 Molecular structure | |
 A volumetric flask of a methylene blue solution | |
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| Trade names | Urelene blue, Provayblue, Proveblue, others[1][2] |
| Other names | CI 52015, basic blue 9[3] |
| AHFS/Drugs.com | Monograph |
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| Routes of administration | By mouth,intravenous |
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| Eliminationhalf-life | 5–24 hours (IVTooltip intravenous therapy)[5] |
| Excretion | Renal In rats: 18%(POTooltip Oral administration), 28% (IV)[8] |
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| CompTox Dashboard(EPA) | |
| ECHA InfoCard | 100.000.469 |
| Chemical and physical data | |
| Formula | C16H18ClN3S |
| Molar mass | 319.85 g·mol−1 |
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Methylthioninium chloride, commonly calledmethylene blue, is asalt used as a dye and as a medication. As a medication, it is mainly used to treatmethemoglobinemia. It has previously been used for treatingcyanide poisoning andurinary tract infections, but this use is no longer recommended.[5]
Methylene blue is typically given byinjection into a vein.[5] Common side effects includeheadache,nausea, andvomiting.
Methylene blue was first prepared in 1876, byHeinrich Caro.[9] It is on theWorld Health Organization's List of Essential Medicines.[10]
Methylene blue is used to treatmethemoglobinemia bychemically reducing theferric iron inhemoglobin toferrous iron.[5][2] Methemoglobinemia can arise from ingestion of certain pharmaceuticals,toxins, orbroad beans in those susceptible.[11] Specifically, it is used to treat methemoglobin levels that are greater than 30% or in which there are symptoms despiteoxygen therapy.[2] Normally, through theNADH- orNADPH-dependentmethemoglobin 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 theheme group frommethemoglobin tohemoglobin. Methylene blue can reduce the half life of methemoglobin from hours to minutes.[12] At high doses, however, methylene blue actually induces methemoglobinemia, reversing this pathway.[12]
Isobutyl nitrite is one of the compounds used aspoppers, aninhalant drug that induces a briefeuphoria.
Isobutyl nitrite is known to causemethemoglobinemia.[13] Severe methemoglobinemia may be treated with methylene blue.[14]
Since its reduction potential is similar to that of oxygen and can be reduced by components of theelectron transport chain, large doses of methylene blue are sometimes used as an antidote topotassium cyanide poisoning, a method first successfully tested in 1933 byMatilda Moldenhauer Brooks in San Francisco,[17] although first demonstrated by Bo Sahlin ofLund University, in 1926.[17][18]
Methylene blue increases blood pressure in people withvasoplegic syndrome (redistributive shock), but does not improve delivery of oxygen to tissues or decrease mortality.[19][20]
Methylene blue has been used incalcium channel blocker toxicity as a possible rescue therapy for distributive shock unresponsive to first line agents. Limited tocase 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.[21]
Methylene blue is used inendoscopicpolypectomy as an adjunct tosaline orepinephrine, and is used for injection into thesubmucosa around thepolyp 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 inchromoendoscopy, and is sprayed onto the mucosa of thegastrointestinal tract in order to identifydysplasia, or pre-cancerous lesions. Intravenously injected methylene blue is readily released into the urine and thus can be used to test theurinary tract for leaks orfistulas.[citation needed]
In surgeries such assentinel lymph node dissections, methylene blue can be used to visually trace the lymphatic drainage of tested tissues. Similarly, methylene blue is added tobone 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 asurgical sealant film, TissuePatch. In fistulas andpilonidal sinuses, it is used to identify the tract for complete excision.[citation needed] It can also be used during gastrointestinal surgeries (such asbowel resection orgastric bypass) to test for leaks.[citation needed]
It is sometimes used incytopathology, in mixtures includingWright-Giemsa andDiff-Quik. It confers a blue color to both nuclei and cytoplasm, and makes the nuclei more visible.[22] When methylene blue is "polychromed" (oxidized in solution or "ripened" by fungal metabolism,[23] 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 areAzure B,Azure A,Azure C, andthionine, respectively.[24] This is the basis of the basophilic part of the spectrum ofRomanowski-Giemsa effect. If only synthetic Azure B andEosin Y is used, it may serve as a standardizedGiemsa 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).[25]
A traditional application of methylene blue is the intravital or supravital staining of nerve fibers, an effect first described byPaul Ehrlich in 1887.[26] 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 ofammonium molybdate. Vital methylene blue was formerly much used for examining the innervation of muscle, skin and internal organs.[27][28][29] The mechanism of selective dye uptake is incompletely understood; vital staining of nerve fibers in skin is prevented byouabain, a drug that inhibits the Na/K-ATPase of cell membranes.[30]
Methylene blue has been used as aplacebo; physicians would tell their patients to expect their urine to change color and view this as a sign that their condition had improved.[31] This same side effect makes methylene blue difficult to use in traditional placebo-controlledclinical 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.[32] However, a low dose does not guarantee inertness.[33]
| Cardiovascular[34][35] | Central nervous system[34][35] | Dermatologic[34][35] | Gastrointestinal[34][35] | Genito-urinary[34][35] | Hematologic[34][35] |
|---|---|---|---|---|---|
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Methylene blue is amonoamine oxidase inhibitor (MAOI)[36] and, if infused intravenously at doses exceeding 5 mg/kg, may result inserotonin syndrome if combined with anyselective serotonin reuptake inhibitors (SSRIs) or other serotonergic drugs (e.g.,duloxetine,sibutramine,venlafaxine,clomipramine,imipramine).[37]
It causeshemolytic anemia in carriers of theG6PD enzymatic deficiency (favism).[38] 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.[39]
While use duringpregnancy may harm the baby, not using it in methemoglobinemia is likely more dangerous.[5][2]
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 overallarea 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.[8]
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 tmax for oral use is 2.2 hours, compared to 0.5 hours for iv use.[40]
Methylene blue is a formal derivative ofphenothiazine. It is a dark green powder that yields a blue solution inwater. 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 ofsodium thiosulfate to give the quinonediiminothiosulfonic acid, reaction with dimethylaniline, oxidation to the indamine, and cyclization to give the thiazine:[41]
A green electrochemical procedure, using onlydimethyl-4-phenylenediamine and sulfide ions has been proposed.[42]
The maximum absorption of light is near 670 nm. The specifics of absorption depend on a number of factors, includingprotonation,adsorption to other materials, andmetachromasy - the formation ofdimers and higher-order aggregates depending on concentration and other interactions:[43]
| Species | Absorption peak | Extinction coefficient (dm3/mol·cm) |
|---|---|---|
| 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 |
Under reducing conditions, the blue-colored methylene blue cation (MB+) gains 1H+ and 2e− to become the electrically neutral and colorlessleucomethylene blue (LMB).[44] The redox midpoint potential E0' is +0.01 V.[45]
The redox properties can be seen in a classical demonstration ofchemical kinetics in general chemistry, the "blue bottle" experiment. Typically, a solution is made ofglucose (dextrose), methylene blue, andsodium 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.[46]
In themitochondrialelectron transport chain reduced methylene blue (MBH2) directly reducescytochrome c rather than to oxygen, limiting the formation ofsuperoxide.[47][48] Methylene blue has been shown to directly accept electrons fromNADH,NADPH, andFADH2.[48]
Methylene blue is widely used as aredox indicator inanalytical chemistry.[46][49][50] Solutions of this substance are blue when in an oxidizing environment, but will turn colorless if exposed to a reducing agent.[51][49]
Methylene blue is also aphotosensitizer used to createsinglet oxygen when exposed to both oxygen and light. It is used in this regard to make organicperoxides by aDiels-Alder reaction which isspin forbidden with normal atmospherictriplet oxygen.[citation needed]
With the help of light, methylene blue can be used to kill some viruses and some bacteria.[52] This kind of photo-disinfection has also been done inside of human bodies (antimicrobial photodynamic therapy).[53] The same process can also be used to disinfect blood plasma.[54]
Methylene blue is theoretically also applicable to other forms ofphotodynamic 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.[55]
The formation of methylene blue after the reaction ofhydrogen sulfide withdimethyl-p-phenylenediamine andiron(III) at pH 0.4 – 0.7 is used to determine byphotometric measurementssulfide concentration in the range 0.020 to 1.50 mg/L (20 ppb to 1.5 ppm).[56] 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 theSpectroquantsulfide test[57] 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 ofsulfate reducing bacteria (SRB). In this colorimetric test, methylene blue is a product formed by the reaction and not a reagent added to the system.[56]
The addition of a strongreducing agent, such asascorbic 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 anion 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.[49]
Methylene blue is a dye behaving as aredox indicator that is commonly used in thefood industry to test the freshness ofmilk anddairy products.[5][58] A few drops of methylene blue solution added to a sample of milk should remain blue (oxidized form in the presence of enough dissolvedO2), otherwise (discoloration caused by the reduction of methylene blue into its colorless reduced form) the dissolvedO2 concentration in the milk sample is low indicating that the milk is not fresh (already abiotically oxidized byO2 whose concentration in solution decreases) or could be contaminated bybacteria also consuming the atmosphericO2 dissolved in the milk.[58] 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.[51]
The adsorption of methylene blue serves as an indicator defining the adsorptive capacity of granularactivated 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.Acolor reaction in an acidified, aqueous methylene blue solution containingchloroform can detectanionic surfactants in a water sample. Such a test is known as anMBAS assay (methylene blue active substances assay).
The MBAS assay cannot distinguish between specific surfactants, however. Some examples of anionic surfactants arecarboxylates,phosphates,sulfates, andsulfonates.[citation needed]
The methylene blue value is defined as the number of milliliter's standard methylene value solution decolorized 0.1 g ofactivated carbon (dry basis).[59]Methylene blue value reflects the amount of clay minerals inaggregate samples.[60] Inmaterials 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.[61]
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In biology, methylene blue is used as adye for a number of different staining procedures, such asWright's stain andJenner's stain. Since it is a temporary staining technique, methylene blue can also be used to examineRNA orDNA under themicroscope or in a gel: as an example, a solution of methylene blue can be used to stain RNA on hybridization membranes innorthern blotting to verify the amount of nucleic acid present. While methylene blue is not as sensitive asethidium bromide, it is less toxic and it does notintercalate in nucleic acid chains, thus avoiding interference with nucleic acid retention on hybridization membranes or with the hybridization process itself.[citation needed]
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]
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.[62] 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.[64]
Methylene blue has been described as "the first fully synthetic drug used in medicine". Methylene blue was first prepared in 1876 by German chemistHeinrich Caro.[65]
Its use in the treatment of malaria was pioneered byPaul Guttmann andPaul 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 thecell 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 World War II, where it was not well-liked by soldiers, who observed, "Even at the loo, we see, we pee,navy blue."[citation needed]
It was discovered to be an antidote tocarbon monoxide poisoning andcyanide poisoning in 1933 byMatilda Brooks.[66]
Methylene blue was the original prototype orlead compound for the design of many antimalarials includingchloroquine, antihistamines, and antipsychotics includingchlorpromazine.[67]
Antimalarial use of the drug has recently (2009) been revived.[68] It simultaneously targets many biological processes in theapicomplexan pathogen[69] though the main mechanism seems to be causing a lethal amount of redox cycling.[39]
A 2018 meta-analysis finds that it has proven effective againstP. falciparum in Africa. It effectively reduces levels of the transmission-stagegametocyte and has synergy with the standardartemisinin-based combination therapy (ACT). Its effects against other malarial species andP. falciparum populations in other locations are unclear.[39]
Another use of methylene blue is to treatifosfamideneurotoxicity. Methylene blue was first reported for treatment andprophylaxis of ifosfamide neuropsychiatric toxicity in 1994. A toxic metabolite of ifosfamide,chloroacetaldehyde (CAA), disrupts the mitochondrialrespiratory chain, leading to an accumulation ofnicotinamide adenine dinucleotide hydrogen (NADH). Methylene blue acts as an alternativeelectron acceptor, and reverses the NADH inhibition of hepaticgluconeogenesis while also inhibiting the transformation of chloroethylamine into chloroacetaldehyde, and inhibits multiple amine oxidase activities, preventing the formation of CAA.[70][71]
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.[72] 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.[73] 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.[74]
Methylene blue inhibitsmonoamine oxidase, inhibits theglutamatergic system (via inhibition ofNO synthase andsoluble guanylate cyclase), modulates mitochondrial function (by acting as an electron acceptor), and decreases the activation ofinflammasomesNLRP3 andNLRC4. As a result, it's been considered potentially useful inneuropsychiatric disorders. In humans it has been tried for (listed in decreasing order of evidence quality):bipolar disorder (especiallydepressive symptoms),Alzheimer's disease,claustrophobia, ifosfamide encephalopathy, andschizophrenia. With methylene blue, a higher dose does not necessarily work better than a lower dose.[33]
In the late 2010s and early 2020s, asocial mediatrend emerged promoting the use of methylene blue for various medical purposes, includinganti-aging, metabolism enhancement, cognitive improvement, cancer treatment, andCOVID-19 treatment.[75][76][77][78] Currently there is no scientific consensus on, and no FDA approval for, its effectiveness and safety for these purposes.[75][76][78] Medical experts cautioned that methylene blue can be toxic in high doses and mayinteract with other medications, potentially reducing their effectiveness or causing unforeseen side effects. Therefore, it should only be used under a doctor's prescription.[75][76]
This trend probably started following the publication of a few scientific papers exploring the potential of methylene blue for treating some medical conditions,[75] such asprogeria,[79] andskin aging.[80] It was also explored as part of anticancerphotodynamic therapy using lasers.[81] One systematic review of the studies expresses optimism but emphasizes the need for more extensive research to confirm methylene blue's clinical applications.[82] 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".[83]
In January 2025,Robert F. Kennedy Jr., then theU.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.[77][84][85]
What is Giemsa's stain and how does it color blood cells, bacteria and chromosomes?
