevier Health Sciences| year =2005 }} At high doses, however, methylene blue actually induces methemoglobinemia, reversing this pathway.

Isobutyl nitrite is one of the compounds used as poppers, an inhalant drug that induces a brief euphoria.

Isobutyl nitrite is known to cause methemoglobinemia.{{cite journal | vauthors = Taylor GM, Avera RS, Strachan CC, Briggs CM, Medler JP, Pafford CM, Gant TB | title = Severe methemoglobinemia secondary to isobutyl nitrite toxicity: the case of the 'Gold Rush' | journal = Oxford Medical Case Reports | volume = 2021 | issue = 2 | pages = omaa136 | date = February 2021 | pmid = 33614047 | pmc = 7885148 | doi = 10.1093/omcr/omaa136 }} Severe methemoglobinemia may be treated with methylene blue.{{cite journal | vauthors = Modarai B, Kapadia YK, Kerins M, Terris J | title = Methylene blue: a treatment for severe methaemoglobinaemia secondary to misuse of amyl nitrite | journal = Emergency Medicine Journal | volume = 19 | issue = 3 | pages = 270–271 | date = May 2002 | pmid = 11971852 | pmc = 1725875 | doi = 10.1136/emj.19.3.270 }}

{{excerpt|Hyoscyamine/hexamethylenetetramine/phenyl salicylate/methylene blue/benzoic acid|paragraphs=1}}

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 Matilda Moldenhauer Brooks in San Francisco, although first demonstrated by Bo Sahlin of Lund University, in 1926.{{Cite journal | title = Methylene blue as an antidote for cyanide and carbon monoxide poisoning | vauthors = Brooks MM | journal = The Scientific Monthly | volume = 43 | issue = 6 | year = 1936 | pages = 585–586| jstor =16280| bibcode = 1936SciMo..43..585M }}{{cite journal | doi = 10.1001/jama.1933.02740050053028| title = Methylene Blue As Antidote for Cyanide Poisoning| journal = JAMA| volume = 100| issue = 5| page = 357| date = 4 February 1933| vauthors = Hanzlik PJ }}

Methylene blue increases blood pressure in people with vasoplegic syndrome (redistributive shock), but does not improve delivery of oxygen to tissues or decrease mortality.{{cite journal | vauthors = Hosseinian L, Weiner M, Levin MA, Fischer GW | title = Methylene Blue: Magic Bullet for Vasoplegia? | journal = Anesthesia and Analgesia | volume = 122 | issue = 1 | pages = 194–201 | date = January 2016 | pmid = 26678471 | doi = 10.1213/ANE.0000000000001045 | s2cid = 26114442 }}{{cite journal | vauthors = Stawicki SP, Sims C, Sarani B, Grossman MD, Gracias VH | title = Methylene blue and vasoplegia: who, when, and how? | journal = Mini Reviews in Medicinal Chemistry | volume = 8 | issue = 5 | pages = 472–490 | date = May 2008 | pmid = 18473936 | doi = 10.2174/138955708784223477 | url = http://www.bentham-direct.org/pages/content.php?MRMC/2008/00000008/00000005/0006N.SGM | access-date = 2020-04-01 | url-status = usurped | archive-url = https://web.archive.org/web/20170918205928/http://www.bentham-direct.org/pages/content.php?MRMC%2F2008%2F00000008%2F00000005%2F0006N.SGM | archive-date = 2017-09-18 }}

Methylene blue has been used in calcium channel blocker toxicity as a possible rescue therapy for distributive shock unresponsive to first line agents. Limited to case reports, a 2024 review found low-quality evidence that methylene blue may reduce short-term mortality, duration of the need for vasopressors, and length of hospital stay.{{cite journal | vauthors = Fernando SM, Tran A, Soliman K, Flynn B, Oommen T, Wenzhe L, Adhikari NK, Kanji S, Seely AJ, Fox-Robichaud AE, Wax RS, Cook DJ, Lamontagne F, Rochwerg B | title = Methylene Blue in Septic Shock: A Systematic Review and Meta-Analysis | journal = Critical Care Explorations | volume = 6 | issue = 7 | pages = e1110 | date = July 2024 | pmid = 38904978 | pmc = 11196076 | doi = 10.1097/CCE.0000000000001110 }}

File:Human Cheek Cells.jpg

File:Methylene blue Crystals.jpg

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.{{Citation needed|date=January 2021}}

In surgeries such as sentinel lymph node dissections, methylene blue can be used to visually trace the lymphatic drainage of tested 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. In fistulas and pilonidal sinuses, it is used to identify the tract for complete excision.{{citation needed|date=April 2018}} It can also be used during gastrointestinal surgeries (such as bowel resection or gastric bypass) to test for leaks.{{Citation needed|date=January 2021}}

It is sometimes used in cytopathology, in mixtures including Wright-Giemsa and Diff-Quik. It confers a blue color to both nuclei and cytoplasm, and makes the nuclei more visible.{{cite journal | vauthors = Hu X, Laguerre V, Packert D, Nakasone A, Moscinski L | title = A Simple and Efficient Method for Preparing Cell Slides and Staining without Using Cytocentrifuge and Cytoclips | journal = International Journal of Cell Biology | volume = 2015 | issue = | page = 813216 | year = 2015 | pmid = 26664363 | pmc = 4664808 | doi = 10.1155/2015/813216 | doi-access = free }} When methylene blue is "polychromed" (oxidized in solution or "ripened" by fungal metabolism,{{Cite book | vauthors = Kiernan JA | chapter = Chapter 19: On Chemical Reactions and Staining Mechanisms | series = Education Guide | veditors = Kumar GL, Kiernan JA | title = Special Stains and H & E | date = 2010 | edition = Second | location = Carpinteria, California | publisher = Dako North America, Inc. |chapter-url= http://www.dako.com/08066_12may10_webchapter19.pdf |archive-url= https://web.archive.org/web/20120513002342/http://www.dako.com/08066_12may10_webchapter19.pdf | quote = What is Giemsa's stain and how does it color blood cells, bacteria and chromosomes? | page = 172 |archive-date=May 13, 2012}} as originally noted in the thesis of Dr. D. L. Romanowsky in the 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.{{cite journal | vauthors = Wilson TM | title = On the Chemistry and Staining Properties of Certain Derivatives of the Methylene Blue Group When Combined With Eosin | journal = The Journal of Experimental Medicine | volume = 9 | issue = 6 | pages = 645–670 | date = November 1907 | pmid = 19867116 | pmc = 2124692 | doi = 10.1084/jem.9.6.645 }} 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 also enhance the staining of nucleoli and polychromatophilic RBCs (reticulocytes).{{cite book | vauthors = Lewis SM, Bain BK, Bates I, Dacie JV |title=Dacie and Lewis practical haematology |date=2006 |publisher=Churchill Livingstone/Elsevier |location=Philadelphia, PA |isbn=978-0-443-06660-3 |edition=10th | page = 61 }}

File:Gross pathology of normal brain and brain of patient treated with methylene blue before death.jpg

A traditional application of methylene blue is the intravital or supravital staining of nerve fibers, an effect first described by Paul Ehrlich in 1887.{{cite journal | vauthors = Ehrlich P | date = 1887 | title = Ueber die Methylenblau Reaktion der lebenden Nerven Substanz. | trans-title = About the methylene blue reaction of the living nerve substance. | language = German | journal =Biologisches Zentralblatt | trans-journal = Biological Central Journal | volume = 6 | pages = 214–224 | url = https://www.pei.de/SharedDocs/Downloads/DE/institut/veroeffentlichungen-von-paul-ehrlich/1886-1896/1887-ueber-methylenblaureaktion-lebender-nervensubstanz.pdf?__blob=publicationFile&v=2 }} cited by {{cite book | vauthors = Baker JR | title = Principles of biological microtechnique. A study of fixation and dyeing. | date = 1958 | location = London | publisher = Methuen }} 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.{{cite journal | vauthors = Wilson JG | title = Intra vitam staining with methylene blue. | journal = The Anatomical Record | date = July 1910 | volume = 4 | issue = 7 | pages = 267–277 | doi = 10.1002/ar.1090040705 | s2cid = 86242668 | url = https://zenodo.org/record/2478243 }}{{cite journal | vauthors = Schabadasch A | title = Untersuchungen zur Methodik der Methylenblaufärbung des vegetativen Nervensystems. | trans-title = Investigations on the methodology of methylene blue staining of the autonomic nervous system. | language = German | journal = Zeitschrift für Zellforschung und Mikroskopische Anatomie | trans-journal = Journal of Cell Research and Microscopic Anatomy | date = January 1930 | volume = 10 | issue = 2 | pages = 221–243 | doi = 10.1007/BF02450696 | s2cid = 36940327 }}{{cite book | vauthors = Zacks SI | date = 1973 | title = The Motor Endplate | edition = 2nd | location = Huntington, NY | publisher = Krieger }} 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.{{cite journal | vauthors = Kiernan JA | title = Effects of metabolic inhibitors on vital staining with methylene blue | journal = Histochemistry | volume = 40 | issue = 1 | pages = 51–57 | date = June 1974 | pmid = 4136702 | doi = 10.1007/BF00490273 | s2cid = 23158612 }}

Methylene blue is highly lipophilic enabling it to readily cross the blood–brain barrier.{{cite journal |vauthors=Xue H, Thaivalappil A, Cao K |title=The Potentials of Methylene Blue as an Anti-Aging Drug |journal=Cells |volume=10 |issue=12 |page=3379 |date=2021 |doi=10.3390/cells10123379 |doi-access=free |pmc=8699482 |pmid=34943887}} Methylene blue becomes more highly concentrated in the brain than in the plasma and has a high affinity for mitochondria. Many studies have indicated beneficial effects on mitochondrial dysfunction and neurodegenerative disease, which are closely related.

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.{{Cite web| url =http://www.sciencebasedmedicine.org/?p=29| title =The ethics of deception in medicine| access-date =2008-01-24| vauthors = Novella S | work =Science Based Medicine| date =23 January 2008| url-status =live| archive-url =https://web.archive.org/web/20080129230034/http://www.sciencebasedmedicine.org/?p=29| archive-date =2008-01-29}} This same side effect makes methylene blue difficult to use in traditional placebo-controlled clinical studies, including those testing for its efficacy as a treatment. One approach is to use a low dose, just enough to turn urine blue, as the placebo group.{{ClinicalTrialsGov|NCT00214877|Methylene blue for cognitive dysfunction in bipolar disorder}} However, a low dose does not guarantee inertness.

Methylene blue is a monoamine oxidase inhibitor (MAOI)

{{cite journal | vauthors = Ramsay RR, Dunford C, Gillman PK | title = Methylene blue and serotonin toxicity: inhibition of monoamine oxidase A (MAO A) confirms a theoretical prediction | journal = British Journal of Pharmacology | volume = 152 | issue = 6 | pages = 946–951 | date = November 2007 | pmid = 17721552 | pmc = 2078225 | doi = 10.1038/sj.bjp.0707430 }} and, if infused intravenously at doses exceeding 5 mg/kg, may result in serotonin syndrome if combined with any selective serotonin reuptake inhibitors (SSRIs) or other serotonergic drugs (e.g., duloxetine, sibutramine, venlafaxine, clomipramine, imipramine).{{cite journal | vauthors = Gillman PK | title = Methylene blue implicated in potentially fatal serotonin toxicity | journal = Anaesthesia | volume = 61 | issue = 10 | pages = 1013–1014 | date = October 2006 | pmid = 16978328 | doi = 10.1111/j.1365-2044.2006.04808.x | s2cid = 45063314 | doi-access = }}

It causes hemolytic anemia in carriers of the G6PD enzymatic deficiency (favism).{{cite journal | vauthors = Rosen PJ, Johnson C, McGehee WG, Beutler E | title = Failure of methylene blue treatment in toxic methemoglobinemia. Association with glucose-6-phosphate dehydrogenase deficiency | journal = Annals of Internal Medicine | volume = 75 | issue = 1 | pages = 83–86 | date = July 1971 | pmid = 5091568 | doi = 10.7326/0003-4819-75-1-83 }} The actual degree of this danger is a subject of controversy as the association was made based on very few cases. A 2018 meta-analysis on clinical trials against malaria in Africa, where the moderate A minus type of G6PD deficiency is prevalent, show no association between MB and hemolysis in such patients. There was, however, a clinically insignificant reduction in hemoglobin.

While use during pregnancy may harm the baby, not using it in methemoglobinemia is likely more dangerous.

After intravenous administration in humans, methylene blue shows a multiphasic change in concentration, with a terminal half-life of 5.25 hours. The initial disappearance from blood actually reflects its movement into organs, with brain, liver, and bile all showing significantly higher concentrations than blood in rats. The overall area under the curve in oral (dry gelatin capsule) administration is only 6.5% of the AUC for iv administration; judging from rat studies, the significantly altered organ distribution plays a key role in this difference.

Administration as an oral solution (500 mg in 200 mL) greatly increases the bioavailbility to 72.3±23.9%. In this newer study, the terminal half-lives were reported as 18.5±11.8 hours for iv use and 18.3±7.2 for oral use. The t_max for oral use is 2.2 hours, compared to 0.5 hours for iv use.{{cite journal | vauthors = Walter-Sack I, Rengelshausen J, Oberwittler H, Burhenne J, Mueller O, Meissner P, Mikus G | title = High absolute bioavailability of methylene blue given as an aqueous oral formulation | journal = European Journal of Clinical Pharmacology | volume = 65 | issue = 2 | pages = 179–189 | date = February 2009 | pmid = 18810398 | doi = 10.1007/s00228-008-0563-x | url = https://hal.archives-ouvertes.fr/hal-00477926/file/PEER_stage2_10.1007%252Fs00228-008-0563-x.pdf }}

Methylene blue is a formal derivative of phenothiazine. It is a dark green powder that yields a blue solution in water. The hydrated form has 3 molecules of water per unit of methylene blue.

This compound is prepared by oxidation of 4-aminodimethylaniline in the presence of sodium thiosulfate to give the quinonediiminothiosulfonic acid, reaction with dimethylaniline, oxidation to the indamine, and cyclization to give the thiazine:{{cite encyclopedia| vauthors = Berneth H |chapter=Azine Dyes |encyclopedia= Ullmann's Encyclopedia of Industrial Chemistry |year=2012 |publisher=Wiley-VCH |place=Weinheim |doi=10.1002/14356007.a03_213.pub3|isbn=978-3-527-30673-2}}

:File:Synthesis of methylene blue.png

A green electrochemical procedure, using only dimethyl-4-phenylenediamine and sulfide ions has been proposed.{{Cite journal | vauthors = Maleki A, Nematollahi D |date=December 2009 |title=An efficient electrochemical method for the synthesis of methylene blue |url=https://linkinghub.elsevier.com/retrieve/pii/S1388248109004846 |journal=Electrochemistry Communications |language=en |volume=11 |issue=12 |pages=2261–2264 |doi=10.1016/j.elecom.2009.09.040}}

File:Methylene blue absorption spectrum.png (base 10 logarithm). In this dataset a peak absorbance of 1.7 (i.e. 98% of transmitted light absorbed) was observed with 665 nm light passing through 1 cm of 10 micromolar methylene blue solution.]]

The maximum absorption of light is near 670 nm. The specifics of absorption depend on a number of factors, including protonation, adsorption to other materials, and metachromasy - the formation of dimers and higher-order aggregates depending on concentration and other interactions:{{cite journal|title=Visible spectroscopy of methylene blue on hectorite, laponite B, and barasym in aqueous suspension| vauthors = Cenens J, Schoonheydt RA |journal=Clays and Clay Minerals |volume=36 |number=3 |pages=214–224 |year=1988 |doi=10.1346/ccmn.1988.0360302 |bibcode=1988CCM....36..214C | s2cid = 3851037 |doi-access=}}

File:Methylene Blue Redox Reaction.svg

Under reducing conditions, the blue-colored methylene blue cation (MB⁺) gains 1H⁺ and 2e⁻ to become the electrically neutral and colorless leucomethylene blue (LMB).{{cite book |chapter=Methylene Blue | chapter-url = https://publications.iarc.fr/_publications/media/download/6707/581359b111699ead293649c15000080016dce9f3.pdf |title=IARC Monographs on the Evaluation of Carcinogenic Risks to Humans |pages= 155–183 (166) |volume=108|date=2016}} The redox midpoint potential E{{sub|0}}' is +0.01 V.{{cite book | vauthors = Jakubowski H | date = 2016 | chapter = Chapter 8: Oxidation/Phosphorylation B: Oxidative Enzymes | title = Biochemistry Online | chapter-url = https://employees.csbsju.edu/hjakubowski/classes/ch331/oxphos/oloxidativeenzymes.html }}

The redox properties can be seen in a classical demonstration of chemical kinetics in general chemistry, the "blue bottle" experiment. Typically, a solution is made of glucose (dextrose), methylene blue, and sodium hydroxide. Upon shaking the bottle, oxygen oxidizes methylene blue, and the solution turns blue. The dextrose will gradually reduce the methylene blue to its colorless, reduced form. Hence, when the dissolved dextrose is entirely consumed, the solution will turn blue again.

In the mitochondrial electron transport chain reduced methylene blue (MBH₂) directly reduces cytochrome c rather than to oxygen, limiting the formation of superoxide.{{cite journal | vauthors = Atamna H, Nguyen A, Ames BN | title = Methylene blue delays cellular senescence and enhances key mitochondrial biochemical pathways | journal = The FASEB Journal | volume = 22 | issue=3 | pages = 703–712 | date = 2008 |url=https://d1wqtxts1xzle7.cloudfront.net/41715377/fj.07-9610com.full-libre.pdf?1454057116=&response-content-disposition=inline%3B+filename%3DMethylene_blue_delays_cellular_senescenc.pdf&Expires=1743199680&Signature=ItIIdTkGebvWdx5Xp7QM4EAb1FWD6ytz5jmUrmR6r25dQwDj53ZGRMgXjUvkX7xhMJCwYSS8LNqQ0TT5oiLJWgvdQyEAiyMoTkdCLInanRGNP6xW5GhYlLWfwIwILUbKZyKOMF-fjc-PTTtjvZNgyiR3yJIhxLBOfooWCg9i10w4-6qZu5oTulqcwUQmBbSBzdtETJBxRemlydYjJDSMqnpzOUPDfdJsF1OhJJ0kTbWJ3nh18RQvH68~9InEZsCMgP5EYTd64K5dF~ss-uYwnDoDTTDIdr48nWxYo1hfprrS9aLymKlk6OpPW1IZ9Pk4oDS4xg1TPCQ4zK7InhD0RQ__&Key-Pair-Id=APKAJLOHF5GGSLRBV4ZA | doi = 10.1096/fj.07-9610com | doi-access = free | pmid = 17928358 }}{{cite journal | vauthors = Poteet E, Winters A, Yang S | title = Neuroprotective actions of methylene blue and its derivatives | journal = PLOS One | volume = 7 | issue = 10 | pages = e48279 | date = 2012 | doi = 10.1371/journal.pone.0048279 | doi-access = free | pmc = 3485214 | pmid = 23118969 | bibcode = 2012PLoSO...748279P }}

Methylene blue has been shown to directly accept electrons from NADH, NADPH, and FADH₂.

Methylene blue is widely used as a redox indicator in analytical chemistry.{{cite journal | vauthors = Cook AG, Tolliver RM, Williams JE |title=The Blue Bottle experiment revisited: How Blue? How Sweet? |journal=Journal of Chemical Education |date=February 1994 |volume=71 |issue=2 |page=160 |doi=10.1021/ed071p160|bibcode=1994JChEd..71..160C }}{{cite journal | vauthors = Anderson L, Wittkopp SM, Painter CJ, Liegel JJ, Schreiner R, Bell JA, Shakhashiri BZ |title=What is happening when the Blue Bottle bleaches: An Investigation of the methylene blue-catalyzed air oxidation of glucose |journal=Journal of Chemical Education |date=9 October 2012 |volume=89 |issue=11 |pages=1425–1431 |doi=10.1021/ed200511d|bibcode=2012JChEd..89.1425A }}{{cite journal | vauthors = Rajchakit U, Limpanuparb T |title=Rapid Blue Bottle experiment: Autoxidation of benzoin catalyzed by redox indicators |journal=Journal of Chemical Education |date=9 August 2016 |volume=93 |issue=8 |pages=1490–1494 |doi=10.1021/acs.jchemed.6b00018|bibcode=2016JChEd..93.1490R }} Solutions of this substance are blue when in an oxidizing environment, but will turn colorless if exposed to a reducing agent.{{cite journal | vauthors = Harvey EN | title = The relation between the oxygen concentration and rate of reduction of methylene blue by milk | journal = The Journal of General Physiology | volume = 1 | issue = 4 | pages = 415–419 | date = March 1919 | pmid = 19871757 | pmc = 2140316 | doi = 10.1085/jgp.1.4.415 }}

Methylene blue is also a photosensitizer used to create singlet oxygen when exposed to both oxygen and light. It is used in this regard to make organic peroxides by a Diels-Alder reaction which is spin forbidden with normal atmospheric triplet oxygen.{{Citation needed|date=January 2021}}

With the help of light, methylene blue can be used to kill some viruses and some bacteria.{{cite web |title=Methylene Blue: The Little-Known Disinfectant |url=https://asm.org/articles/2022/march/methylene-blue-the-little-known-disinfectant |website=ASM.org |language=en}} This kind of photo-disinfection has also been done inside of human bodies (antimicrobial photodynamic therapy).{{cite journal | vauthors = Boltes Cecatto R, Siqueira de Magalhães L, Fernanda Setúbal Destro Rodrigues M, Pavani C, Lino-Dos-Santos-Franco A, Teixeira Gomes M, Fátima Teixeira Silva D | title = Methylene blue mediated antimicrobial photodynamic therapy in clinical human studies: The state of the art | journal = Photodiagnosis and Photodynamic Therapy | volume = 31 | page = 101828 | date = September 2020 | pmid = 32473398 | doi = 10.1016/j.pdpdt.2020.101828 }} The same process can also be used to disinfect blood plasma.{{cite journal | vauthors = Lachert E |title=Methods of pathogen inactivation in whole blood and red blood cells: current state of knowledge |journal=Acta Haematologica Polonica |date=31 August 2021 |volume=52 |issue=4 |pages=406–411 |doi=10.5603/AHP.2021.0076 |url=https://journals.viamedica.pl/acta_haematologica_polonica/article/download/AHP.2021.0076/63958|doi-access=free }}

Methylene blue is theoretically also applicable to other forms of photodynamic therapy, i.e. the use of oxygen, light, and a photosentizer to kill cells. Research on using it to locally kill cancer cells is in a preclinical stage.{{cite journal | vauthors = Taldaev A, Terekhov R, Nikitin I, Melnik E, Kuzina V, Klochko M, Reshetov I, Shiryaev A, Loschenov V, Ramenskaya G | title = Methylene blue in anticancer photodynamic therapy: systematic review of preclinical studies | journal = Frontiers in Pharmacology | volume = 14 | page = 1264961 | date = 28 September 2023 | pmid = 37841915 | pmc = 10568458 | doi = 10.3389/fphar.2023.1264961 | doi-access = free }}

The formation of methylene blue after the reaction of hydrogen sulfide with dimethyl-p-phenylenediamine and iron(III) at pH 0.4 – 0.7 is used to determine by photometric measurements sulfide concentration in the range 0.020 to 1.50 mg/L (20 ppb to 1.5 ppm).{{cite journal | vauthors = Siegel LM | title = A direct microdetermination for sulfide | journal = Analytical Biochemistry | volume = 11 | issue = 1 | pages = 126–132 | date = April 1965 | pmid = 14328633 | doi = 10.1016/0003-2697(65)90051-5 }} The test is very sensitive and the blue coloration developing upon contact of the reagents with dissolved H₂S is stable for 60 min. Ready-to-use kits such as the Spectroquant sulfide test{{cite web|url=http://photometry.merck.de/|title=Analytik und Probenvorbereitung|archive-url=https://web.archive.org/web/20070315204844/http://photometry.merck.de/|archive-date=2007-03-15 | publisher = Merck KGaA | location = Darmstadt, Germany }} facilitate routine analyses. The methylene blue sulfide test is a convenient method often used in soil microbiology to quickly detect in water the metabolic activity of sulfate reducing bacteria (SRB). In this colorimetric test, methylene blue is a product formed by the reaction and not a reagent added to the system.

The addition of a strong reducing agent, such as ascorbic acid, to a sulfide-containing solution is sometimes used to prevent sulfide oxidation from atmospheric oxygen. Although it is certainly a sound precaution for the determination of sulfide with an ion selective electrode, it might however hamper the development of the blue color if the freshly formed methylene blue is also reduced, as described here above in the paragraph on redox indicator.

Methylene blue is a dye behaving as a redox indicator that is commonly used in the food industry to test the freshness of milk and dairy products.{{cite journal | vauthors = Thornton H |title=Studies on Oxidation-Reduction in Milk: The Methylene Blue Reduction Test |journal=Journal of Dairy Science |date=May 1930 |volume=13 |issue=3 |pages=221–245 |publisher=Elsevier Inc. |doi=10.3168/jds.S0022-0302(30)93520-5 |doi-access=free }} A few drops of methylene blue solution added to a sample of milk should remain blue (oxidized form in the presence of enough dissolved {{O2}}), otherwise (discoloration caused by the reduction of methylene blue into its colorless reduced form) the dissolved {{O2}} concentration in the milk sample is low indicating that the milk is not fresh (already abiotically oxidized by {{O2}} whose concentration in solution decreases) or could be contaminated by bacteria also consuming the atmospheric {{O2}} dissolved in the milk. In other words, aerobic conditions should prevail in fresh milk and methylene blue is simply used as an indicator of the dissolved oxygen remaining in the milk.

{{Further|MBAS assay}}

The adsorption of methylene blue serves as an indicator defining the adsorptive capacity of granular activated carbon in water filters. Adsorption of methylene blue is very similar to adsorption of pesticides from water, this quality makes methylene blue serve as a good predictor for filtration qualities of carbon. It is as well a quick method of comparing different batches of activated carbon of the same quality.

A color reaction in an acidified, aqueous methylene blue solution containing chloroform can detect anionic surfactants in a water sample. Such a test is known as an MBAS assay (methylene blue active substances assay).

The MBAS assay cannot distinguish between specific surfactants, however. Some examples of anionic surfactants are carboxylates, phosphates, sulfates, and sulfonates.{{citation needed|date=July 2022}}

The methylene blue value is defined as the number of milliliter's standard methylene value solution decolorized 0.1 g of activated carbon (dry basis).{{cite web | title = Test Methods for Activated Carbon | url = https://activatedcarbon.org/images/Test_method_for_Activated_Carbon_86.pdf | work = European Council of Chemical Manufacturers' Federations | date = April 1986 }}

Methylene blue value reflects the amount of clay minerals in aggregate samples.{{cite report |url=http://www.astm.org/Standards/C1777.htm| id = ASTM C1777 | title = Standard Test Method for Rapid Determination of the Methylene Blue Value for Fine Aggregate or Mineral Filler Using a Colorimeter | date = 7 July 2020 | location = West Conshohocken, PA | publisher = ASTM (American Society for Testing and Material) International |url-status=live |archive-url= https://web.archive.org/web/20140228153812/http://www.astm.org/Standards/C1777.htm |archive-date=2014-02-28 | doi = 10.1520/C1777-20 }} In materials science, methylene blue solution is successively added to fine aggregate which is being agitated in water. The presence of free dye solution can be checked with stain test on a filter paper.{{Cite web | author = Standing Committee on Concrete Technology (SCCT) |url= https://www.cedd.gov.hk/filemanager/eng/content_78/cs3_2013_original.pdf |title=Construction Standard CS3:2013 – Aggregates for Concrete| publisher = The Government of the Hong Kong Special Administrative Region | date = May 2013 }}

{{cleanup section|reason=Duplicate of preceding stain section, just broader and "less medical".|date=February 2025}}

In biology, methylene blue is used as a dye for a number of different staining procedures, such as Wright's stain and Jenner's stain. Since it is a temporary staining technique, methylene blue can also be used to examine RNA or DNA under the microscope or in a gel: as an example, a solution of methylene blue can be used to stain RNA on hybridization membranes in northern blotting to verify the amount of nucleic acid present. While methylene blue is not as sensitive as ethidium bromide, it is less toxic and it does not intercalate in nucleic acid chains, thus avoiding interference with nucleic acid retention on hybridization membranes or with the hybridization process itself.{{Citation needed|date=January 2021}}

It can also be used as an indicator to determine whether eukaryotic cells such as yeast are alive or dead. The methylene blue is reduced in viable cells, leaving them unstained. However dead cells are unable to reduce the oxidized methylene blue and the cells are stained blue. Methylene blue can interfere with the respiration of the yeast as it picks up hydrogen ions made during the process.{{Citation needed|date=January 2021}}

• Methylene blue is used in aquaculture and by tropical fish hobbyists as a treatment for fungal infections. With the help of light it is also effective against bacteria and viruses.
• It can also be effective in treating fish infected with the parasitic protozoa Ichthyophthirius multifiliis (ich), although a combination of malachite green and formaldehyde is far more effective against it.
• Methylene blue also works against nitrite poisoning as it treats the resulting methemoglobinemia. Like in humans, it also treats cyanide poisoning.
• Non-professional sources also claim that it works for ammonia poisoning,{{cite web |title=Methylene Blue as an Anti-Fungal & Anti-Parasitic Drug for Aquarium Fish |url=https://www.thesprucepets.com/methylene-blue-1379926 |website=The Spruce Pets |language=en}} but there is little medical literature to back this up.

It is usually used to protect newly laid fish eggs from being infected by fungus. This is useful when the hobbyist wants to artificially hatch the fish eggs.{{Cite web | title = Methylene Blue |url=https://www.3lfish.com/fish-medicants/#methylene-blue | work = 3 Little Fish Sdn Bhd |date=10 September 2021 | location = Kelana Jaya, Selangor, Malaysia }} For poisoning, injury (prevention of infection), or sickness, methylene blue is given as a "medicated bath" for the fish.

Methylene blue is not without side effects to fish.{{cite journal | vauthors = Nipu N, Wei L, Hamilton L, Lee H, Thomas J, Mennigen JA | title = Methylene blue at recommended concentrations alters metabolism in early zebrafish development | journal = Communications Biology | volume = 8 | issue = 1 | page = 120 | date = January 2025 | pmid = 39856203 | pmc = 11760885 | doi = 10.1038/s42003-025-07471-8 }}

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.Heinrich Caro was an employee of the Badische Anilin- und Sodafabrik, BASF, of Mannheim, Germany, which received a patent for methylene blue in 1877:

• Badische Anilin- und Sodafabrik, BASF, of Mannheim, Germany, "Verfahren zur Darstellung blauer Farbstoffe aus Dimethylanilin und anderen tertiaren aromatischen Monaminen" [Method for preparation of blue dyes from dimethylaniline and other tertiary aromatic monoamines], Deutsches Reich Patent no. 1886 (issued: December 15, 1877).
• Available on-line at: {{cite book | vauthors = Friedlaender P |title=Fortschritte der Theerfarbenfabrikation und verwandter Industriezweige |trans-title=Progress of the manufacture of coal-tar dyes and related branches of industry |date=1888 |publisher=Julius Springer |location=Berlin, Germany |volume=1 |pages=247–249 |url=https://books.google.com/books?id=3akMAAAAYAAJ&pg=PA247 |language=German |access-date=2016-10-12 |archive-date=2015-03-21 |archive-url=https://web.archive.org/web/20150321021246/http://books.google.com/books?id=3akMAAAAYAAJ&pg=PA247 }}

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. Changing the cell membrane of pathogens is in fact how various drugs work, so the theory was partially correct, although far from complete. 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."{{citation needed|date=February 2025}}

It was discovered to be an antidote to carbon monoxide poisoning and cyanide poisoning in 1933 by Matilda Brooks.{{cite journal | doi = 10.1001/jama.1933.02740010061028| title = Methylene Blue As Antidote for Cyanide and Carbon Monoxide Poisoning| journal = JAMA| volume = 100| page = 59| date = January 1933| vauthors = Brooks MM }}

Methylene blue was the original prototype or lead compound for the design of many antimalarials including chloroquine, antihistamines, and antipsychotics including chlorpromazine.{{cite journal | vauthors = Wainwright M |title=Dyes in the development of drugs and pharmaceuticals |journal=Dyes and Pigments |date=January 2008 |volume=76 |issue=3 |pages=582–589 |doi=10.1016/j.dyepig.2007.01.015}}

Antimalarial use of the drug has recently (2009) been revived.{{cite journal | vauthors = Coulibaly B, Zoungrana A, Mockenhaupt FP, Schirmer RH, Klose C, Mansmann U, Meissner PE, Müller O | title = Strong gametocytocidal effect of methylene blue-based combination therapy against falciparum malaria: a randomised controlled trial | journal = PLOS ONE | volume = 4 | issue = 5 | pages = e5318 | year = 2009 | pmid = 19415120 | pmc = 2673582 | doi = 10.1371/journal.pone.0005318 | doi-access = free | bibcode = 2009PLoSO...4.5318C }} It simultaneously targets many biological processes in the apicomplexan pathogen{{cite journal | vauthors = Calderón M, Weitzel T, Rodriguez MF, Ciapponi A |title=Methylene blue for treating malaria |journal=Cochrane Database of Systematic Reviews (Protocol) |date=20 October 2017 |volume=2022 |issue=6 |pages=CD012837 |doi=10.1002/14651858.CD012837 |pmc=6485827}} though the main mechanism seems to be causing a lethal amount of redox cycling.

A 2018 meta-analysis finds that it has proven effective against P. falciparum in Africa. It effectively reduces levels of the transmission-stage gametocyte and has synergy with the standard artemisinin-based combination therapy (ACT). Its effects against other malarial species and P. falciparum populations in other locations are unclear.{{cite journal | vauthors = Lu G, Nagbanshi M, Goldau N, Mendes Jorge M, Meissner P, Jahn A, Mockenhaupt FP, Müller O | title = Efficacy and safety of methylene blue in the treatment of malaria: a systematic review | journal = BMC Medicine | volume = 16 | issue = 1 | page = 59 | date = April 2018 | pmid = 29690878 | pmc = 5979000 | doi = 10.1186/s12916-018-1045-3 | doi-access = free }}

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.

{{cite journal | vauthors = Alici-Evcimen Y, Breitbart WS | title = Ifosfamide neuropsychiatric toxicity in patients with cancer | journal = Psycho-Oncology | volume = 16 | issue = 10 | pages = 956–960 | date = October 2007 | pmid = 17278152 | doi = 10.1002/pon.1161 | s2cid = 27433170 | doi-access = free }}{{cite journal | vauthors = Abahssain H, Moukafih B, Essangri H, Mrabti H, Meddah B, Guessous F, Fadhil FZ, Souadka A, Errihani H | title = Methylene blue and ifosfamide-induced encephalopathy: Myth or reality? | journal = Journal of Oncology Pharmacy Practice | volume = 27 | issue = 1 | pages = 143–149 | date = January 2021 | pmid = 33153383 | doi = 10.1177/1078155220971843 }}

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.

{{cite journal | vauthors = Patel PN | title = Methylene blue for management of Ifosfamide-induced encephalopathy | journal = The Annals of Pharmacotherapy | volume = 40 | issue = 2 | pages = 299–303 | date = February 2006 | pmid = 16391008 | doi = 10.1345/aph.1G114 | s2cid = 21124635 }} Alternatively, it has been suggested that intravenous methylene blue every six hours for prophylaxis during ifosfamide treatment in people with history of ifosfamide neuropsychiatric toxicity.

{{cite journal | vauthors = Dufour C, Grill J, Sabouraud P, Behar C, Munzer M, Motte J, Oberlin O, Paci A, Hartmann O | title = [Ifosfamide induced encephalopathy: 15 observations] | language = fr | journal = Archives de Pédiatrie | volume = 13 | issue = 2 | pages = 140–145 | date = February 2006 | pmid = 16364615 | doi = 10.1016/j.arcped.2005.10.021 }} 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.

{{cite journal | vauthors = Aeschlimann C, Cerny T, Küpfer A | title = Inhibition of (mono)amine oxidase activity and prevention of ifosfamide encephalopathy by methylene blue | journal = Drug Metabolism and Disposition | volume = 24 | issue = 12 | pages = 1336–1339 | date = December 1996 | pmid = 8971139 | doi = 10.1016/S0090-9556(25)08471-5 }}

Methylene blue inhibits monoamine oxidase, inhibits the glutamatergic system (via inhibition of NO synthase and soluble guanylate cyclase), modulates mitochondrial function (by acting as an electron acceptor), and decreases the activation of inflammasomes NLRP3 and NLRC4. As a result, it's been considered potentially useful in neuropsychiatric disorders. In humans it has been tried for (listed in decreasing order of evidence quality): bipolar disorder (especially depressive symptoms), Alzheimer's disease, claustrophobia, ifosfamide encephalopathy, and schizophrenia. With methylene blue, a higher dose does not necessarily work better than a lower dose.{{cite journal | vauthors = Alda M | title = Methylene Blue in the Treatment of Neuropsychiatric Disorders | journal = CNS Drugs | volume = 33 | issue = 8 | pages = 719–725 | date = August 2019 | pmid = 31144270 | doi = 10.1007/s40263-019-00641-3 }}

In the late 2010s and early 2020s, a social media trend emerged promoting the use of methylene blue for various medical purposes, including anti-aging, metabolism enhancement, cognitive improvement, cancer treatment, and COVID-19 treatment.{{Cite web | vauthors = Whitehead J |date=2022-03-18 |title=Medical expert issues warning about fish tank disinfectant craze |url=https://www.the-independent.com/life-style/health-and-families/fish-tank-cleaner-anti-aging-doctor-methylene-blue-b2038679.html |access-date=2025-03-12 |website=The Independent |language=en}}{{Cite web | vauthors = Tiller N |date=2022-04-20 |title=When Medicines Go Rogue, Part 1: Methylene Blue |url=https://skepticalinquirer.org/exclusive/when-medicines-go-rogue-methylene-blue/ |access-date=2025-03-12 |website=Skeptical Inquirer |language=en-US}}{{Cite web | vauthors = Liddell J |date=2025-02-06 |title=What was RFK Jr putting in his drink mid-flight? |url=https://www.independent.co.uk/news/world/americas/us-politics/rfk-jr-drinking-flight-methylene-blue-b2693495.html |access-date=2025-03-12 |website=The Independent |language=en}}{{Cite web | vauthors = Stebbing J |date=2025-01-27 |title=From methylene blue to vitamin E, here's why health and wellness supplements are no silver bullet for cancer |url=https://theconversation.com/from-methylene-blue-to-vitamin-e-heres-why-health-and-wellness-supplements-are-no-silver-bullet-for-cancer-247847 |access-date=2025-03-12 |website=The Conversation |language=en-US}} Currently there is no scientific consensus on, and no FDA approval for, its effectiveness and safety for these purposes. Medical experts cautioned that methylene blue can be toxic in high doses and may interact with other medications, potentially reducing their effectiveness or causing unforeseen side effects. Therefore, it should only be used under a doctor's prescription.

This trend probably started following the publication of a few scientific papers exploring the potential of methylene blue for treating some medical conditions, such as progeria,{{cite journal | vauthors = Xiong ZM, Choi JY, Wang K, Zhang H, Tariq Z, Wu D, Ko E, LaDana C, Sesaki H, Cao K | title = Methylene blue alleviates nuclear and mitochondrial abnormalities in progeria | journal = Aging Cell | volume = 15 | issue = 2 | pages = 279–290 | date = April 2016 | pmid = 26663466 | pmc = 4783354 | doi = 10.1111/acel.12434 }} and skin aging.{{cite journal | vauthors = Xiong ZM, O'Donovan M, Sun L, Choi JY, Ren M, Cao K | title = Anti-Aging Potentials of Methylene Blue for Human Skin Longevity | journal = Scientific Reports | volume = 7 | issue = 1 | page = 2475 | date = May 2017 | pmid = 28559565 | pmc = 5449383 | doi = 10.1038/s41598-017-02419-3 | bibcode = 2017NatSR...7.2475X }} It was also explored as part of anticancer photodynamic therapy using lasers.{{cite journal | vauthors = Taldaev A, Terekhov R, Nikitin I, Melnik E, Kuzina V, Klochko M, Reshetov I, Shiryaev A, Loschenov V, Ramenskaya G | title = Methylene blue in anticancer photodynamic therapy: systematic review of preclinical studies | journal = Frontiers in Pharmacology | volume = 14 | page = 1264961 | date = 2023-09-28 | pmid = 37841915 | pmc = 10568458 | doi = 10.3389/fphar.2023.1264961 | doi-access = free }} One systematic review of the studies expresses optimism but emphasizes the need for more extensive research to confirm methylene blue's clinical applications.{{cite journal | vauthors = Xue H, Thaivalappil A, Cao K | title = The Potentials of Methylene Blue as an Anti-Aging Drug | journal = Cells | volume = 10 | issue = 12 | page = 3379 | date = December 2021 | pmid = 34943887 | pmc = 8699482 | doi = 10.3390/cells10123379 | doi-access = free }} Another review takes a more critical stance, stating that "it is obvious that the clinical use of MB represents a rather controversial problem given the heterogeneity of available data and the lack of preclinical data, which is in conflict with standards of safe use of such substances in human medicinal practice".{{cite journal | vauthors = Bužga M, Machytka E, Dvořáčková E, Švagera Z, Stejskal D, Máca J, Král J | title = Methylene blue: a controversial diagnostic acid and medication? | journal = Toxicology Research | volume = 11 | issue = 5 | pages = 711–717 | date = October 2022 | pmid = 36337249 | pmc = 9618115 | doi = 10.1093/toxres/tfac050 }}

In January 2025, Robert F. Kennedy Jr., then the U.S. health secretary nominee, was filmed adding droplets of an unidentified blue liquid to his drink during a flight. While many have speculated that it was methylene blue, Kennedy has not addressed the claims.{{Cite news | vauthors = Mahdawi A |date=2025-02-11 |title=Did RFK Jr really drink fish medicine? He definitely has weird ideas about 'making America healthy again' |url=https://www.theguardian.com/commentisfree/2025/feb/11/did-rfk-jr-really-drink-fish-medicine-definitely-has-weird-ideas-making-america-healthy-again |access-date=2025-03-12 |work=The Guardian |language=en-GB |issn=0261-3077}}{{Cite news |date=2025-02-06 |title=Robert F. Kennedy Jr.'s viral video sparks mystery over blue liquid. What Is Methylene Blue? |url=https://economictimes.indiatimes.com/magazines/panache/rfk-jr-s-viral-video-sparks-mystery-over-blue-liquid-what-is-methylene-blue/articleshow/117987350.cms?from=mdr |access-date=2025-03-12 |work=The Economic Times |issn=0013-0389}}

{{Reflist}}

• {{cite web | url = https://druginfo.nlm.nih.gov/drugportal/name/methylthioninium%20chloride | publisher = U.S. National Library of Medicine | work = Drug Information Portal | title = Methylene blue }}
• {{cite web | title=Methylene blue test | website=MedlinePlus | url=https://medlineplus.gov/ency/article/003412.htm }}

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