Quote: Glutathione is required for the methylation of B12,
methjylb12 doesn't require methylation, obviously assuming inactive b12.
This reference seems to contradict that gluathione is antagonistic to B12 absorption as I've read on this thread??
My experience was that glutathione (precursors) antagonized methylfolate producing immediate hard folate deficiency and immediately increased inflammation and inflammatory response throughout the body. Further it aqppeared to (and according to Rich biochemically
I would like to comment on the interaction of glutathione and vitamin B12, both in the normal metabolism and in freddd's metabolism, as I understand it. I hope everyone understands that I am trying to be constructive in doing so, not trying to be argumentative.
In the normal metabolism, glutathione is used by the cells to remove alkyl ligands, such as methyl or adenosyl, from incoming cobalamins. This has been shown in a recent study, the abstract of which is pasted below. Later in the normal processing of B12 in the cells, either a methyl group is reattached to the cobalamin after it is bound to the enzyme methionine synthase, or an adenosyl group is attached to it after it is transported into the mitochondria.
In addition, although it is not yet clear in detail, it appears that between these parts of the normal processing, glutathione binds with cobalamin to form glutathionylcobalamin, and this serves to protect the cobalamin from reacting with other substances and being lost from its normal uses in the cells. Evidence for the presence of glutathionylcobalamin in cells has been reported in a recent paper by Hannibal et al., the abstract of which is also pasted below, and the reaction between aquocobalamin and glutathione has been found to be quite rapid (abstract below by Xia et al.). So in the normal metabolism of B12, glutathione plays a significant, positive role.
Based on this, in the GD--MCB hypothesis I have suggested that CFS onset results when glutathione is sufficiently lowered by a variety of possible causes in a person with the appropriate genomic predisposition that B12 is sufficiently lost from production of methyl B12 so that a partial block in the methylation cycle occurs. This then reflects back on the ability to produce glutathione, keeping it depleted and causing CFS to be a chronic illness.
freddd has reported that in his case, he found glutathione or its precursors to be deleterious. He has also reported information suggesting that he has inherited a mutation or inborn error of metabolism in his B12 processing enzymes. I suggest that based on his experience with glutathione, this mutation must be located in an enzyme that is involved in a step beyond the use of glutathione to remove the methyl group from cobalamin. Apparently, his cells are not able to replace the methyl group on cobalamin after it has been removed, or to add an adenosyl group to cobalamin. Thus, he has found that the only way his cells can get enough methylcobalamin and adenosylcobalamin is to supply these active, coenzyme forms directly as supplements, taken in large dosages. This is what might be referred to in the physical sciences as a "brute force" approach. I can understand that it is the only thing that works in a case like his, and I'm glad that he discovered it.
However, I want to emphasize that this biochemical behavior is unusual, and it is not likely to be the case in most people who have CFS, because the mutations that can cause this are rare in the human population. Many people with CFS have reported that they have found glutathione to be beneficial to them. There are also some who have not reacted well to it. It isn't clear that this is because of interference with B12 utilization, as in freddd's case, however. It may result from utilization of glutathione by yeasts in the gut, mobilization of toxins into the blood by improved operation of the detox system, stimulation of the immune system, producing a Herxheimer effect, or breakdown of some of the glutathione to form sulfite, which is more than the sulfite oxidase enzyme can handle. I don't think we know which of these mechanisms could be involved in a given case.
In order for freddd's approach to work, it must be true that by putting large concentrations of the coenzyme forms of B12 into the blood, enough of each of them diffuses into the cells and survives removal of their ligands to give the cells enough methyl B12 and adenosyl B12 directly to satisfy their needs for them. As far as I know, this is not described in the current research literature. Over the past weekend, I had the opportunity to talk personally to Prof. Barry Shane at a medical conference. He is from U.C.-Berkeley, and is an authority on folate and B12 metabolism. I asked him whether B12 could enter cells without being bound to transcobalamin, and he initially said no. Then I told him that there appears to be evidence that if large dosages are used, some unbound methyl B12 and adenosyl B12 are able to enter cells. He then conceded that if the concentrations were high enough, this might be possible. He compared this to the known ability of the body to absorb 1 % of B12 that passes through the gut, even without intrinsic factor. However. he said that occurs by diffusion through the tight junctions between the enterocytes that line the intestine, and that would be different from entry through a cell membrane. Anyway, this conversation confirmed to me that reserachers have not yet studied the direct transport of B12 through the cell membranes, which is apparently what happens in freddd's treatment. I don't doubt that it occurs. It just doesn't seem to have been studied.
freddd has described, and the experiences reported by others here seem to confirm, that his treatment approach will work on people who have a wide variety of B12-related problems, including those who have CFS, which involves a partial block in the methylation cycle. This is understandable, because freddd's treatment bypasses all of the normal processes of absorption by the gut, transport by transcobalamin, and processing of B12 within the cells to form the coenzyme forms of B12, and it does this by supplying them directly in large dosages.
However, I suggest (and freddd may not agree) that this approach is actually "overkill" for most people with CFS. I suggest that the reason is that most people with CFS have normal absorption, transport and processing of B12, except for glutathione depletion, which allows their B12 to be hijacked within the cells by reaction with other substances, including toxins. This is why hydroxocobalamin in high dosages, together with 5-methyl tetrahydrofolate to compensate for the methyl trap that resulted when their availability of methyl B12 decreased due to this hijacking, works for most of them. I suggest that the reason it did not work for nearly a third of them is not that hydroxocobalamin is not effective for them, but that they have had other issues that have prevented the methylation cycle function from being restored, such as depletion of vitamin and mineral cofactors for this part of the metabolism (such as zinc, magnesium, or other B-vitamins) or depletion of amino acids, including methionine, or high body burdens of toxic metals that block the enzymes in this part of the biochemistry.
I want to reiterate that according to the literature, mutations in the intracellular B12 processing enzymes are rare. The most recent paper I've found reports that only about 400 people have been identified worldwide as having these mutations. Of course, there must be people who have not been identified, especially in the underdeveloped countries, but nevertheless, the evidence is that these mutations are rare. When this number is compared to the much larger number of people who have CFS (of the order of a million in the U.S. alone), it is clear that most people with CFS do not have these mutations. According to Prof. Shane, more of the problems with B12 involve transcobalamin deficiency, so that B12 cannot be properly transported from the gut to the cells. freddd's treatment should also work for people who have this problem, but I don't think they would respond in a negative way to glutathione, as he did, because very few of them would be expected to also have a mutation in their intracellular B12 processing enzymes.
Another issue that continues to be discussed here is whether it is better to "push through" the symptoms and continue with large dosages of the coenzyme forms of B12, or whether it is better to start with lower dosages of folate and B12 ( such as hydroxocobalamin) as in the Simplified Treatment Approach that I have suggested, and work up as tolerated. I don't have a hard and fast answer to this, and it may depend on the individual and their body burden of toxins.
I know that some people have been able to more or less continue with freddd's protocol and have been reporting benefits from doing so. I also know that there are people who have found this approach to be intolerable in terms of the symptoms that have arisen, and they have dropped back and have taken a slower approach.
I don't think I am able to judge what people should do about this, because unless I were "in their skins," I have no way to know how severe their symptoms are. I also don't know if mobilizing toxins (such as mercury) at higher levels can move them to less desirable locations in the body, such as the brain, or whether large dosages of methyl B12 can eventually remove them from the brain.
In view of these unknowns, my tendency is to advise caution, and to favor the more gradual approach, but I don't have personal experience with this, as freddd does, and I can't argue with his personal experience. He also reports that there have been a number of others who have had similar experiences, though I'm not aware of controlled studies with these higher dosages.
The best data I have for the more gradual approach is from the clinical study that Dr. Nathan and I carried out with 30 women. Two-thirds of them improved, some markedly. Two of them were able to return to full-time work, and the 84-year-old was able to join her friends for a trip to Paris.
Best regards,
Rich
J Biol Chem. 2009 Nov 27;284(48):33418-24. Epub 2009 Oct 2.
A human vitamin B12 trafficking protein uses glutathione transferase activity for processing alkylcobalamins.
Kim J, Hannibal L, Gherasim C, Jacobsen DW, Banerjee R.
Department of Biological Chemistry, University of Michigan Medical Center, Ann Arbor, Michigan 48109-5066, USA.
Pathways for tailoring and processing vitamins into active cofactor forms exist in mammals that are unable to synthesize these cofactors de novo. A prerequisite for intracellular tailoring of alkylcobalamins entering from the circulation is removal of the alkyl group to generate an intermediate that can subsequently be converted into the active cofactor forms. MMACHC, a cytosolic cobalamin trafficking chaperone, has been shown recently to catalyze a reductive decyanation reaction when it encounters cyanocobalamin. In this study, we demonstrate that this versatile protein catalyzes an entirely different chemical reaction with alkylcobalamins using the thiolate of glutathione for nucleophilic displacement to generate cob(I)alamin and the corresponding glutathione thioether. Biologically relevant thiols, e.g. cysteine and homocysteine, cannot substitute for glutathione. The catalytic turnover numbers for the dealkylation of methylcobalamin and 5'-deoxyadenosylcobalamin by MMACHC are 11.7 +/- 0.2 and 0.174 +/- 0.006 h(-1) at 20 degrees C, respectively. This glutathione transferase activity of MMACHC is reminiscent of the methyltransferase chemistry catalyzed by the vitamin B(12)-dependent methionine synthase and is impaired in the cblC group of inborn errors of cobalamin disorders.
PMID: 19801555 [PubMed - indexed for MEDLINE]
Clin Chem Lab Med. 2008;46(12):1739-46.
Accurate assessment and identification of naturally occurring cellular cobalamins.
Hannibal L, Axhemi A, Glushchenko AV, Moreira ES, Brasch NE, Jacobsen DW.
Department of Cell Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA.
BACKGROUND: Accurate assessment of cobalamin profiles in human serum, cells, and tissues may have clinical diagnostic value. However, non-alkyl forms of cobalamin undergo beta-axial ligand exchange reactions during extraction, which leads to inaccurate profiles having little or no diagnostic value. METHODS: Experiments were designed to: 1) assess beta-axial ligand exchange chemistry during the extraction and isolation of cobalamins from cultured bovine aortic endothelial cells, human foreskin fibroblasts, and human hepatoma HepG2 cells, and 2) to establish extraction conditions that would provide a more accurate assessment of endogenous forms containing both exchangeable and non-exchangeable beta-axial ligands. RESULTS: The cobalamin profile of cells grown in the presence of [ 57Co]-cyanocobalamin as a source of vitamin B12 shows that the following derivatives are present: [ 57Co]-aquacobalamin, [ 57Co]-glutathionylcobalamin, [ 57Co]-sulfitocobalamin, [ 57Co]-cyanocobalamin, [ 57Co]-adenosylcobalamin, [ 57Co]-methylcobalamin, as well as other yet unidentified corrinoids. When the extraction is performed in the presence of excess cold aquacobalaminacting as a scavenger cobalamin (i.e. "cold trapping"), the recovery of both [ 57Co]-glutathionylcobalamin and [ 57Co]-sulfitocobalamin decreases to low but consistent levels. In contrasts, the [ 57Co]-nitrocobalamin observed in the extracts prepared without excess aquacobalamin is undetected in extracts prepared with cold trapping. CONCLUSION: This demonstrates that beta-ligand exchange occur with non-covalently bound beta-ligands. The exception to this observation is cyanocobalamin with a non-exchangeable CN- group. It is now possible to obtain accurate profiles of cellular cobalamin.
PMID: 18973458 [PubMed - indexed for MEDLINE]
Inorg Chem. 2004 Oct 18;43(21):6848-57.
Studies on the formation of glutathionylcobalamin: any free intracellular aquacobalamin is likely to be rapidly and irreversibly converted to glutathionylcobalamin.
Xia L, Cregan AG, Berben LA, Brasch NE.
Research School of Chemistry, Australian National University, Canberra ACT 0200, Australia.
A decade ago Jacobsen and co-workers reported the first evidence for the presence of glutathionylcobalamin (GSCbl) in mammalian cells and suggested that it could in fact be a precursor to the formation of the two coenzyme forms of vitamin B(12), adenosylcobalamin and methylcobalamin (Pezacka et al. Biochem. Biophys. Res. Commun. 1990, 169, 443). It has also recently been proposed by McCaddon and co-workers that GSCbl may be useful for the treatment of Alzheimer's disease (McCaddon et al. Neurology 2002, 58, 1395). Aquacobalamin is one of the major forms of vitamin B(12) isolated from mammalian cells, and high concentrations of glutathione (1-10 mM) are also found in cells. We have now determined observed equilibrium constants, K(obs)(GSCbl), for the formation of GSCbl from aquacobalamin and glutathione in the pH range 4.50-6.00. K(obs)(GSCbl) increases with increasing pH, and this increase is attributed to increasing amounts of the thiolate forms (RS(-)) of glutathione. An estimate for the equilibrium constant for the formation of GSCbl from aquacobalamin and the thiolate forms of glutathione of approximately 5 x 10(9) M(-1) is obtained from the data. Hence, under biological conditions the formation of GSCbl from aquacobalamin and glutathione is essentially irreversible. The rate of the reaction between aquacobalamin/hydroxycobalamin and glutathione for 4.50 < pH < 11.0 has also been studied and the observed rate constant for the reaction was found to decrease with increasing pH. The data were fitted to a mechanism in which each of the 3 macroscopic forms of glutathione present in this pH region react with aquacobalamin, giving k(1) = 18.5 M(-1) s(-1), k(2) = 28 +/- 10 M(-1) s(-1), and k(3) = 163 +/- 8 M(-1) s(-1). The temperature dependence of the observed rate constant at pH 7.40 ( approximately k(1)) was also studied, and activation parameters were obtained typical of a dissociative process (DeltaH++ = 81.0 +/- 0.5 kJ mol(-1) and DeltaS++ = 48 +/- 2 J K(-1) mol(-1)). Formation of GSCbl from aquacobalamin is rapid; for example, at approximately 5 mM concentrations of glutathione and at 37 degrees C, the half-life for formation of GSCbl from aquacobalamin and glutathione is 2.8 s. On the basis of our equilibrium and rate-constant data we conclude that, upon entering cells, any free (protein-unbound) aquacobalamin could be rapidly and irreversibly converted to GSCbl. GSCbl may indeed play an important role in vitamin B(12)-dependent processes.
PMID: 15476387 [PubMed - indexed for MEDLINE]
