Methylene blue

Side effects

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). 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, shows no association between MB and hemolysis in such patients. There was, however, a clinically insignificant reduction in hemoglobin.

Pregnancy

While use during pregnancy may harm the fetus, 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 t_max 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:

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 midpoint potential E₀' is +0.01 V.

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

Other uses

Redox indicator

Methylene blue is widely used as a redox indicator in analytical chemistry. Solutions of this substance are blue when in an oxidizing environment, but will turn colorless if exposed to a reducing agent.

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. This kind of photo-disinfection has also been done inside of human bodies (antimicrobial photodynamic therapy). The same process can also be used to disinfect blood plasma.

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. Its cytotoxicity may be related to its ability to inhibit tubulin polymerization.

Sulfide analysis

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). 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 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.

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. 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.

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 carboxylates, phosphates, sulfates, and sulfonates.

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). Methylene blue value reflects the amount of clay minerals in aggregate samples. 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 a stain test on a filter paper.

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 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.

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

• 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, 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. 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.

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.

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. It simultaneously targets many biological processes in the apicomplexan pathogen 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.

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.

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.

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 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.

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. 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, and skin aging. It was also explored as part of anticancer photodynamic therapy using lasers. One systematic review of the studies expresses optimism but emphasizes the need for more extensive research to confirm methylene blue's clinical applications. Another review takes a more critical stance, stating that "it is obvious that the clinical use of [methylene blue] 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".

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.