Modulation of Nitrergic Transmission by Methylcobalamin and Hydroxocobalamin or Increases in Observed Km's via Sequestration of Endogenous Cobalamins?

This article is really important in the context of the supposed therapeutic uses of high-dose (I'm talking about very high doses, such as the bizarrely-high doses used in research in the treatment of amyotrophic lateral sclerosis (ALS), not the high doses that are used to overcome endogenous inhibitors of B12-dependent enzymes in people who do not have neurodegenerative diseases, etc.) methylcobalamin (MeCbl) or hydroxocobalamin (OHCbl) (forms of vitamin B12 that are not "equivalent." in terms of their effects, to cyanocobalamin) [Oh et al., 1999: (http://www.ncbi.nlm.nih.gov/pubmed/10071959)]. The authors found that extracellular OHCbl scavenged (bound, either reversibly or irreversibly) nitric oxide (NO) and thereby decreased the release of glutamate in response to the activation of NMDA receptors by exogenous N-methyl-D-aspartate (a drug ligand for NMDA glutamate receptors). As far as I can tell, this is the only paper that has provided reasonably-direct evidence of the modulation of glutamatergic transmission, by NO scavenging or "NO buffering," by OHCbl. Other researchers have shown that high concentrations of methylcobalamin (usually 10 uM) can produce either excitation or attenuation of glutamatergic transmission [(http://scholar.google.com/scholar?num=100&hl=en&lr=&q=methylcobalamin+NMDA+OR+glutamate); (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=hydroxocobalamin+NMDA+OR+glutamate); (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=%22nitric+oxide%22+hydroxocobalamin+OR+methylcobalamin)], and the modulation by MeCbl or OHCbl of nitrergic neurotransmission has been shown in the contexts of other neurotransmitter systems, etc. [Colpaert and Lefebvre, 2000: (http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1571952&blobtype=pdf)(http://www.ncbi.nlm.nih.gov/pubmed/10725269)]. Colpaert and Lefebvre (2000) discuss the capacity of OHCbl to either reversibly or irreversibly bind NO to form nitrosocobalamin (NOCbl) and other cobalamins (superoxocobalamin, formed by a reaction of superoxide with Cbl, can also bind NO, and the character of the binding essentially depends on the oxidation state of the cobalt atom of Cbl. These might be some other articles by that group (http://scholar.google.com/scholar?q=Colpaert+cobalamin+OR+hydroxocobalamin+OR+methylcobalamin&num=100&hl=en&lr=).

The paper by Oh et al. (1999) is a really important paper, and it's conceivable that lower concentrations of OHCbl or MeCbl would produce some of this effect. This NO-buffering effect of either MeCbl or OHCbl has been shown in many other articles, and the most commonly-observed net effect of MeCbl or OHCbl is essentially to prolong the action of NO while reducing the amplitude of the initial response, such as the excitatory postsynaptic potential or calcium influx or smooth muscle cell contraction or relaxation. Colpaert and Lefebvre (2000) discuss that type of thing.

A lot of authors have written articles about the NO-scavenging effect of methylcobalamin and the potential relevance to inflammatory conditions that have been associated with "B12-responsiveness." I tend to think these effects on glutamatergic transmission, resulting from NO-buffering (as opposed to, for example, a B12-induced disinhibition of the tricarboxylic acid cycle (TCA cycle) in response to a decrease in intracellular or intramitochondrial methylmalonic acid (MMA) levels), would mainly occur at very high doses (much higher than those used in all but a few clinical trials). I say that because the concentration used in those articles has typically been 10 uM, which is much higher than the serum or extracellular fluid concentrations of Cbl's that are seen normally, even in response to 250-500 ug of MeCbl per day, given parenterally. I showed the serum B12 values for very high dose MeCbl in a past posting, and the levels were only about ~34.2-36.7 nM (http://hardcorephysiologyfun.blogspot.com/2009/01/unanswered-questions-about.html). But I do think that endogenously-produced NOCbl or glutathionylcobalamin could increase the observed/effective Km for the binding of 5'-deoxyadenosylcobalamin to methylmalonyl-CoA mutase (MMM) as a cofactor (or the observed Km for the binding of MeCbl to methionine synthase).

It's remarkable that I've never seen a measurement or estimate of the extent to which endogenously-produced NOCbl or other species could increase the effective Km's for the binding of Cbl-derived cofactors (MeCbl and AdoCbl) to their respective enzymes. Some of the data from cell culture studies hint at that effect [similar to the so-called "arginine paradox," in which increases in extracellular arginine can increase the activities of nitric oxide synthase (NOS) enzymes at concentrations up to 500 uM, which is much higher than the Km, for arginine binding to endothelial NOS, derived from research (research that failed to take into account the pronounced in vivo inhibition of NOS enzymes by ADMA and N(omega)-monomethylarginine and other endogenous NOS inhibitors, etc.]. It would be hard to measure the binding of endogenously-produced NO to Cbl's, though, because the binding can be reversible. I think it could be done, though. Peters et al. (1983) [Peters et al., 1983: (http://jn.nutrition.org/cgi/reprint/113/6/1221.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/6854414)] found that propionate uptake (and, by extension, the propionate oxidation rate) increased as the liver cobalamin contents of sheep increased up to 250 ng/g ww. That works out to about 300 nM (250 ng/g ww x 1000)/(1355.38 x 0.615) (http://hardcorephysiologyfun.blogspot.com/2008/12/cell-biology-conversion-factors-for-ngg.html) as an intracellular concentration of total cobalamins in the livers of the sheep. Given that AdoCbl constitutes ~72.8 percent of the total cobalamins in the livers of humans [Yamada et al., 2000: (http://jn.nutrition.org/cgi/reprint/130/8/1894)(http://www.ncbi.nlm.nih.gov/pubmed/10917899?dopt=Abstract)] (and assuming these percentages and Km values are comparable for sheep and humans, an assumption that might be incorrect), the maximal rate of propionate uptake (and, by extension, oxidation) may not occur until the intracellular AdoCbl concentration is ~218 nM. That's about 3.5-4 times the Km for AdoCbl binding to MMM in the human liver. The Km values for AdoCbl binding to human wild-type MMM have been found to be 50 nM or 62.5 nM in the human liver or in human fibroblasts (http://hardcorephysiologyfun.blogspot.com/2009/01/km-values-for-adocbl-binding-to-mmm-and.html), and the provision of B12 from standard diets in animals or humans, in the absence of supplementation, is likely to produce intracellular concentrations of AdoCbl that are far below the Km for AdoCbl binding to MMM (http://hardcorephysiologyfun.blogspot.com/2009/01/methylcobalamin-and-other-forms-of.html). It's possible that the sheep Km is higher, but I still tend to think endogenous inactivation or "sequestration" of Cbl's (as glutathionylcobalamin or NOCbl) could explain the ongoing findings that higher doses of MeCbl sometimes lower homocysteine levels more effectively than more commonly-used dosages. Also, ischemia can upregulate MMM expression and could increase the effective/observed Km values. When one considers that NO itself (and undoubtedly other endogenous inhibitors) can inhibit MMM activity, it's not unreasonable to think that the observed Km values for Cbl-dependent enzymes might be higher than the strictly-defined values found in the absence of inflammation during in vitro experiments, etc.