Methylthioninium chloride, commonly called methylene blue, is a salt used as a dye and as a medication. As a medication, it is mainly used to treat methemoglobinemia. It has previously been used for treating cyanide poisoning and urinary tract infections, but this use is no longer recommended.[
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Methylene blue is typically given by intravenous.
Methylene blue was first prepared in 1876, by Heinrich Caro.
Medical uses
Methemoglobinemia
Methylene blue is used to treat methemoglobinemia by redox the ferric iron in hemoglobin to ferrous iron.[
] At high doses, however, methylene blue actually induces methemoglobinemia, reversing this pathway.
Isobutyl nitrite toxicity
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.
In a combination drug: Methylphen
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 Matilda Moldenhauer Brooks in San Francisco,
Shock
Methylene blue increases blood pressure in people with vasoplegic syndrome (redistributive shock). It does not improve delivery of oxygen to tissues or decrease mortality.
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 , 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.
Dye or stain
Methylene blue is used in colonoscopy 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 is a high risk for perforation. Methylene blue is also used as a dye in endoscopy, and is sprayed onto the mucosa of the gastrointestinal tract 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 .
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 several medical devices, including a surgical sealant film, TissuePatch. In fistulas and , it is used to identify the tract for complete excision. It can also be used during gastrointestinal surgeries (such as bowel resection or gastric bypass) to test for leaks.
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.
A traditional application of methylene blue is the intravital or supravital staining of nerve fibers, an effect first described by Paul Ehrlich in 1887.[ cited by ] 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.
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.[
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Side effects
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Staining of skin
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Injection site necrosis (SC)
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Discoloration of urine (doses over 80 μg)
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Bladder irritation
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Methylene blue is a monoamine oxidase inhibitor (MAOI)[
] 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).
It causes hemolytic anemia in carriers of the G6PD enzymatic deficiency (favism).[
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Pregnancy
While use during pregnancy may harm the baby, not using it in methemoglobinemia is likely more dangerous.
Pharmacokinetics
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 bioavailability 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 hours for oral use. The tmax for oral use is 2.2 hours, compared to 0.5 hours for iv use.
Chemistry
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.
Preparation
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:
A green electrochemical procedure, using only dimethyl-4-phenylenediamine and sulfide ions has been proposed.
Light absorption properties
The maximum absorption of light is near 670 nm. The specifics of absorption depend on several factors, including protonation, adsorption to other materials, and metachromasy – the formation of dimers and higher-order aggregates depending on concentration and other interactions:
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| MB+ (solution) | 664 | 95000 |
| MBH2+ (solution) | 741 | 76000 |
| (MB+)2 (solution) | 605 | 132000 |
| (MB+)3 (solution) | 580 | 110000 |
| MB+ (adsorbed on clay) | 673 | 116000 |
| MBH2+ (adsorbed on clay) | 763 | 86000 |
| (MB+)2 (adsorbed on clay) | 596 | 80000 |
| (MB+)3 (adsorbed on clay) | 570 | 114000 |
Redox properties
Under reducing conditions, the blue-colored methylene blue cation (MB+) gains 1H+ and 2e− to become the electrically neutral and colorless leucomethylene blue (LMB).
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 Mitochondrion electron transport chain, reduced methylene blue (MBH2) directly reduces cytochrome c rather than to oxygen, limiting the formation of superoxide.
Other uses
Redox indicator
Methylene blue is widely used as a redox indicator in analytical chemistry.
Photosensitizer
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.
With the help of light, methylene blue can be used to kill some viruses and some bacteria.
Methylene blue is theoretically also applicable to other forms of photodynamic therapy, i.e., the use of oxygen, light, and a photosensitizer to kill cells. Research on using it to kill cancer cells locally is in a preclinical stage.
Sulfide analysis
The formation of methylene blue after the reaction of hydrogen sulfide with dimethyl-p-phenylenediamine and Ferric 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).
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.
Test for milk freshness
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.
Water testing
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 also 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 , , , and .
Methylene blue value of fine aggregate
The methylene blue value is defined as the number of milliliters of standard methylene blue solution decolorized 0.1 g of activated carbon (dry basis).
Biological staining
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 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.
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.
Aquaculture
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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.
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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.
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Methylene blue also works against nitrite poisoning as it treats the resulting methemoglobinemia. Like in humans, it also treats cyanide poisoning.
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Non-professional sources also claim that it works for ammonia poisoning,
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.
Methylene blue is not without side effects to fish.
History
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:
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Badische Anilin- und Sodafabrik, BASF, of Mannheim, Germany, "Verfahren zur Darstellung blauer Farbstoffe aus Dimethylanilin und anderen tertiaren aromatischen Monaminen" Method, Deutsches Reich Patent no. 1886 (issued: December 15, 1877).
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Available on-line at:
Its use in the treatment of malaria was pioneered by Paul Guttmann and Paul Ehrlich in 1891. During this period before World War I, 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 how various drugs work, so the theory was partially correct, although far from complete. Methylene blue continued to be used in World War II, where it was not well-liked by soldiers, who observed, "Even at the loo, we see, we pee, navy blue."
It was discovered to be an antidote to carbon monoxide poisoning and cyanide poisoning in 1933 by Matilda Brooks.
Methylene blue was the original prototype or lead compound for the design of many antimalarials including chloroquine, antihistamines, and antipsychotics including chlorpromazine.
Research
Malaria
Antimalarial use of the drug has recently (2009) been revived.[
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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.
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.[
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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, has resulted in improvement of symptoms within 10 minutes to several days.[
] 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.[
] 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.[
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Neuropsychiatric disorders
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 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.
Society and culture
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.
This trend probably started following the publication of a few scientific papers exploring the potential of methylene blue for treating some medical conditions,
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.
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