• Log in with Facebook Log in with Twitter Log In with Google      Sign In    
  • Create Account
  LongeCity
              Advocacy & Research for Unlimited Lifespans

Photo
* * * * - 2 votes

Methylene Blue and MAO Inhibition


  • Please log in to reply
114 replies to this topic

#31 Isochroma

  • Guest
  • 791 posts
  • 1
  • Location:Earth

Posted 20 July 2011 - 03:26 AM

MB orally is a very different species than by IV. The injectable's been shown to cause brain damage @ 5mg/kg, but no study has found damage with oral.

It's the high peak level attained by IV that causes problems. GI absorption's slow enough that even 60-120mg per dose is just fine. IV administration causes a greater than 10-fold higher blood concentration than the same dose taken orally..

I also have a gutty feeling that oral use results in a very different reduced/oxidized ratio of the molecule in blood than IV. The reduced form, leuco-methylene blue may have a very different MAO interaction than the oxidized form.

"In early experiments, oral MB was found to be innocuous in 15kg dogs at 2g per day, but an intravenous dose of 0.5g was lethal (Agell and Agell, 1929)."

Again, context is everything: the study above shows more than a 4:1 increase in toxicity when used by IV rather than orally. Since no deaths were measured in the 2g oral dosing the minimum dose for oral toxicity isn't even revealed, but we do know it's higher than 4:1 vs. IV.

In-vitro and IV studies are a world away from oral dosing. There is not one study reporting MAO-inhibition related toxicity from oral dosing. They're all IV or in-vitro using concentrations that would be very difficult to obtain orally.

Finally, MAO inhibition is more closely correlated with peak concentration rather than the total AUC (Area Under the Curve) aka. total dose. Since MB is water-soluble the peak concentration attained will be a function primarily of absorption rate.

Absorption rate of IV is instant - compared to oral which is very much slower. Putting the problem in context shows the difference between modes of administration of the exact same dose makes the difference between life and death.

The fear-mongering is a waste of threadspace and is already giving me a placebo-headache. I'd like to see some more reports on actual effects or lack thereof.

Edited by Isochroma, 20 July 2011 - 03:45 AM.

  • like x 4

#32 aaron43

  • Guest
  • 143 posts
  • 13
  • Location:California

Posted 20 July 2011 - 06:35 AM

as I, nor anyone else (correct me if Im wrong) does not know

- excuse my grammar error
- I said I don't know, correct me if I'm wrong. I only say what I know to my current knowledge, if it's an unfounded thought but is a connection I have found, i'll always have "in my opinion". I will always note if it is the case that I don't know the subject material well enough to comment. What I found previously when I was researching it, had myself convinced to avoid adderall/any RI of some kind due to the majority daily use of such drugs. Let me clear that up

As with all biological effects of drugs, DOSE MATTERS. At sub-milligram doses, methylene blue is NOT a significant MAOI or RIMA. Please don't start a new internet paranoia here. If you are going to take a gram of it, then you can worry. There are one million micrograms in a gram.

Yes, but this response, does not tell me why I should change my perception on it. Its the same logic that had me believe that it's ok to take with once, but should avoid due to frequency of use, which is what I thought but summed it up in "should avoid".

Finally, MAO inhibition is more closely correlated with peak concentration rather than the total AUC (Area Under the Curve) aka. total dose.

The IC50 for MB inhibition of MAO-A is more than an order of magnitude higher than you will see with sub-milligram oral dosing.

I must give you credit, but this is all that needed to be said in the first place, as it would of taken away the idea that any MAO-A activity could occur. And, scientifically without question, enforces the idea that Reuptake Inhibitors have no negative effects with Methylene Blue

I must say that the nootropic dose is so small that it most likely doesn't matter.

You must notice that I always say must, and I have always said must, and I shall always say must. It is my form of expression. And I do feel that there is zero fear-mongering here


My main objection is that you are using scary language ("I must warn", "does not know what could happen") about this, with no qualification of it at all, when you should just say you don't know, or say nothing.

If you have a degree, then by all means try to degrade me, but I do wake up every morning and read new information on nootropics, I believe my knowledge of nootropics is more than someone who would not read on them everyday. But I feel I am on par with anyone else, and if you happen to know some information that I don't, then please enlighten me, and I will be inviting it in which i'll say "correct me if I'm wrong"
I am one year from my degree in Nutrition Science, I do know that I am just finishing up the first half of my credits to the science portion of the degree. And I am continuously increasing my knowledge on the subject. I have to learn about "pharmacokinetics and toxicity" on my own, and I do not know how to find that sort of academic information with what I have available for the IC50 of Methylene Blue at the microgram levels or anything of the sort. So I appreciate the expansion of knowledge, but I don't appreciate attempts to undermine credibility with an attack on qualification when I have never claimed myself as an authority, but as an experienced user. I stumbled upon MB recently, and decided to try and expand popular knowledge that MB is a very useful nootropic. . My knowledge prior to becoming knowledgeable on MB, was only so much, but since, it has grown, and my credibility will continue to grow as long as I keep learning the true academic information in the field
I will have the greatest credibility, but it does take time

Edited by aaron43, 20 July 2011 - 06:53 AM.


sponsored ad

  • Advert
Rent this spot in Nootropics Topics to support Longecity (this will replace the google ad).

#33 thedevinroy

  • Guest
  • 1,188 posts
  • 326
  • Location:USA
  • NO

Posted 20 July 2011 - 05:10 PM

As far as a MAO inhibitor goes, Methylene Blue can act as one at the dose I am taking. If you figure that the oral is 10x less than the IV peak level.

http://www.ncbi.nlm....les/PMC2078225/

MB inhibits 50% MAO-A at 164nM.
MB molecular weight is 319.85 g/mol.
Average volume in blood stream is 6L.
Constant of 10x that of IV needed.

164 * (10^(-9)) * 319.85 * 6 * 10 / 1000 = 3.15mg

Don't know all that fancy pharmacology IC50 stuff that well, but if 3.15mg is true, then an equivalent of 3.15mg of competition must exist. Thus, at 1.15mg, the inhibition would yeild...

1.15 / (3.15 + 1.15) = 26.74%

Probably none of that is correct, but one thing I would like to state is that in my personal experience, I can feel the MAOi effects at 1.15mg. The point was to just show that the in vitro effects happened at very low concentrations, in the nanamoles, and that does have some effect in vivo if well absorbed at the milligram dose.

#34 niner

  • Guest
  • 16,276 posts
  • 2,000
  • Location:Philadelphia

Posted 20 July 2011 - 06:48 PM

As far as a MAO inhibitor goes, Methylene Blue can act as one at the dose I am taking. If you figure that the oral is 10x less than the IV peak level.

http://www.ncbi.nlm....les/PMC2078225/

MB inhibits 50% MAO-A at 164nM.
MB molecular weight is 319.85 g/mol.
Average volume in blood stream is 6L.
Constant of 10x that of IV needed.

164 * (10^(-9)) * 319.85 * 6 * 10 / 1000 = 3.15mg

Don't know all that fancy pharmacology IC50 stuff that well, but if 3.15mg is true, then an equivalent of 3.15mg of competition must exist. Thus, at 1.15mg, the inhibition would yeild...

1.15 / (3.15 + 1.15) = 26.74%

Probably none of that is correct, but one thing I would like to state is that in my personal experience, I can feel the MAOi effects at 1.15mg. The point was to just show that the in vitro effects happened at very low concentrations, in the nanamoles, and that does have some effect in vivo if well absorbed at the milligram dose.

Some of this is correct, but the analysis is incorrect in a couple important ways. It's very very hard to predict in vivo concentrations like this, but this sort of analysis at least provides an upper limit. However, it is an upper limit for the maximum concentration that can be reached, which is very transient. Whenever we ingest a drug, the concentration in blood rises fairly quickly to a maximum, then starts falling off. The maximum concentration doesn't last long at all. More important is the shape of the IC50 curve. It's a sigmoid, with %inhibition on the y axis and concentration on the x axis. As a result of this non-linearity, half the concentration of the inhibitor gives a lot less than half the inhibition. When you say that you feel the MAOI effects, what does that feel like? Have you taken MAOIs before?

#35 snuffie

  • Guest
  • 52 posts
  • 10
  • Location:Canada
  • NO

Posted 20 July 2011 - 11:13 PM

Can anyone with journal access acquire and share the full text of these articles? I think the first one might be particularly applicable to our research...

Schwiebert C, Irving C, Gillman PK. Small doses of methylene blue, previously considered safe, can precipitate serotonin toxicity. Anaesthesia. Aug 2009;64(8):924.

Ramsay RR, Dunford C, Gillman PK. Methylene blue and serotonin toxicity:inhibition of monoamine oxidase A (MAO A) confirms a theoretical prediction. BR J Pharmocol. 2007/11;152:946-951.

Rowley M, Riutort K, Shapiro D, Casler J, Festic E, Freeman WD. Methylene blue-associated serotonin syndrome: a 'green' encephalopathy after parathyroidectomy.Neurocrit Care. 2009;11(1):88-93.

Khavandi A, Whitaker J, Gonna H. Serotonin toxicity precipitated by concomitant use of citalopram and methylene blue. Med J Aust. Nov 3 2008;189(9):534-5.

...sigh...journal access is what I miss most about university...

#36 niner

  • Guest
  • 16,276 posts
  • 2,000
  • Location:Philadelphia

Posted 21 July 2011 - 01:10 AM

Can anyone with journal access acquire and share the full text of these articles? I think the first one might be particularly applicable to our research...

Schwiebert C, Irving C, Gillman PK. Small doses of methylene blue, previously considered safe, can precipitate serotonin toxicity. Anaesthesia. Aug 2009;64(8):924.

Ramsay RR, Dunford C, Gillman PK. Methylene blue and serotonin toxicity:inhibition of monoamine oxidase A (MAO A) confirms a theoretical prediction. BR J Pharmocol. 2007/11;152:946-951.

...sigh...journal access is what I miss most about university...

The science publishing houses are inhibiting the progress of science though their greed. Most of the work they publish is publicly funded, but they lock it up for profit. (rant...)

I don't have the full text, except for Ramsay et al., but the following abstract explains what Gillman means by a "small dose" of MB:

J Psychopharmacol. 2011 Mar;25(3):429-36. Epub 2010 Feb 8.
CNS toxicity involving methylene blue: the exemplar for understanding and predicting drug interactions that precipitate serotonin toxicity.
Gillman PK.

PsychoTropical Research, Bucasia, Queensland, Australia. kg@matilda.net.au

Methylene blue has only recently been noted to cause severe central nervous system toxicity. Methylene blue is used for various conditions, including, intravenously, in methemoglobinemia, vasoplegia and as an aid to parathyroidectomy (at doses of 1-7.5 mg kg(-1)). This review of the current evidence concludes that 13 of 14 of the reported cases of CNS toxicity were serotonin toxicity that met the Hunter Serotonin Toxicity Criteria. That has important preventative and treatment implications. Serotonin toxicity is precipitated by the monoamine oxidase inhibitor (MAOI) property of methylene blue interacting with serotonin reuptake inhibitors. Serotonin toxicity is reviewed, using the lessons inherent in the methylene blue story and experience, to illustrate how the mechanisms and potency of serotonergic drugs interact to determine severity. Recent human data showed that an intravenous dose of only 0.75 mg kg(-1) of methylene blue produced a peak plasma concentration of 500 ng ml(-1) (1.6 µM), indicating that the concentration in the central nervous system reaches a level that inhibits monoamine oxidase A. That is consonant with the actual occurrence of severe serotonin toxicity in humans at the dose of only 1 mg kg(-1). It seems that all proposed uses of methylene blue entail levels that block monoamine oxidase, so cessation of serotonin reuptake inhibitors should be very carefully considered before using methylene blue.

PMID: 20142303

The lowest i.v. dose noted, 0.75 mg/kg would correspond to 52.5 mg intravenous for a 70 kg adult. If we accept the common factor of ten difference between oral and iv MB, we would be talking about an oral dose of 525 mg, or eight thousand seven hundred fifty times the 60 mcg nootropic dose.

Methylene Blue has been used with some fairly whopping doses, historically, so 'small' in this context is relative.
  • like x 2

#37 aaron43

  • Guest
  • 143 posts
  • 13
  • Location:California

Posted 21 July 2011 - 10:10 AM

Can anyone with journal access acquire and share the full text of these articles? I think the first one might be particularly applicable to our research...

Schwiebert C, Irving C, Gillman PK. Small doses of methylene blue, previously considered safe, can precipitate serotonin toxicity. Anaesthesia. Aug 2009;64(8):924.

Ramsay RR, Dunford C, Gillman PK. Methylene blue and serotonin toxicity:inhibition of monoamine oxidase A (MAO A) confirms a theoretical prediction. BR J Pharmocol. 2007/11;152:946-951.

Rowley M, Riutort K, Shapiro D, Casler J, Festic E, Freeman WD. Methylene blue-associated serotonin syndrome: a 'green' encephalopathy after parathyroidectomy.Neurocrit Care. 2009;11(1):88-93.

Khavandi A, Whitaker J, Gonna H. Serotonin toxicity precipitated by concomitant use of citalopram and methylene blue. Med J Aust. Nov 3 2008;189(9):534-5.

...sigh...journal access is what I miss most about university...


this is the full text: Its pretty short actually Schwiebert C, Irving C, Gillman PK. Small doses of methylene blue, previously considered safe, can precipitate serotonin toxicity. Anaesthesia. Aug 2009;64(8):924.

Small doses of methylene blue, previously considered safe, can precipitate serotonin toxicity
Potentially life-threatening serotonin toxicity is the result of an adverse drug interaction between monoamine oxi- dase inhibitors (MAOI) and other serotonergic agents, and has previously been the subject of an editorial in this journal [1]. All reported cases have occurred in patients on selective sero- tonin re-uptake inhibitors who received the redox dye methylene blue peri- operatively. A possible pharmacological mechanism underlying this interaction has recently been demonstrated in vitro by confirming methylene blue’s activity as a MAOI [2]. However, as we currently do not know methylene blue’s in vivo potency as a MAOI, we do not know whether there is a dose methylene blue small enough to be considered safe.
Current drug safety advice issued by the Medicines and Healthcare products Regulatory Agency [3] recognises that ‘CNS toxicity’ can be precipitated by methylene blue doses of between 5 and 10 mg.kg)1 against the background of treatment with serotonergic drugs. Indeed, all reported cases of definite serotonin toxicity have so far involved doses of more than 5 mg.kg)1. Doses smaller than 5 mg.kg)1 have been asso- ciated with adverse signs, but these have not allowed the diagnosis of serotonin toxicity, according to the widely accepted Hunter Serotonin Toxicity Criteria [4, 5], in any of the relevant cases.
We would like to add to the record a case from our practice, where definite serotonin toxicity was precipitated by only 1 mg.kg)1 of methylene blue. This case involved a young female with malignant disease of the pelvis, whose
regular medication included the selec- tive serotonin re-uptake inhibitor par- oxetine. She received 1 mg.kg)1 of methylene blue intraoperatively to aid cystoscopic identification of the ureteric ostia. Following emergence from anaes- thesia she exhibited confusion, agita- tion, aphasia, ocular clonus, mydriasis, hyperreflexia and arterial hypertension. In the presence of an selective serotonin re-uptake inhibitor, these signs allow the clear and unambiguous diagnosis of serotonin toxicity.
This case is the first to demonstrate a clear link between definitive serotonin toxicity and an methylene blue dose of < 5 mg.kg)1. It also provides evidence that the small dose of 1 mg.kg)1 pro- vides MAO inhibition in vivo, and risks serotonin toxicity. We suggest that drug safety advice as well as clinical use of methylene blue should be modified accordingly.
C. Schwiebert C. Irving P. K. Gillman Charing Cross Hospital London, UK
Royal Marsden Hospital London, UK Pioneer Valley Private Hospital Mackay, Queensland, Australia E-mail: c.schwiebert@gmail.com
References
1 Gillman PK. Extracting value from case reports: lessons from serotonin toxicity. Anaesthesia 2006; 61: 419–22.
2 Ramsay RR, Dunford C, Gillman PK. Methylene blue and serotonin toxicity: inhibition of monoamine oxidase A (MAO A) confirms a theoretical pre- diction. British Journal of Pharmacology 2007; 152: 946–51.
3 Medicines and Healthcare products Regulatory Agency. Methylthioninium chloride (methylene blue): CNS toxi- city with serotonergic drugs. Drug Safety Update 2008; 1(6): 5.
4 Isbister GK, Buckley NA, Whyte IM. Serotonin toxicity: a practical approach to diagnosis and treatment. Medical Journal of Australia 2007; 187: 361–5.
5 Boyer EW, Shannon M. The Serotonin Syndrome. New England Journal of Medicine 2005; 352: 1112–20.


Ramsay RR, Dunford C, Gillman PK. Methylene blue and serotonin toxicity:inhibition of monoamine oxidase A (MAO A) confirms a theoretical prediction. BR J Pharmocol. 2007/11;152:946-951.

RESEARCH PAPER
Methylene blue and serotonin toxicity: inhibition of monoamine oxidase A (MAO A) confirms a theoretical prediction
RR Ramsay1, C Dunford1 and PK Gillman2 1Centre for Biomolecular Sciences, University of St Andrews, North Haugh, St Andrews, Fife, Scotland, UK and 2PsychoTropical
Research, Bucasia, Queensland, Australia
Background and purpose: Monoamine oxidase inhibitors (MAOI) are known to cause serotonin toxicity (ST) when administered with selective serotonin reuptake inhibitors (SSRI). Methylene blue (methylthionium chloride, MB), a redox dye in clinical use, has been reported to precipitate ST in patients using SSRI. MB was assessed for MAO inhibition and so for its potential to precipitate ST.
Experimental approach: Inhibition of purified human MAO was quantified using kinetic assays and visible spectral changes to study the interactions of MB with MAO A. Key results: MB was a potent (tight binding) inhibitor for MAO A. It also inhibited MAO B but at much higher concentration. Interactions of MB with the active site of MAO A were confirmed by its action both as an oxidising substrate and as a one-electron reductant.
Conclusions and implications: MB is a potent reversible inhibitor of MAO A with implications for gut uptake of amines when administered orally. At concentrations reported in the literature after intravenous administration, MAO B would be partially inhibited but MAO A would be completely inhibited. This inhibition of MAO A would be expected to lead to perturbations of 5-hydroxytryptamine metabolism and hence account for ST occurring when administered to patients on SSRI treatment. British Journal of Pharmacology (2007) 152, 946–951; doi:10.1038/sj.bjp.0707430; published online 27 August 2007
Keywords: methylene blue; methylthionium chloride; serotonin toxicity; monoamine oxidase A; MAOI; tight binding
Abbreviations: MAO, monoamine oxidase; MAOI, monoamine oxidase inhibitor; MB, methylene blue (methylthionium chloride); SSRI, selective serotonin reuptake inhibitor; ST, serotonin toxicity
Introduction
Serotonin toxicity (ST), formerly referred to as serotonin syndrome, is one of very few drug–drug interactions involving usual therapeutic doses of commonly used drugs that are capable of developing rapidly and causing death within 24h. A mixture of therapeutic doses of any drug with significant monoamine oxidase (MAO) inhibitor (MAOI) properties with another drug that has potency as a selective serotonin reuptake inhibitor (SSRI) produces a high risk of precipitating this rapidly worsening interaction (reviewed in Gillman, 2006a). The typical clinical features of ST are (i) neuromuscular hyperactivity: tremor, clonus, myoclonus and hyperreflexia, and, in the advanced stage, pyramidal rigidity; (ii) autonomic hyperactivity: diaphoresis, fever, tachycardia, tachypnoea and mydriasis; and (iii) altered mental status: agitation, excitement,
Correspondence: Dr RR Ramsay, Centre for Biomolecular Sciences, University of St Andrews, North Haugh, St Andrews, Fife, Scotland KY16 9ST, UK. E-mail: rrr@st-and.ac.uk Received 29 May 2007; revised 11 July 2007; accepted 17 July 2007; published online 27 August 2007
with confusion in the advanced stage. There have been several deaths or near deaths, reported recently where a single dose of a SSRI has been inadvertently added to a MAOI (Otte et al., 2003; Cassens et al., 2006; Zonneveld et al., 2006).
In the past the only drugs recognised as possessing these properties were antidepressants used by specialists so that the risk of them being combined inadvertently was small. However, there are now many drugs on the market that act as 5-hydroxytryptaminergic agents (mostly SSRIs), or MAOIs, which are neither advertised, nor generally recognised, as 5-hydroxytryptaminergic drugs (for example, sibutramine and linezolid). Despite the clear understanding of ST interactions that has developed over the past decade, there is still misinformation in standard texts, such as the British National Formulary, and in pharmaceutical company product information (Gillman, 2005). Recent reviews deal with this complex topic in detail (Gillman, 1998, 2006a, b, c; Dunkley et al., 2003; Whyte et al., 2003; Whyte, 2004; Isbister and Buckley, 2005).
+
Figure 1
N H
N S+ N
Methylene blue inhibits monoamine oxidase A
RR Ramsay et al 947
tyrosines so that the amine is close to the N-5 of the flavin. The active site of MAO A is wider than that of MAO B (De Colibus et al., 2005) and, in functional studies, MAO A accommodates larger non-flexible ligands (Efange et al., 1993; Medvedev et al., 1998). Structure–function studies have shown that MAO substrates and inhibitors are some- what lipophilic with an aromatic ring and a nitrogen (or oxygen or sulphur) close to that ring (reviewed in Wouters, 1998; Kalgutkar et al., 2001; Ramsay and Gravestock, 2003). MB fits this description and indeed was an inhibitor and electron acceptor for MAO B (Shumakovich et al., 2004). Other tricyclic heterocycles, such as the b-carbolines (Kim et al., 1997), are also known as effective inhibitors, particularly of MAO A.
The experiments reported below show that MB inhibits both MAO A and MAO B. In particular, it is a tight-binding inhibitor of MAO A that interacts with the active site as shown by its action as an oxidative substrate and by its redox effects on the flavin of MAO A.
Methods
MAO assays
Recombinant human liver MAO A and MAO B were purified as reported previously (Tan and Ramsay, 1993).
MAO A (usually about 50nM) was assayed spectrophoto- metrically at 314 nm in 50 mM potassium phosphate, pH 7.2–0.2%, Triton-X100 at 301C using kynuramine (0.1– 0.9 mM) as the substrate (Tan and Ramsay, 1993). The kinetic constants were calculated by the Shimazu kinetic software. Apparent Km values in the presence of MB (30–200 nM) were also determined. This experiment revealed an apparent Ki of 69 nM, only slightly higher than the enzyme concentration, indicating that Michaelis–Menten analysis could not be used because the concentration of free inhibitor would be depleted by binding to the enzyme. To obtain the Ki, the initial rates in the absence and presence of MB were analysed by the equation developed by Morrison (1968) for tight- binding inhibitors.
MAO B was assayed polarographically using the same buffer as for MAO A (Tan and Ramsay, 1993). Inhibition was measured for 0–20 mM MB in the presence of 0.6 mM benzylamine (2 􏰁 Km ) as shown in Figure 2. Further analysis of the kinetics of interaction with MAO B would require varying both the amine and oxygen substrates, because the Km for oxygen is 0.28mM (Husain et al., 1982; Ramsay et al., 1987).
Spectrophotometric studies of MAO
Spectral data were obtained using MAO A in 50 mM potassium phosphate, pH 7.2, 0.05% Brij, as in previous work (Ramsay, 1991; Jones et al., 2005). The enzyme was prepared for the experiments by centrifugal gel filtration, diluted to about 15mM, and its concentration determined from the absor- bance at 456 nm (extinction coefficient, 12 300 M􏰀1 cm􏰀1). For anaerobic experiments, oxygen was replaced with argon by cycling the custom-made anaerobic cuvette between vacuum and argon.
2H+ + 2e
2H+ + 2e
N
Blue
Colourless
NH S N
Oxidised and reduced forms of methylene blue.
The spectrum concept of ST predicts clearly that severe, life-threatening, degrees of toxicity are likely to develop only after the co-administration of SSRIs and MAOIs. A case report of neurological toxicity associated with the administration of methylene blue (methylthionium chloride, MB), which precipitated the work in this article (Rosenbaum, 2006) seemed an exception. However, the patients in that and subsequently uncovered reports (Gillman, 2006c) had all been taking an SSRI antidepressant before surgery involving the intravenous infusion of MB. All these patients experienced severe toxicity of the degree expected to result only from a combination of MAOI and SSRI, and too severe to be the result of an SSRI alone. A search of the literature revealed no report of such toxicity from MB alone. This led to the prediction that MB (Figure 1) must possess significant MAOI potency.
Existing clinical literature (reviewed in Gillman, 2006c), including a report of antidepressant effect of MB (Naylor et al., 1987), appeared to support the possibility that MB would inhibit MAO, as did the well-known properties of MB as an electron acceptor. Effective inhibition of amine oxidases, including MAO, was reported to play a role in the prevention by MB of ifosfamide encephalopathy (Aeschlimann et al., 1996; Kupfer et al., 1996). However, the only reported Ki for inhibition of MAO B was rather high at 5.6mM (Bachurin et al., 2001; Shumakovich et al., 2004). This is within the range for whole-blood concentration immediately after a 100 mg oral dose in humans, and intra- venous administration gave more than 10-fold higher sustained concentrations (Peter et al., 2000). In another study, the concentration of MB in rat brain was estimated at 0.5 mM from 1 mg kg􏰀1 intraperitoneally (Callaway et al., 2004).
The role of MAO in the brain, its involvement in disease and the therapeutic potential of MAOIs has recently been reviewed in the light of new structural information (Youdim et al., 2006). Crystal structures of MAO A (De Colibus et al., 2005) and MAO B (Binda et al., 2002) have shown that the active site is a long, narrow hydrophobic cavity penetrating deep into the molecule where the substrate is aligned by two
British Journal of Pharmacology (2007) 152 946–951
Methylene blue inhibits monoamine oxidase A
RR Ramsay et al
Dose–response curves for the inhibition of MAO A (left) and MAO B (right) by methylene blue (MB). MAO A (122 nM) was assayed spectrophotometrically with 0.3 mM (2 􏰁 Km) kynuramine, and 25 concentrations of MB (some points omitted for clarity). MAO B was assayed polarographically with 0.6 mM (2 􏰁 Km) benzylamine.
Data analysis
Parameters are shown as the values calculated7s.e. from the non-linear regression fit. The calculation of Ki used the equation for tight binding inhibitors (Morrison, 1968) in PRISM (from Graphpad Software, Inc., http://www.graphpad. com/curvefit/). Ki values are the mean7s.d. mean of values from four experiments.
Materials
All chemicals were purchased from Sigma-Aldrich (Poole, Dorset, UK). The purity of the MB was checked by determining the ratio of the absorbance at 664–610nm using 10 mM MB in water (Fujimoto et al., 1994). The ratio was 2.02 so the MB was used as supplied. It should be noted that the molar absorbance of MB varies with concentration (Lewis et al., 1943). The oxidised and reduced forms of MB are shown in Figure 1.
Results
MAO A and MAO B have very different sensitivity to MB
Figure 2 shows the inhibition of the two forms of MAO by MB, each assayed at subsaturating substrate concentration (2 􏰁 Km). The IC50 value for MB inhibition of MAO A was 16478 nM under this specific condition with 122 nM MAO A. In contrast, no inhibition of MAO B was seen in the spectrophotometric assay at 100nM. Data from the polaro- graphic assays of MAO B are shown in Figure 2 (the right- hand curve) and the IC50 value for MAO B inhibition by MB was 5.571.7 mM.
MB is a tight-binding inhibitor of MAO A
The IC50 value of 16478nM (Figure 2) was obtained using 122 nM MAO A in the assays. Clearly, the normal Michaelis– Menten assumption that the inhibitor concentration would not be depleted by the enzyme did not hold. When
948
Figure 2
both kynuramine and MB were varied, the IC50 value increased as the substrate concentration increased (data not shown), consistent with competitive inhibition (Henderson, 1972). Tight-binding analysis of data for MAO A according to Morrison (1968) gave a Ki value of 2773 nM (mean7s.d.; n 1⁄4 4).
MB binds to the active site of MAO A
Inhibitors binding in the active site of MAO A influence the visible spectrum of the covalently bound cofactor flavin adenine dinucleotide (FAD) (Hynson et al., 2003). When MAO A was titrated with MB (Figure 3), only a very small and featureless change in the difference spectrum of the MAO A FAD (MAO A with MB—MAO A alone) at around 450 nm was detected (Figure 3b). In previous work, the KD for inhibitor binding was calculated from inhibitor-induced changes in the flavin spectrum (Hynson et al., 2003). However, the influence of MB on the flavin spectrum was too small to analyse accurately, with a maximum decrease of 0.01 that was compromised by the large adjacent absorbance peaks of the MB.
In contrast, the absorbance of MB was greatly (about 50%) decreased in the presence of enzyme (Figure 3b) compared with the value of 61 500 M􏰀1 cm􏰀1 determined for the conditions used here, an aqueous solution of 50mM potassium phosphate, pH7.4, containing 0.05% Brij. The molar extinction coefficient for MB is influenced by the concentration at which it is determined (Lewis et al., 1943; Bergmann and O’Konski, 1963), by the salt concentration in aqueous solution (Fujimoto et al., 1994), and by the presence of detergent (Yamamoto et al., 2007). Binding to or association with surfaces or other compounds, such as serum albumin (Hu et al., 2005), also alters the absorption spectrum. Thus, the decrease in absorbance of MB induced by enzyme was likely to be due to nonspecific absorption rather than specific insertion into the hydrophobic active site and was thus not examined further.
MB acts as an oxidising substrate for MAO A
Proximity of MB to the flavin was confirmed by the reduction of MB by kynuramine in the presence of MAO A under anaerobic conditions (Figure 4). MAO A with 10mM MB was made anaerobic and excess kynuramine (0.3mM final concentration) was added from the sidearm. Spectra were collected every 30 min overnight (Figure 4a). The time courses for the absorbance of MB (664 nm) and for the flavin (456nm) show that the flavin was rapidly reduced by the excess of substrate, then reoxidised until MB reduction reached steady state (Figure 4b). The steady-state rate of reduction of MB over 12 h (Figure 4b) was 0.35 nmol h􏰀1 and the rate of oxidation of kynuramine was 0.39nmolh􏰀1. To compare this rate with that where oxygen is the electron acceptor, as in Figure 2, the observed velocities were converted to rate constants to normalise for the different enzyme concentrations. The rate constant for the oxidation of kynuramine by MB was 0.045h􏰀1, extremely slow compared to the rate constant with oxygen at
British Journal of Pharmacology (2007) 152 946–951
Methylene blue inhibits monoamine oxidase A
RR Ramsay et al 949
Figure 3 Titration of MAO A with methylene blue (MB). Increments of MB (1 mM) were added to MAO A (7.6 mM) and the spectra recorded for MAO A with final concentrations of 0–10 mM MB as indicated. Arrows indicate the increase at 665 nm due to MB and the decrease in the flavin region. (a) The difference between the spectrum for MAO A with 6 mM or 10 mM MB and the spectrum for MAO A alone is shown in (b). © The difference spectra are the same as (b) but on an expanded scale to show the change in the absorbance of the MAO A flavin.
Figure 4 Methylene blue (MB) acts as the oxidising substrate for MAO A under anaerobic conditions. (a) MAO A (8 mM) with 10 mM MB was made anaerobic and 0.3 mM kynuramine added at time 0. The absorbance of MB at 665 nm decreased with time as indicated by the arrow. The flavin absorbance at 456 nm decreased during the mixing time but returned to its starting value over 3 h. (b) The steady-state rate of oxidation of kynuramine (314 nm) and of reduction of MB (665 nm) were compared between 4 and 12 h when the flavin absorbance at 456 nm was constant.
4950 h􏰀1. Others have previously reported that MB gave a 500-fold slower rate than oxygen with MAO B (Shumakovich et al., 2004).
MB in the presence of reducing agents reduces MAO A
MB is commonly used as a photosensitiser. Light excites MB into a triplet state that can extract electrons from amines
British Journal of Pharmacology (2007) 152 946–951
Methylene blue inhibits monoamine oxidase A
RR Ramsay et al
Figure 5 Reduction of MAO A by methylene blue (MB) in the presence of D-amphetamine and dithiothreitol (DTT). MAO A was diluted eightfold from stock to 27 mM so that the concentration of D-amphetamine was 60mM and of DTT about 0.35mM, was made anaerobic, and 30 mM MB added both to the MAO A cuvette and to the blank cuvette. The spectral change after the addition showed some reduction of MB (decrease at 665 nm) and appearance of the features of the MAO A anionic semiquinone (increase at 380 and 412nm, decrease at 456nm). After 2h at 201C, the spectrum indicated almost complete conversion of MAO A to the semiquinone form. No further change was observed even 24 h later.
generating reduced MB (Kim and Scranton, 2004). Thiol- reducing agents are often used to produce reduced MB (for example, Jonnalagadda and Tshabalala, 1992). When MB was added anaerobically to MAO A-containing dithiothreitol (normally present for storage) at about 0.3 mM and the inhibitor amine, D-amphetamine, at 50mM (Ki for MAO A is 20 mM (Ramsay and Hunter, 2002)), reduction of MAO A was observed. Figure 5 shows the decrease at 456nm and the appearance of the 412 nm peak typical of the anionic flavosemiquinone in MAO A in the presence of 30mM MB. After 2 h, the one-electron reduction of MAO A was complete. No further reduction to the dihydroflavin was observed because of the presence of D-amphetamine that stabilises the MAO semiquinone form (Ramsay and Hunter, 2002). Only about 1% radical was generated in the absence of dithiothrei- tol, so the radical generation requires the thiol reagent.
Discussion
MB has been in medical use for many years with few reports of toxic effects. In early experiments, oral MB was found to be innocuous in 15 kg dogs at 2 g per day, but an intravenous dose of 0.5 g was lethal (Agell and Agell, 1929). The recent case reports of neurological sequelae following MB infusions summarised by Gillman (2006c) confirm that MB is not an innocuous substance. Indeed, MB is cytotoxic to bacteria and has been reported to increase oxidative stress (May et al., 2003). Furthermore, its reactive redox chemistry led to its use as a photosensitiser. However, the particular clinical observa- tions in ST and their occurrence only in patients on SSRIs indicated that MB must also influence the 5-hydroxytrypta- mine system.
The high Ki value for MAO B of 5.6mM (Bachurin et al., 2001), confirmed by the IC50 reported here, was not
950
British Journal of Pharmacology (2007) 152 946–951
consistent with MAO B as a target for the action of MB. Despite this, in rats, where the brain concentration of MB was estimated to be about 0.5 mM (Callaway et al., 2004), both MAO inhibition and increase of 5-hydroxytryptamine as a result of MB treatment have been reported (Lerch et al., 2006). If MAO B is not inhibited, then MAO A could be the target. At the molecular level, both the crystal structure (Binda et al., 2002) and the inhibitor profile for MAO B (Efange et al., 1993) indicate that the active site is relatively narrow. In contrast, MAO A accommodates (De Colibus et al., 2005) and is strongly inhibited by larger molecules such as b-carbolines (Kim et al., 1997) and oxazolidinones such as linezolid (Jones et al., 2005). The data presented here establish that MB is a very potent inhibitor of MAO A with a Ki of 27 nM and that it binds in the active site of MAO A.
The prediction based on the clinical observations, that MAO A would be inhibited by MB, has been proven. It is consistent with structural information about the two MAO isoforms and is supported by information from studies on rat brain. MB is an effective MAO A inhibitor and thus will influence 5-hydroxytryptamine levels at doses expected during MB infusion. For comparison, linezolid, for which the risk of ST in patients receiving SSRIs is established (Morales-Molina et al., 2005), has a Ki for MAO A of 20 mM (Jones et al., 2005). We conclude that the use of MB by infusion in combination with other 5-hydroxytryptaminergic agents could lead to the ST syndrome as a result of MAO A inhibition.
Acknowledgements
Some of the experiments formed part of CD’s undergraduate thesis (University of St Andrews). Some initial experiments were performed by Mr Aleksey Zholobenko, University of St Andrews, during summer work experience and were reported in a poster presented at the 12th Amine Oxidase Workshop, August 2006 and at the Biochemical Society focus meeting on Health Implications of Dietary Amines, October 2006 (http://www.biochemis...ings/postertoc/ SA062/default.htm).


Edited by aaron43, 21 July 2011 - 10:13 AM.

  • like x 3
  • dislike x 1

#38 snuffie

  • Guest
  • 52 posts
  • 10
  • Location:Canada
  • NO

Posted 21 July 2011 - 02:11 PM

Can anyone with journal access acquire and share the full text of these articles? I think the first one might be particularly applicable to our research...

Schwiebert C, Irving C, Gillman PK. Small doses of methylene blue, previously considered safe, can precipitate serotonin toxicity. Anaesthesia. Aug 2009;64(8):924.

Ramsay RR, Dunford C, Gillman PK. Methylene blue and serotonin toxicity:inhibition of monoamine oxidase A (MAO A) confirms a theoretical prediction. BR J Pharmocol. 2007/11;152:946-951.

...sigh...journal access is what I miss most about university...

The science publishing houses are inhibiting the progress of science though their greed. Most of the work they publish is publicly funded, but they lock it up for profit. (rant...)

I don't have the full text, except for Ramsay et al., but the following abstract explains what Gillman means by a "small dose" of MB:

J Psychopharmacol. 2011 Mar;25(3):429-36. Epub 2010 Feb 8.
CNS toxicity involving methylene blue: the exemplar for understanding and predicting drug interactions that precipitate serotonin toxicity.
Gillman PK.

PsychoTropical Research, Bucasia, Queensland, Australia. kg@matilda.net.au

Methylene blue has only recently been noted to cause severe central nervous system toxicity. Methylene blue is used for various conditions, including, intravenously, in methemoglobinemia, vasoplegia and as an aid to parathyroidectomy (at doses of 1-7.5 mg kg(-1)). This review of the current evidence concludes that 13 of 14 of the reported cases of CNS toxicity were serotonin toxicity that met the Hunter Serotonin Toxicity Criteria. That has important preventative and treatment implications. Serotonin toxicity is precipitated by the monoamine oxidase inhibitor (MAOI) property of methylene blue interacting with serotonin reuptake inhibitors. Serotonin toxicity is reviewed, using the lessons inherent in the methylene blue story and experience, to illustrate how the mechanisms and potency of serotonergic drugs interact to determine severity. Recent human data showed that an intravenous dose of only 0.75 mg kg(-1) of methylene blue produced a peak plasma concentration of 500 ng ml(-1) (1.6 µM), indicating that the concentration in the central nervous system reaches a level that inhibits monoamine oxidase A. That is consonant with the actual occurrence of severe serotonin toxicity in humans at the dose of only 1 mg kg(-1). It seems that all proposed uses of methylene blue entail levels that block monoamine oxidase, so cessation of serotonin reuptake inhibitors should be very carefully considered before using methylene blue.

PMID: 20142303

The lowest i.v. dose noted, 0.75 mg/kg would correspond to 52.5 mg intravenous for a 70 kg adult. If we accept the common factor of ten difference between oral and iv MB, we would be talking about an oral dose of 525 mg, or eight thousand seven hundred fifty times the 60 mcg nootropic dose.

Methylene Blue has been used with some fairly whopping doses, historically, so 'small' in this context is relative.


Thanks!:) That's excellent. (and I fully agree re. journal access...*shakes fist*)

#39 thedevinroy

  • Guest
  • 1,188 posts
  • 326
  • Location:USA
  • NO

Posted 21 July 2011 - 02:20 PM

As far as a MAO inhibitor goes, Methylene Blue can act as one at the dose I am taking. If you figure that the oral is 10x less than the IV peak level.

http://www.ncbi.nlm....les/PMC2078225/

MB inhibits 50% MAO-A at 164nM.
MB molecular weight is 319.85 g/mol.
Average volume in blood stream is 6L.
Constant of 10x that of IV needed.

164 * (10^(-9)) * 319.85 * 6 * 10 / 1000 = 3.15mg

Don't know all that fancy pharmacology IC50 stuff that well, but if 3.15mg is true, then an equivalent of 3.15mg of competition must exist. Thus, at 1.15mg, the inhibition would yeild...

1.15 / (3.15 + 1.15) = 26.74%

Probably none of that is correct, but one thing I would like to state is that in my personal experience, I can feel the MAOi effects at 1.15mg. The point was to just show that the in vitro effects happened at very low concentrations, in the nanamoles, and that does have some effect in vivo if well absorbed at the milligram dose.

Some of this is correct, but the analysis is incorrect in a couple important ways. It's very very hard to predict in vivo concentrations like this, but this sort of analysis at least provides an upper limit. However, it is an upper limit for the maximum concentration that can be reached, which is very transient. Whenever we ingest a drug, the concentration in blood rises fairly quickly to a maximum, then starts falling off. The maximum concentration doesn't last long at all. More important is the shape of the IC50 curve. It's a sigmoid, with %inhibition on the y axis and concentration on the x axis. As a result of this non-linearity, half the concentration of the inhibitor gives a lot less than half the inhibition. When you say that you feel the MAOI effects, what does that feel like? Have you taken MAOIs before?

It feels like coffee without the jitters. It feels like a mood lift. It feels like I can smile easier.

Yeah I figured it was incorrect... I just can't find a good vitro to vivo conversion chart, table, calculator, etc. and I'm just sick of people saying, "I don't know," or "I can't give you an estimate," or whatever form of fear of taking an educated stab at something. At least I tried and not just pulled the result out of a black hole in my imagination... well, not completely, anyway.

#40 X_Danny_X

  • Guest
  • 344 posts
  • -2

Posted 21 July 2011 - 03:07 PM

this is the full text: Its pretty short actually <snip>


this is so much to read and understand. basically MB can become serotonin toxic with inhibiting MAOI too much, even in low doses?

Edited by chrono, 09 October 2011 - 01:29 AM.

  • dislike x 3

#41 thedevinroy

  • Guest
  • 1,188 posts
  • 326
  • Location:USA
  • NO

Posted 21 July 2011 - 03:37 PM

this is so much to read and understand. basically MB can become serotonin toxic with inhibiting MAOI too much, even in low doses?

No, you would need a dose of about a half a gram. Please read niner's post on a page or two back for more of an explanation.

#42 aaron43

  • Guest
  • 143 posts
  • 13
  • Location:California

Posted 21 July 2011 - 06:31 PM

this is so much to read and understand. basically MB can become serotonin toxic with inhibiting MAOI too much, even in low doses?


no no snuff just asked for the full study, i can get access to most of the studies

#43 niner

  • Guest
  • 16,276 posts
  • 2,000
  • Location:Philadelphia

Posted 21 July 2011 - 06:51 PM


this is so much to read and understand. basically MB can become serotonin toxic with inhibiting MAOI too much, even in low doses?


no no snuff just asked for the full study, i can get access to most of the studies

Please don't paste full journal articles into a post when you can post a link instead. The Ramsay article has already been linked several times in this thread.
  • dislike x 1
  • like x 1

#44 snuffie

  • Guest
  • 52 posts
  • 10
  • Location:Canada
  • NO

Posted 21 July 2011 - 11:54 PM

Can anyone with journal access acquire and share the full text of these articles? I think the first one might be particularly applicable to our research...
Schwiebert C, Irving C, Gillman PK. Small doses of methylene blue, previously considered safe, can precipitate serotonin toxicity. Anaesthesia. Aug 2009;64(8):924.
Ramsay RR, Dunford C, Gillman PK. Methylene blue and serotonin toxicity:inhibition of monoamine oxidase A (MAO A) confirms a theoretical prediction. BR J Pharmocol. 2007/11;152:946-951.
....

this is the full text: Its pretty short actually Schwiebert C, Irving C, Gillman PK. Small doses of methylene blue, previously considered safe, can precipitate serotonin toxicity. Anaesthesia. Aug 2009;64(8):924.....


hey thanks for the full text, that's awesome, i'll have a read....

p.s., re. him/her/it, i am a 'her'...although in the morning i often resemble an 'it' :laugh:

@niner, sorry for inadvertently clogging up the thread! next time i will ask if someone can email it / paste into a linked google doc / something.

Edited by snuffie, 22 July 2011 - 12:00 AM.


#45 niner

  • Guest
  • 16,276 posts
  • 2,000
  • Location:Philadelphia

Posted 22 July 2011 - 12:18 AM

Niner: I can't post the link, because I go to a university, and if I did, all you guys would just get the abstract because your not logged in. And even if it was already posted in the thread, snuff asked for it, so Im giving it to her/him/it, as well as the other study that is not provided in this forum. Please stop trying to tell me what I can and cannot do, I won't listen because I don't care, the only thing I'm interested in is the academic information. It will be a waste of threadspace to try and critique my posts, and is weird since your all about not wasting threadspace. And this nitpicking needs to stop because again this person asked for the full study, something that I have access too, and I gave it to them. I don't need to know your personal opinion on the way I post, but would much rather here about what you think about the information that is included in the posts. If you have nothing to say on the subject that is in the post, just like you tried to tell me, then you don't need to say anything

Aaron, the full text of that article is available for free, and has been posted in this thread multiple times, including in a response to snuffie, not to mention in a response to one of your posts, which I take it you didn't read. Sadly, as moderator, it's my job to tell you what you can and cannot do. There is no "right" to post here, and we particularly frown on misinformation.

#46 aaron43

  • Guest
  • 143 posts
  • 13
  • Location:California

Posted 22 July 2011 - 04:10 AM

Niner: I can't post the link, because I go to a university, and if I did, all you guys would just get the abstract because your not logged in. And even if it was already posted in the thread, snuff asked for it, so Im giving it to her/him/it, as well as the other study that is not provided in this forum. Please stop trying to tell me what I can and cannot do, I won't listen because I don't care, the only thing I'm interested in is the academic information. It will be a waste of threadspace to try and critique my posts, and is weird since your all about not wasting threadspace. And this nitpicking needs to stop because again this person asked for the full study, something that I have access too, and I gave it to them. I don't need to know your personal opinion on the way I post, but would much rather here about what you think about the information that is included in the posts. If you have nothing to say on the subject that is in the post, just like you tried to tell me, then you don't need to say anything

Aaron, the full text of that article is available for free, and has been posted in this thread multiple times, including in a response to snuffie, not to mention in a response to one of your posts, which I take it you didn't read. Sadly, as moderator, it's my job to tell you what you can and cannot do. There is no "right" to post here, and we particularly frown on misinformation.


your superhero moderator response is useless, notice how you didn't comment on any of the actual information in my post, like I said useless.
and misinformation for providing two studies and for giving information to my knowledge and even saying "correct me if I'm wrong"? I take it you didn't read.

Edited by aaron43, 22 July 2011 - 04:13 AM.

  • dislike x 2

#47 Nootr

  • Guest
  • 180 posts
  • -9
  • Location:Spain

Posted 22 July 2011 - 07:55 AM

I am also taking melatonin, GABA and 5-HTP. I hope I won't have any problems if I take M.B. also.

#48 thedevinroy

  • Guest
  • 1,188 posts
  • 326
  • Location:USA
  • NO

Posted 22 July 2011 - 08:42 PM

I am also taking melatonin, GABA and 5-HTP. I hope I won't have any problems if I take M.B. also.

Methylene Blue doesn't act as an MAOi at microgram doses, so you can stay on the MAO-B inhibitor if you want. Just take less than a milligram.

EDIT: And I see no other interactions. The only known interactions at the nootropic dose are from CoQ-10 and Idebenone.

Edited by devinthayer, 22 July 2011 - 09:03 PM.


#49 MrHappy

  • Guest, Moderator
  • 1,815 posts
  • 404
  • Location:Australia

Posted 15 August 2011 - 10:13 AM

This substance seems really really interesting. I'm on escitalopram and mirtazapine (as well as dexamphetamine which I however could skip) though so I'm not sure I want to take it..
Could anyone tell me:
How well do you find MB to improve focus in comparison to amphetamine?
How effective do you find the lower dose range to be (around 20 mg or so) ?

First, 20mg is NOT a lower dose - that's a bloody large dose! Most people start on 60ug and work up to 1mg.
Second, according to the FDA and a lot of other literature, don't go anywhere near this stuff while you are taking SRIs unless you want to die.
Third and last, are you known to be G6PD deficient? If so, don't touch it either - you'll probably die, also.

Other than that, it's worth a look, I think.

#50 nupi

  • Guest
  • 1,532 posts
  • 108
  • Location:Switzerland

Posted 15 August 2011 - 11:13 AM

Several posters claim the 60ug are far below the doses required to activate MB's MAOI properties but so far I have stayed away from it for exactly that fear, even though I am not technically on an SRI (in general, I feel there is somewhat murky understanding what exactly Bupropion does so best not to temp fate). Mixing any RI with a potential MAOI seems like a bad idea ™.

#51 MrHappy

  • Guest, Moderator
  • 1,815 posts
  • 404
  • Location:Australia

Posted 15 August 2011 - 11:29 AM

Several posters claim the 60ug are far below the doses required to activate MB's MAOI properties but so far I have stayed away from it for exactly that fear, even though I am not technically on an SRI (in general, I feel there is somewhat murky understanding what exactly Bupropion does so best not to temp fate). Mixing any RI with a potential MAOI seems like a bad idea ™.


Sure... but most people seem to have shuffled onto 1mg doses, too. Not sure how much 60ug does, really.

#52 niner

  • Guest
  • 16,276 posts
  • 2,000
  • Location:Philadelphia

Posted 15 August 2011 - 03:27 PM

First, 20mg is NOT a lower dose - that's a bloody large dose! Most people start on 60ug and work up to 1mg.
Second, according to the FDA and a lot of other literature, don't go anywhere near this stuff while you are taking SRIs unless you want to die.
Third and last, are you known to be G6PD deficient? If so, don't touch it either - you'll probably die, also.

20mg is not a low dose by our standards, although it is if you compare it to other uses of MB, where oral doses of hundreds of milligrams are used. However, I have to take issue with your use of words like "unless you want to die". No one is going to die from serotonin syndrome at anywhere near the doses we are talking about. In the literature that brought the MAOI problem to our attention, it occurred with an IV dose of 50mg, which corresponds to an oral dose of 500mg. An oral dose of one or two milligrams just isn't enough to cause a problem. The IC50 for MB with MAO-A is 164 nM. While that is in the ballpark of levels we're trying to hit, it's only 50% inhibition. To generate a toxic serotonin syndrome you would need to seriously shut down your MAOs for a long time, and we aren't doing that at these doses. There would be a lot of very noticeable symptoms from serotonin excess well before lethality, so you could stop dosing MB, which would wash out soon enough (half life 5-6 hrs)

G6PD deficiency comes in various degrees of severity. If you have this X-linked genetic disease, you probably know it, or have the less-severe form. To say that we will "probably die" at these doses is hyperbole.
  • like x 1

#53 MrHappy

  • Guest, Moderator
  • 1,815 posts
  • 404
  • Location:Australia

Posted 15 August 2011 - 10:12 PM

First, 20mg is NOT a lower dose - that's a bloody large dose! Most people start on 60ug and work up to 1mg.
Second, according to the FDA and a lot of other literature, don't go anywhere near this stuff while you are taking SRIs unless you want to die.
Third and last, are you known to be G6PD deficient? If so, don't touch it either - you'll probably die, also.

20mg is not a low dose by our standards, although it is if you compare it to other uses of MB, where oral doses of hundreds of milligrams are used. However, I have to take issue with your use of words like "unless you want to die". No one is going to die from serotonin syndrome at anywhere near the doses we are talking about. In the literature that brought the MAOI problem to our attention, it occurred with an IV dose of 50mg, which corresponds to an oral dose of 500mg. An oral dose of one or two milligrams just isn't enough to cause a problem. The IC50 for MB with MAO-A is 164 nM. While that is in the ballpark of levels we're trying to hit, it's only 50% inhibition. To generate a toxic serotonin syndrome you would need to seriously shut down your MAOs for a long time, and we aren't doing that at these doses. There would be a lot of very noticeable symptoms from serotonin excess well before lethality, so you could stop dosing MB, which would wash out soon enough (half life 5-6 hrs)

G6PD deficiency comes in various degrees of severity. If you have this X-linked genetic disease, you probably know it, or have the less-severe form. To say that we will "probably die" at these doses is hyperbole.


Well, fair enough, I didn't mean instant death.. more the kind of thing that self-experimenting, unchecked, for a number of weeks with variable dosage could lead to - http://www.aafp.org/...otonergics.html .

I have also been reading conflicting mentions of the half-life of MB. Some say 4-5.5 hours, others are saying 10-12 hours. Could leading to excessive dosing.

Additionally, I would like to know from everyone else on here experimenting .. call it a 'poll':
How many of you have 'sore' or 'tight' feeling necks and what dosage are you taking?

#54 MrHappy

  • Guest, Moderator
  • 1,815 posts
  • 404
  • Location:Australia

Posted 31 August 2011 - 04:25 AM

The antidepressant effect is probably due to MB being a MAOI. Elevated dopamine levels in the ventral tegmental area would simulate a 'reward' response in the brain.
  • dislike x 1

#55 MrHappy

  • Guest, Moderator
  • 1,815 posts
  • 404
  • Location:Australia

Posted 31 August 2011 - 10:08 PM

I have a question.If methylene blue really works as a nootropic.How could it not be used as a treatment for Dementia.
Although MB has been widely used by doctor for anoyher purpose like antidote or placebo for decades.
If it really work, it should be researched more ealier.Because someone of the patient wii notice the effect on mental.
Why? I really wanna know.


It has been in clinical trials for a number of years. Google "methylene blue alzheimer's". 2 products are Rember from TauRx and ProveBlue from ProvePharm. The dosage is a key factor.

@everyone: I'm concerned (for healthy younger people) that as a MAO-I, the common 1mg dose we've been experimenting with (and enjoying a feeling of wellbeing from) will lead to increased tolerance to dopamine. This could lead to mental health or dependancy issues longer term. If the nootropic effect is best realised at 3nM / 60mcg, that would be a safer dose level and avoid the 'antidepressant effect' and risk. Edit:typos

Edited by MrHappy, 31 August 2011 - 10:09 PM.


#56 longevitynow

  • Guest
  • 266 posts
  • 31
  • Location:Mexico City

Posted 03 September 2011 - 12:57 AM

It has been in clinical trials for a number of years. Google "methylene blue alzheimer's". 2 products are Rember from TauRx and ProveBlue from ProvePharm. The dosage is a key factor.

@everyone: I'm concerned (for healthy younger people) that as a MAO-I, the common 1mg dose we've been experimenting with (and enjoying a feeling of wellbeing from) will lead to increased tolerance to dopamine. This could lead to mental health or dependancy issues longer term. If the nootropic effect is best realised at 3nM / 60mcg, that would be a safer dose level and avoid the 'antidepressant effect' and risk. Edit:typos


Using MB for Alzheimer disease and in studies for Bipolar disorder, I believe the dose was around 50 mg 3x a day. I'm skeptical that the 1 mg dose that a lot of people are experimenting with will affect dopamine or have a significantly large MAO-I effect (i.e. comparable to drugs). I don't doubt that this low dose may have an effect on these systems in the body (psychiatric drugs are typically given at much higher doses than necessary, IMO), but at a dose of 1 mg the numerical effect on these systems compared to meds is minimal (and therefore probably very safe).

#57 MrHappy

  • Guest, Moderator
  • 1,815 posts
  • 404
  • Location:Australia

Posted 03 September 2011 - 06:50 AM


Using MB for Alzheimer disease and in studies for Bipolar disorder, I believe the dose was around 50 mg 3x a day. I'm skeptical that the 1 mg dose that a lot of people are experimenting with will affect dopamine or have a significantly large MAO-I effect (i.e. comparable to drugs). I don't doubt that this low dose may have an effect on these systems in the body (psychiatric drugs are typically given at much higher doses than necessary, IMO), but at a dose of 1 mg the numerical effect on these systems compared to meds is minimal (and therefore probably very safe).


Well, I can tell you from personal experience, having just decided to see what coming off 3x1mg dose is like that the MAO-I effect is very noticeable. It's been 4 days and I'm still not back to normal brain chemistry. Feels like coming down after a week long bender of dance parties.. Empty headed.

#58 FDA Approved

  • Guest
  • 101 posts
  • 14
  • Location:UK

Posted 04 September 2011 - 08:31 AM

Even from 60mcg?

What colour was the glass of water after you put 1mL of the 'intermediate' solution into it?


I didnt make it with one ml in 330 like you suggested. I made it by putting one ml in 20 and then one ml from that in 15ml. In the cup I drink it changes just barely, but it doesnt really become too blue(its a bit hard to judge, because I am using blue plastic cups). There was a photo earlier in the thread of 600mcg in a glass of water and mine didnt seem to come close to that. I might do it in a glass later to check how much it changes. And the detachment effect was really significant - I wasnt even feeling scared/freaked out when watching something relatively scary last night, nor was I getting as angry at things that usually get me angry. Also I wasnt feeling exactly tired, but still it made me go to sleep an hour and a half earlier and I slept at least an hour more.
FWIW I am taking it with my regular dose of 3x800-900mg Piracetam + 200-300mg Cdp-Choline and Ginkgo Biloba. Also I took a Multi-vitamin immediately after drinking the Blue.

Edited by FDA Approved, 04 September 2011 - 08:49 AM.


#59 MrHappy

  • Guest, Moderator
  • 1,815 posts
  • 404
  • Location:Australia

Posted 04 September 2011 - 09:28 PM

Hmmm well Ginkgo is already a MAO-I, so that explains it. Taking 2 MAO-Is without a 2 week separation is a bit risky. It will be hard to estimate your monoamine levels. Edit: always make typos from using my phone!

Edited by MrHappy, 04 September 2011 - 09:30 PM.


sponsored ad

  • Advert
Rent this spot in Nootropics Topics to support Longecity (this will replace the google ad).

#60 FDA Approved

  • Guest
  • 101 posts
  • 14
  • Location:UK

Posted 05 September 2011 - 08:33 AM

Hmmm well Ginkgo is already a MAO-I, so that explains it. Taking 2 MAO-Is without a 2 week separation is a bit risky. It will be hard to estimate your monoamine levels. Edit: always make typos from using my phone!


Good catch, I forgot that Ginkgo is a mild MAO-I. I will stop both Ginkgo and Methylene Blue for a few days and then continue only with Methylene Blue. Thanks for the help, MrHappy.




5 user(s) are reading this topic

0 members, 5 guests, 0 anonymous users