from: (http://hmg.oxfordjournals.org/cgi/content/full/6/9/1457 pubmed: http://www.ncbi.nlm.nih.gov/pubmed/9285782?dopt=Abstract) (Janata et al., 1997)
Km = 62.5 nM
(from cited reference 4 and normalized from protein content in reference 17)
Km = 50 nM [value from Janata et al. (1997)]
On the surface, this, together with the estimates of the concentrations of intracellular total cobalamins (iCbl) in the livers of rats (and, by extension, humans) fed "standard" (nonsupplemented) doses of Cbl(http://hardcorephysiologyfun.blogspot.com/2009/01/methylcobalamin-and-other-forms-of.html), would seem to suggest that one would get "less mileage," in terms of enhancement of MMM activity, out of an increase in the itCbl concentration in a cell. The reasoning would be that the intracellular AdoCbl levels would be closer to the Km for AdoCbl binding to MMM. But here's an article showing that ischemia quadrupled the MMM mRNA content [implying that there would be more binding sites to take advantage of a higher intracellular AdoCbl (potentially increasing the effective Km for AdoCbl binding) thereby maximizing anaplerosis and ATP production via the propionate pathway]:
[Purnima Narasimhan et al., 1996: (http://www.jneurosci.org/cgi/content/full/16/22/7336) (pubmed: http://www.ncbi.nlm.nih.gov/pubmed/8929440?dopt=Abstract)
The main concern with higher serum Cbl levels would probably be with the kidneys, but renal toxicity has never been reported in the literature, to my knowledge, in response to large i.v. doses of Cbls. And most of those studies have been done in patients with renal failure. If the high serum Cbl levels were causing renal toxicity, one would expect to see reports of worsened kidney function. Also, many cell culture studies have used 10 uM extracellular methylcobalamin, even in cultures of neurons, and shown cytoprotective or neuroprotective effects and no apparent cytotoxicity or neurotoxicity (such as in Kikuchi et al., 1997: http://www.iovs.org/cgi/content/abstract/38/5/848 or pubmed: http://www.ncbi.nlm.nih.gov/pubmed/9112980?dopt=Abstract). But, as Birn et al. (2003) [(http://ndt.oxfordjournals.org/cgi/content/full/18/6/1095) (pubmed: http://www.ncbi.nlm.nih.gov/pubmed/12748340?dopt=Abstract)] noted, most of the iCbl in cells outside the kidneys is protein-bound, and cells in the kidneys accumulate iCbl as free Cbl's in lysosomes/endosomes.
Nonetheless, Birn et al. (2003) used cyanocobalamin, and many articles have shown that methylcobalamin and hydroxocobalamin (basically because these forms are "not cyanocobalamin") have substantially higher bioavailabilities and stronger therapeutic or "functional" effects than cyanocobalamin has. Zeitlin et al. (1985) [(http://bloodjournal.hematologylibrary.org/cgi/content/abstract/66/5/1022)(pubmed: http://www.ncbi.nlm.nih.gov/pubmed/4052627?dopt=Abstract)] found evidence that hydroxocobalamin was binding to albumin in a patient with no transcobalamin II binding proteins and noted that the apparently poor or nonexistent binding of cyanocobalamin probably helps to account for its poor bioavailability and poor retention. More specifically, the limited binding of cyanocobalamin to albumin, such as in humans with serum Cbl levels in excess of the transcobalamin binding capacity of <1.4 nM, suggests that this may account for its poor retention by the kidneys. Albumin-bound cobalamins other than cyanocobalamin could be reabsorbed in the proximal tubules. But it seems more likely, as suggested by Birn et al. (2003), that the CNCbl in the kidneys had accumulated in lysosomes, as free CNCbl, following dissociation from transcobalamin proteins. Hall et al. (1984) found that CNCbl was less efficiently transported into cells and that a lower percentage of CNCbl than OHCbl was bound to a protein whose MW implied it was albumin. The authors also cited articles that show that transport of CNCbl into mitochondria is poor in comparison to the transport of OHCbl. Hall et al. (1984) [(http://bloodjournal.hematologylibrary.org/cgi/content/abstract/63/2/335) (pubmed: http://www.ncbi.nlm.nih.gov/pubmed/6692038?dopt=Abstract)] suggested that the binding of Cbl's to serum proteins might serve as a reservoir and that desaturation of transcobalamin II. The authors noted that injections of Cbl's cause increases in the release of apo-transcobalamin II into the blood and cause the circulating TCII, with Cbl bound, to be removed from the circulation. Then, as suggested by Hall et al. (1984), the Cbl's bound to other proteins in the serum would be released and enter cells as Cbl-TCII complexes. Hoffer et al. (2005a) found that OHCbl at 1 mg i.v. weekly produced a 40-fold elevation in serum Cbl, and the use of the same regimen with CNCbl only increased serum Cbl levels 10-fold (http://www.ncbi.nlm.nih.gov/pubmed/16154437). Although those authors also showed comparable functional effects, in terms of reductions in homocysteine, serum homocysteine, the articles have generally shown methylcobalamin and hydroxocobalamin to produce stronger functional responses, higher serum Cbl values, and higher degrees of tissue retention.
It seems clear that cyanocobalamin is less efficiently-retained than OHCbl or MeCbl, and the apparently-saturable reabsorption of CNCbl [Alejandro Nava-Ocampo et al., 2005: (http://www.nature.com/clpt/journal/v75/n2/abs/clpt2004328a.html)(pubmed: http://www.ncbi.nlm.nih.gov/pubmed/15730428)] and high tissue levels of iCbl in the kidneys [Linnell et al., 1983: (http://www.ncbi.nlm.nih.gov/pubmed/6822883?dopt=Abstract) could suggest that the levels of iCbl in the kidneys would reach a plateau as the serum Cbl levels are increased. This would tend to suggest that the kidneys will not accumulate "supraphysiological" concentrations of free iCbl.
It also seems unusual that Birn et al. (2003) found that 10 ug/d of subcutaneous CNCbl [this scales to 14 ug/kg bw/d s.c. for a 70-kg human, using the 0.1 kg weight for a newly-weaned rat and a scaling factor of 7.14 = (70/0.10)^(0.3)] did not elevate serum Cbl significantly, in comparison to the standard Cbl diet of 41 ug/kg diet [which scales to ~40 ug/d for a 70-kg human, if I use the young-rat weight of 0.1 kg, the 0.100 kg diet/kg bw conversion factor, and the 7.14 scaling factor (http://hardcorephysiologyfun.blogspot.com/2009/01/methylcobalamin-and-other-forms-of.html)]. This large dose of s.c. CNCbl produced a serum Cbl level of ~1.26 nM [the authors expressed the concentration as "log(serum Cbl in fM) = ~ 6.1," meaning that fM = 10^6.1 = ~1,260,000 fM or 1.26 nM] (Hoffer et al., 2005b: http://www.ncbi.nlm.nih.gov/pubmed/15931623). In contrast, an intravenous dose in humans of 1 mg CNCbl every week (averages to 143 ug/d or 2 ug/kg bw/d for a 70-kg human) produced a mean serum CNCbl of 4.8 nM (4,805 pM). Additionally, the mean intracellular concentration of B12 in the kidneys following the daily CNCbl injections was still only ~5,000 nM [(2,000 pmol/g ww) x (2,500/1,000)].
One might explain some of this fourfold-higher serum concentration in humans given one-seventh the scaled dose in terms of an effect of their renal failure, given that people on dialysis have been shown to clear different forms of Cbl more slowly than humans with normal renal function. But the discrepancies seem rather extreme, especially since the rats that had been fed the standard diet (this scales to 40 ug/d for a 70-kg human) had roughly the same ~1.26 nM serum Cbl levels as did the rats that had been fed the standard diet and had also gotten the injections. A human ingesting 40 ug/d of CNCbl would not be expected to get a serum Cbl level nearly as high as 1.26 nM, and a human getting 40 ug/d of CNCbl and 14 ug/kg bw/d of s.c. CNCbl would be expected to have a serum Cbl much higher than 1.26 nM. The rats had roughly equivalent serum Cbl values in response to both regimens.
The results of other articles suggest that the endogenous formation of NOCbl may not be a major issue, in relation to the kidneys, even assuming that this process occurs to a significant extent (I'll try to find research showing the percentage of iCbl that is normally NOCbl, but I think it's normally only a small percentage). Brouwer et al. (1996) cites some examples of articles discussing the inactivation of cobalamins by nitrous oxide in anesthetic gas mixtures (it's still being used in anesthesia), but large amounts of nitrous oxide are required, in a person with low Cbl stores, to completely deplete the functional Cbl stores [Brouwer et al., 1996: (http://bloodjournal.hematologylibrary.org/cgi/content/abstract/88/5/1857)(pubmed: http://www.ncbi.nlm.nih.gov/pubmed/8781445?dopt=Abstract)]. The article by Brouwer et al. (1996) also cites some references showing that methylcobalamin can block NO-mediated cytotoxicity in cultured neuronal cells (Kikuchi et al., 1997: http://www.iovs.org/cgi/content/abstract/38/5/848 or pubmed: http://www.ncbi.nlm.nih.gov/pubmed/9112980?dopt=Abstract) and that hydroxocobalamin and cobalamin analogues (cobinamide is one) have been used to treat NO-mediated toxicity in models of sepsis. Also, this article (Gerth et al., 2006: http://www.ncbi.nlm.nih.gov/pubmed/16990191) discusses the way hydroxocobalamin is used to treat acute cyanide poisoning, and the dosages used for that application are very large. Especially in the case of the nitric oxide binding to Cbl's, I think that redox cycling with NOCbl or other forms would tend to preclude their use in sepsis, in particular. If there were that effect, to a major degree, one would not expect to see the usefulness in animal models of sepsis that one does see. The iNOS-mediated output of NO is thought to be massive in sepsis, and this type of in vivo situation would reasonably be expected to unmask those types of theoretical, redox cycling reactions that NO-bound Cbl's might participate in. Bauer et al. (2002) [(http://jnci.oxfordjournals.org/cgi/content/full/94/13/1010)(pubmed: http://www.ncbi.nlm.nih.gov/pubmed/12096086)] found that the IC50's for the induction of apoptosis by NOCbl, in cells that are not malignant, range from 50-130 uM, and that's the extracellular concentration. That would suggest that even the most "toxic" forms of cobalamin (and NOCbl would probably only constitute a small percentage of the iCbl's) would have to be at very high extracellular concentrations to accumulate at high levels intracellularly and produce cytotoxic effects.
I think there's some room for dosage increases, but I'm not suggesting that one would want a really high serum Cbl value. It's an important issue, though, because methylcobalamin can drastically enhance the effectiveness of homcysteine reduction strategies (and also exert some protective effects by reducing methylmalonic acid levels). The literature shows a lot of variation, both between studies and between individuals, in the serum Cbl responses to the same dosages of different forms of oral Cbl. Some of the issues I haven't addressed are discussed in this article: (Solomon, 2007: http://www.ncbi.nlm.nih.gov/pubmed/16814909 or http://home.hetnet.nl/~hindrikdejong/Solomon-B12-2006.pdf).
One of these days, I'll put up the links to some of the dose-response data for forms of oral methylcobalamin, cyanocobalamin, and hydroxocobalamin. But I've scarcely ever seen a serum Cbl value much higher than around 3,000 pg/mL (~2.2 nM), even in response to dosages higher than 3 mg/d. That doesn't mean it's not possible to increase them more, but there isn't/aren't a lot of data to look at. It's difficult to find dose-response data for oral forms of Cbl.
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