Methylene Blue

Research Overview

Methylene Blue (MB) serves as a valuable research tool for investigating cellular respiration, mitochondrial function, and oxidative stress mechanisms in laboratory settings. First synthesized in 1876 by German chemist Heinrich Caro, Methylene Blue represents one of the earliest synthetic dyes and has found diverse research applications spanning over 140 years. Beyond its historical use as a biological stain, modern research focuses on MB’s unique redox properties and its effects on mitochondrial electron transport, making it an essential compound for investigating cellular energy metabolism and neuroprotection mechanisms.

Laboratory studies utilize Methylene Blue to examine electron transport chain function, mitochondrial respiratory capacity, oxidative stress responses, and cellular redox balance. Research protocols investigate MB’s concentration-dependent effects, with low doses (nanomolar to low micromolar) enhancing mitochondrial function while higher concentrations may exhibit different biological activities. The compound’s ability to exist in both oxidized (blue) and reduced (colorless leucomethylene blue) forms enables real-time monitoring of cellular redox status in experimental systems.

Methylene Blue research demonstrates several distinct biological activities that vary significantly with concentration. At low concentrations (0.01-4 μM), MB functions as an electron transport enhancer and mitochondrial respiratory stimulant. At moderate concentrations (10-50 μM), it exhibits antimicrobial properties through oxidative stress generation. At higher concentrations (>50 μM), MB may inhibit certain cellular processes including monoamine oxidase, nitric oxide synthase, and guanylate cyclase. This concentration-dependent activity profile requires careful experimental design to investigate specific mechanisms.

Molecular Characteristics

Complete Specifications:

• Chemical Name: 3,7-bis(dimethylamino)phenothiazin-5-ium chloride
• IUPAC Name: [7-(dimethylamino)phenothiazin-3-ylidene]-dimethylazanium chloride
• CAS Registry Number: 61-73-4
• Molecular Weight: 319.85 Da (anhydrous), 373.90 Da (trihydrate)
• Molecular Formula: C16H18ClN3S (anhydrous)
• PubChem CID: 6099
• ChEMBL ID: CHEMBL458934
• Classification: Phenothiazine dye, redox-active compound
• Appearance: Dark green crystalline powder (appears blue in solution)
• Solubility: Water (43.6 mg/mL at 25°C), ethanol, chloroform
• UV-Vis Absorption Maximum: 664 nm (aqueous solution)
• Molar Extinction Coefficient: 95,000 M⁻¹cm⁻¹ at 664 nm

The molecular structure of Methylene Blue consists of a phenothiazine core with two dimethylamino substituents at the 3 and 7 positions. This symmetric structure creates a conjugated system that enables electron delocalization, contributing to both the compound’s intense blue color and its redox activity. The positive charge on the central thiazine ring allows electrostatic interaction with negatively charged cellular components including mitochondrial membranes and nucleic acids.

Redox Chemistry and Mechanism of Action

Redox Cycling Properties:

Methylene Blue’s primary research value stems from its reversible redox chemistry:

• Oxidized Form (MB+): Blue-colored cationic form, standard reduction potential E₀’ = +11 mV at pH 7
• Reduced Form (MBH2): Colorless leucomethylene blue, produced by accepting 2 electrons and 2 protons
• Redox Couple: MB+ + 2e⁻ + 2H+ ⇌ MBH2

This reversible electron transfer occurs at physiologically relevant potentials, positioning Methylene Blue between NAD+/NADH (E₀’ = -320 mV) and cytochrome c (E₀’ = +254 mV) in the electron transport chain. This intermediate redox potential enables MB to accept electrons from NADH (via various reductases) and donate them to cytochrome c, effectively bypassing Complex I (NADH dehydrogenase) and Complex III (cytochrome bc1 complex).

Mitochondrial Electron Transfer Enhancement:

At low concentrations (0.1-2 μM), Methylene Blue functions as an alternative electron carrier:

1. MB accepts electrons from NADH via various enzymes including diaphorase, NADH-cytochrome b5 reductase, and potentially other NADH-dependent reductases
2. The reduced form (leucomethylene blue) diffuses within mitochondrial membranes
3. MBH2 donates electrons directly to cytochrome c, bypassing Complex I and III
4. This alternative pathway maintains electron flow when standard respiratory complexes are impaired
5. Enhanced electron transfer increases ATP production efficiency while potentially reducing reactive oxygen species (ROS) generation from Complex I and III

This mechanism makes Methylene Blue particularly valuable for investigating mitochondrial dysfunction associated with Complex I impairment, which occurs in various neurodegenerative conditions, aging, and metabolic disorders.

Pharmacokinetic Profile in Research Models

Methylene Blue pharmacokinetics have been characterized in various preclinical research models and clinical studies:

Absorption and Bioavailability:

• Oral bioavailability: 50-90% in mammalian models (high for small molecules)
• Rapid gastrointestinal absorption with peak plasma levels at 1-2 hours
• Multiple administration routes investigated: oral, IV, IP, intranasal
• Blood-brain barrier penetration demonstrated in research models
• CNS penetration enables neurological research applications

Distribution:

• Volume of distribution: 5-10 L/kg (wide tissue distribution)
• High tissue accumulation, particularly in mitochondria-rich organs
• Brain penetration: Crosses blood-brain barrier efficiently
• Mitochondrial accumulation: Selective accumulation in mitochondria due to cationic charge and membrane potential
• Protein binding: 95% bound to plasma proteins

Metabolism and Elimination:

• Primary metabolism: Reduction to leucomethylene blue by various reductases
• Secondary metabolism: Demethylation to azure B, azure C, and azure A (active metabolites)
• Elimination half-life: 5-6 hours (may vary with dose and species)
• Excretion: Primarily urinary (>50%), some biliary excretion
• Notable: Urine and other body fluids turn blue-green due to MB excretion

These pharmacokinetic characteristics inform research protocol design, particularly regarding dosing strategies, timing of measurements, and appropriate experimental windows.

Research Applications

Mitochondrial Function and Bioenergetics Research

Methylene Blue serves as a primary research tool for investigating mitochondrial respiratory function and cellular energy metabolism:

• Electron Transport Chain Studies: Investigation of electron transfer mechanisms, respiratory complex function, and alternative electron pathways
• ATP Production Research: Analysis of oxidative phosphorylation efficiency, mitochondrial membrane potential, and energy metabolism
• Mitochondrial Dysfunction Models: Studies examining Complex I deficiency, mitochondrial disease models, and aging-related mitochondrial impairment
• Oxygen Consumption Analysis: Measurement of mitochondrial respiration rates using Clark electrode, Seahorse analyzer, or other respirometry methods
• Mitochondrial Membrane Potential: Investigation of ΔΨm maintenance and effects on calcium handling and ROS generation
• Mitochondrial Biogenesis: Research on PGC-1α activation and mitochondrial number/function enhancement

Research protocols typically employ isolated mitochondria, permeabilized cells, intact cell cultures, or tissue preparations to measure oxygen consumption, ATP production, and mitochondrial function parameters in the presence of various MB concentrations.

Neuroprotection and Neurodegenerative Disease Research

Substantial research investigates Methylene Blue’s neuroprotective properties and potential mechanisms:

• Tau Aggregation Inhibition: Studies examining MB’s ability to prevent tau protein aggregation and neurofibrillary tangle formation
• Alzheimer’s Disease Models: Research using APP/PS1 mice, tau transgenic models, and cellular models of amyloid and tau pathology
• Parkinson’s Disease Research: Investigation of Complex I enhancement and neuroprotection in MPTP and rotenone models
• Cognitive Function Studies: Examination of learning, memory consolidation, and cognitive performance in various behavioral paradigms
• Neuronal Survival Research: Analysis of neuroprotective mechanisms against oxidative stress, excitotoxicity, and metabolic impairment
• Synaptic Function: Studies on neurotransmitter systems, synaptic plasticity, and neuronal signaling

Experimental models include primary neuronal cultures, brain slice preparations, organotypic cultures, and various transgenic mouse models of neurodegenerative disease. Outcome measures include cognitive testing, histological analysis of protein aggregates, neuronal survival assays, and electrophysiological recordings.

Oxidative Stress and Antioxidant Research

Methylene Blue’s concentration-dependent oxidative properties enable diverse redox research:

• Antioxidant Mechanisms (Low Dose): Investigation of ROS scavenging at low concentrations through enhanced mitochondrial function and reduced electron leak
• Pro-oxidant Effects (High Dose): Studies examining ROS generation and oxidative stress induction at higher concentrations
• Redox Cycling Research: Analysis of MB’s electron cycling between oxidized and reduced forms
• Oxidative Damage Models: Research on lipid peroxidation, protein oxidation, and DNA damage in various oxidative stress conditions
• Antioxidant Enzyme Modulation: Investigation of effects on superoxide dismutase, catalase, and glutathione systems
• Redox Signaling Pathways: Studies on Nrf2 activation, HIF-1α modulation, and other redox-sensitive transcription factors

Research designs must carefully control MB concentrations to achieve desired oxidative effects, as the compound exhibits biphasic concentration-dependent activities.

Antimicrobial Research Applications

At moderate to high concentrations, Methylene Blue exhibits antimicrobial properties investigated in various research contexts:

• Photodynamic Antimicrobial Therapy Research: Studies combining MB with light activation for enhanced antimicrobial effects
• Bacterial Cell Research: Investigation of MB uptake, intracellular accumulation, and bactericidal mechanisms
• Viral Inactivation Studies: Research on MB-mediated photoinactivation of viruses in blood products and other applications
• Fungal and Parasitic Research: Examination of antifungal and antiparasitic mechanisms
• Biofilm Studies: Investigation of MB penetration and efficacy against bacterial biofilms
• Resistance Mechanisms: Research on bacterial resistance to MB and combination therapy approaches

Antimicrobial research typically employs higher MB concentrations (10-100 μM) with or without light activation, examining kill kinetics, minimal inhibitory concentrations, and mechanisms of microbial death.

Cognitive Enhancement and Memory Research

Laboratory investigations examine Methylene Blue’s effects on cognitive processes:

• Memory Consolidation Studies: Research on encoding, consolidation, and retrieval processes in various memory paradigms
• Working Memory Research: Investigation of prefrontal cortex-dependent cognitive tasks
• Attention and Executive Function: Studies examining cognitive control and attentional processing
• Learning Mechanisms: Research on hippocampal-dependent learning and synaptic plasticity
• Cerebral Blood Flow: Investigation of MB effects on regional brain perfusion and neurovascular coupling
• Metabolic Enhancement: Studies examining brain glucose metabolism and oxidative metabolism changes

Behavioral paradigms include Morris water maze, novel object recognition, radial arm maze, fear conditioning, and various operant conditioning tasks in rodent models.

Cellular Signaling Research

Methylene Blue modulates multiple cellular signaling pathways at various concentrations:

• Nitric Oxide Pathway: Investigation of NO synthase inhibition and cGMP pathway modulation
• Guanylate Cyclase Research: Studies on soluble guanylate cyclase inhibition and cGMP reduction
• Monoamine Oxidase Inhibition: Research on MAO-A/B inhibition and effects on neurotransmitter metabolism
• Protein Kinase Pathways: Investigation of effects on protein phosphorylation and kinase signaling
• Autophagy Research: Studies examining MB effects on autophagy induction and mitophagy
• Apoptosis Pathway Investigation: Research on cell death signaling and survival pathway modulation

These diverse signaling effects require careful experimental design to isolate specific mechanisms of interest.

Laboratory Handling and Storage Protocols

Powder Storage:

• Store at room temperature in tightly sealed container
• Protect from light exposure (store in amber glass containers or wrap in foil)
• Protect from moisture (hygroscopic)
• Stability: Multiple years when properly stored
• Desiccated storage recommended in humid environments

Solution Preparation:

• Dissolve in sterile water, PBS, or appropriate buffer
• Common stock concentration: 10 mM in water or DMSO
• Vortex or sonicate briefly to ensure complete dissolution
• MB is highly soluble in water (43.6 mg/mL = 136 mM)
• Filter sterilize solutions through 0.22 μm filter if sterility required
• Final pH typically 5.5-7.0 depending on buffer system

Solution Storage:

• Aqueous stock solutions: Store at 4°C protected from light (stable for several months)
• DMSO stock solutions: Store at -20°C (stable for extended periods)
• Working solutions: Prepare fresh or store at 4°C for up to 1 week
• Protect all solutions from light exposure (wrap tubes/bottles in foil)
• Avoid repeated freeze-thaw cycles of stock solutions

Concentration Guidelines for Research:

• Mitochondrial enhancement: 0.01-2 μM (nanomolar to low micromolar)
• Neuroprotection studies: 0.1-10 μM
• Antimicrobial research: 10-100 μM
• Cell signaling inhibition: 1-50 μM depending on target
• Always perform dose-response studies to determine optimal concentrations

Light Sensitivity Considerations:
Methylene Blue exhibits photosensitivity and can generate reactive oxygen species upon light exposure. For photodynamic studies, this property is utilized. For non-photodynamic research, protect solutions from light to prevent unwanted photochemical reactions.

Quality Assurance and Analytical Testing

Each Methylene Blue batch undergoes comprehensive analytical characterization:

Purity Analysis:

• High-Performance Liquid Chromatography (HPLC): ≥98% purity
• Analytical method: Reversed-phase HPLC with UV detection at 664 nm
• Multiple wavelength detection to identify related compounds and impurities
• Peak purity assessment using photodiode array detection

Structural Verification:

• UV-Visible Spectroscopy: Confirms characteristic absorption maximum at 664 nm with appropriate extinction coefficient
• Mass Spectrometry (ESI-MS or MALDI-TOF): Confirms molecular weight 319.85 Da
• Nuclear Magnetic Resonance (NMR): Structural confirmation of phenothiazine core and substituents
• Infrared Spectroscopy (IR): Confirms functional groups

Contaminant Testing:

• Heavy metals: ICP-MS analysis for lead, arsenic, mercury, cadmium
• Related dyes: Azure A, Azure B, Azure C (demethylation products) quantified
• Residual solvents: GC-MS analysis for manufacturing solvents
• Water content: Karl Fischer titration
• Chloride content: Ion chromatography or argentometric titration

Physical Properties:

• Melting point determination
• Loss on drying
• Appearance and color assessment
• Particle size analysis (for powders)

Documentation:

• Certificate of Analysis (COA) provided with each batch
• Spectroscopic data (UV-Vis, MS) included with COA
• Batch-specific QC results traceable by lot number
• Third-party analytical verification available upon request

Research Considerations

Experimental Design Factors:

Researchers should consider several critical factors when designing Methylene Blue experiments:

1. Concentration Selection: MB exhibits biphasic and multiphasic concentration-dependent effects. Low concentrations (nanomolar to low micromolar) enhance mitochondrial function, while higher concentrations may inhibit various enzymes or generate oxidative stress. Always perform concentration-response studies.

2. Light Exposure Control: MB is photosensitive and generates ROS upon light activation. For non-photodynamic studies, conduct experiments under subdued lighting or wrap culture vessels/tubes in foil. For photodynamic studies, carefully control light wavelength, intensity, and duration.

3. Vehicle Considerations: MB can be dissolved in water, PBS, or DMSO. Keep DMSO concentration minimal (typically <0.1% final concentration) to avoid vehicle effects. Water or PBS is preferred for most cell culture and tissue studies.

4. Time Course Analysis: MB reduction by cellular enzymes occurs rapidly (seconds to minutes). Mitochondrial enhancement effects may manifest within minutes to hours. Long-term effects on gene expression and protein levels require longer exposure periods (hours to days).

5. Redox State Monitoring: In cell-free systems or tissue homogenates, MB redox state can be monitored by absorbance at 664 nm (oxidized form) or by reduction to colorless leucoform. Cellular reducing systems rapidly convert MB to MBH2.

6. Interference with Assays: MB’s intense blue color and redox activity can interfere with colorimetric assays, fluorescence assays, and absorbance-based measurements. Consider assay compatibility when designing experiments. May need to remove MB before measurements or use alternative assays.

7. Species and Cell Type Differences: Cellular reducing capacity varies across cell types and species. Neurons, hepatocytes, and other metabolically active cells reduce MB more rapidly than less active cell types.

Mechanism Investigation:

Methylene Blue’s multiple mechanisms of action require systematic investigation:

• Mitochondrial Effects: Isolate mitochondria or use permeabilized cells to study direct effects on respiratory chain. Measure oxygen consumption with different respiratory substrates to identify affected complexes.
• Enzyme Inhibition Studies: Examine specific enzyme targets (MAO, NOS, guanylate cyclase) using purified enzyme preparations or specific activity assays.
• Cellular vs. Mitochondrial Effects: Compare effects in intact cells vs. isolated mitochondria to distinguish direct mitochondrial effects from cellular signaling effects.
• Photodynamic vs. Non-photodynamic Effects: Conduct parallel experiments with and without light exposure to separate these mechanisms.

Control Experiments:

Essential controls for Methylene Blue research:

• Vehicle controls (water, PBS, or DMSO at same concentration)
• Light exposure controls (when relevant)
• Concentration-response curves including concentrations above and below expected active range
• Time course analysis to determine optimal timing
• Parallel experiments with structurally related compounds (Azure B, Azure C) to assess structure-activity relationships

Compliance and Safety Information

Regulatory Status:
Methylene Blue is provided as a research chemical for laboratory studies and preclinical research. While Methylene Blue has FDA-approved medical applications (methemoglobinemia treatment), this research-grade product is not manufactured under GMP conditions and is not approved for human administration or therapeutic use.

Intended Use:

• In-vitro cell culture studies
• In-vivo preclinical research in approved animal models
• Biochemical and enzymatic assays
• Mitochondrial function studies
• Academic and institutional research applications
• Teaching and demonstration purposes

NOT Intended For:

• Human consumption or administration
• Therapeutic treatment or diagnosis
• Dietary supplementation
• Self-administration or biohacking applications
• Veterinary therapeutic use without appropriate oversight

Safety Protocols:

Researchers should follow standard chemical safety practices when handling Methylene Blue:

• Personal Protective Equipment: Lab coat, nitrile gloves, safety glasses (MB stains skin and clothing)
• Ventilation: Use in well-ventilated areas; fume hood not typically required for standard handling
• Skin Contact: Wash immediately with soap and water (staining will fade over several days)
• Eye Contact: Rinse immediately with water for 15 minutes; seek medical attention
• Ingestion: Seek immediate medical attention (while MB has medical uses, research-grade material should not be ingested)
• Spills: Absorb with paper towels or absorbent material; clean surfaces with water; MB stains porous surfaces
• Waste Disposal: Dispose according to institutional chemical waste protocols; consider as chemical waste
• First Aid: Consult Safety Data Sheet (SDS) for complete first aid procedures

Drug Interactions and Research Considerations:

Methylene Blue inhibits monoamine oxidase and should not be combined with serotonergic drugs in research models to avoid serotonin syndrome. This interaction is well-documented and requires consideration in research design involving neurotransmitter systems.

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