Some of these articles that give serum cobalamin concentrations contain typos, and that last article I cited does contain some. I corrected the values in the last posting, but I think the issue has to do with the lack of conversion of a lowercase "m," during the journal article formatting process, into the Greek letter "mu" (I'm expressing it with a u instead of the mu character). But a lowercase m is milli-, and the mu means micro-. If someone didn't know the reference ranges for serum B12 and read the articles, the typos would be confusing. Here's the conversion factor (using the molar mass of cyanocobalamin, because everyone still assumes that cyanocobalamin is vitamin B12). Note: the molar masses of MeCbl and Cbl and CNCbl are not different enough from one another to affect the calculations, given the large molar masses of cobalamins:
(Y pg/mL cobalamin/CNCbl/MeCbl)/1355.38 = Z nM cobalamin [or (1,000)(Z) pM]
Here's a good article that discusses the way in which B12 reabsorption by the proximal tubule cells, in the kidneys, becomes saturated at serum B12 levels in excess of about 12,000 pg/mL (12 ng/mL), or 8 nM. At serum B12 concentrations in excess of about 8 nM, the urinary excretion rate of B12 would be greatly increased and correlate positively with the glomerular filtration rate (the authors cite references 99 and 147):
http://ajprenal.physiology.org/cgi/content/full/291/1/F22
(pubmed: http://www.ncbi.nlm.nih.gov/pubmed/16760376?dopt=Abstract) (Henrik Birn, 2006)
The laboratory reference range for serum B12 is 100-900 pg/mL (0.0738-0.664 nM, or 738-6640 pM). Those references on the quantitative aspects of the reabsorption of B12 help to explain that article in that last posting. It's only one factor that makes homocysteine reduction so difficult to achieve in people with renal failure or kidney disease, but that article on the kidneys in folate and B12 homeostasis helps show the way, for example, the failure of the reabsorption of cobalamin or of reduced folates could drastically affect homocysteine reduction strategies. But part of it is the uremic toxins that interfere with one-carbon metabolism in cells throughout the body.
On page F24, the authors cite references 7 and 102 and state that the standard diet fed to rats causes rats to have serum B12 levels of about 1.1 nM. The authors note that the average serum B12 values in humans range from 0.3-0.5 nM (on two separate pages of the article), and that's an interesting point. I actually hadn't thought to look for articles on the normal serum B12 in rats. I thought that either no article had collected "sample data" on rat serum B12 distributions, in the "rat population," or that the serum B12 values would be comparable to humans' serum B12 values. The mean level in rats is comparable but is 2-3 times higher than the value in humans, and this is telling in view of other articles that have provided data on the tissue content of B12 (meaning methylcobalamin and 5'-deoxyadenosylcobalamin, aquacobalamin, etc.). Here are some of the data:
The "complete diet" used by John Linnell et al. (1983) (http://jn.nutrition.org/cgi/content/abstract/113/1/124 pubmed: http://www.ncbi.nlm.nih.gov/pubmed/6822883?dopt=Abstract) provided the laboratory rats with 50 ug CNCbl/kg diet, which scales (http://hardcorephysiologyfun.blogspot.com/2008/12/equations-for-animal-food-intake-and.html) to 2.5 ug/kg bw, or .53079 ug/kg bw for a 70-kg human. That's ~37 ug per day for a 70-kg human. That scaling gives a very low dose of B12, but it's actually more than ten times the RDA of 2.4 ug for a human.
Here are the conversions for the intracellular total cobalamins and intracellular methylcobalamin values from the Linnell article [based on the (nmol/g ww) x 2500 = Z nM conversion factor that I showed here: http://hardcorephysiologyfun.blogspot.com/2008/12/cell-biology-conversion-factors-for-ngg.html]:
For rats fed the standard B12 diet used by Linnell et al. (1983), the intracellular (mean) total cobalamins (aquacobalamin + 5'-deoxyadenosylcobalamin + methylcobalamin (+nitrosocobalamin/superoxocobalamin/glutathionylcobalamin/sulfitocobalamin, as minor contributors):
67 pg/mg ww liver = 0.04943 nmol/g ww = 123.6 nM (~124 nM)
738 pg/mg ww kidneys = (~1,361 nM)
23.2 pg/mg ww spleen = (~42.8 nM)
268 pg/mg ww adrenals = (~494 nM)
(Note that those are crude calculations, but the conversion factors for those tissues are probably not drastically different from the liver-based conversion factors.)
Here are the methylcobalamin tissue contents and calculated intracellular concentrations from the data of Linnell et al. (1983) [note: I'm using the 1,335.38 g/mol molar mass to convert pg MeB12 to nmol MeB12, even though that's the molar mass of CNB12. I'm dividing Y pg/mg ww by 1335.38, to get nmol/g ww, and then multiplying that by 2500 (the same as I did above)]:
1.6 pg/mg ww liver = 0.00119816 nmol/g ww = (~0.66 nM MeB12)
0.3 pg/mg ww spleen = (~0.57 nM MeB12)
26.9 pg/mg ww adrenals = (~50.4 nM MeB12)
107.6 pg/mg ww kidneys = (~201 nM MeB12)
In another article (Henrik 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), Birn et al. (2003) found that rats fed 41 ug/kg diet (scales to a human dose of 0.435 ug/kg bw or ~30 ug/d for a 70-kg human) cyanocobalamin had these tissue levels of total cobalamins (converted by me into intracellular total cobalamins):
kidneys: 710 pmol/g ww = 0.710 nmol/g ww = (~1,780 nM)
liver: 15 pmol/g ww = 0.015 nmol/g ww = (~38 nM)
Those values (38 nM and 124 nM) for the intracellular concentrations of total cobalamins in the liver are not very far off the estimate that Yamada et al. (2000) (http://www.ncbi.nlm.nih.gov/pubmed/10917899?dopt=Abstract) made for the intracellular total cobalamin concentration in the rat liver (211 nM) in response to the standard rat diet (which, in the other two studies I cited, was 41-50 ug/kg diet and scaled to 30-37 ug/d for a 70-kg human).
Yamada et al. (2000) also used this value to estimate that the intracellular methylcobalamin concentration in the rat liver is ~8.2 nM. Yamada et al. (2000) made that estimation by using the data from this article, by Linnell et al. (1974) (http://www.ncbi.nlm.nih.gov/pubmed/4206133), showing that the total cobalamin pool in the liver of a human is made up of about 3.9 percent methylcobalamin and 72.8 percent 5'-deoxyadenosylcobalamin. Yamada et al. (2000) noted that the Km for the binding of methylcobalamin to methionine synthase is 340 nM. Given the estimates of the intracellular methylcobalamin concentrations (8.2 and 0.66 nM) in the livers of rats fed standard diets, the Km for methylcobalamin binding to methionine synthase can be estimated to be 41 to 515 times the intracellular methylcobalamin concentration.
The Km for the binding a substrate or cofactor, like methylcobalamin, is the concentration of the substrate that, upon its availability for binding to the enzyme, produces an initial reaction rate, by the enzyme, that is half-maximal (Vmax/2). The Km is also sometimes applied to analyses of the "velocities" of transporters and to other ligand-receptor interactions, etc. When the concentration of a substrate is very far below the Km for its binding to an enzyme, this can imply that the concentration of the substrate or cofactor (methylcobalamin, in this case) is abnormally low or, in the absence of any subjective determination of concentrations that are normal or "abnormal," that the activity of the enzyme might be increased by an increase in the concentration of the substrate (methylcobalamin) that is available to bind to the enzyme. The intracellular methylcobalamin levels in a rat or human on a "standard-rat-or-human diet" are likely to be "really low."
When one takes into account the fact that the standard rat diets produce mean serum B12 concentrations that are 2-3 times those of humans (see citation early in this posting), the intracellular concentrations of methylcobalamin in the livers of many humans may be even lower. I don't feel like applying that percentage calculation across the other tissue values.
One article showed that elderly people required up to 200 times the RDA of cyanocobalamin to correct a deficiency state (http://scholar.google.com/scholar?num=100&hl=en&lr=&cluster=91363613711915479), and that's not even addressing the broader dosage range. I do think, though, that the accumulation of free cobalamins in the kidneys could be undesirable. Even though cobalamins can bind superoxide and "scavenge" it (thereby allowing superoxocobalamin to reversibly bind NO), there's evidence that cobalamins may be able to participate in redox cycling reactions to some extent. Even though these types of estimates on intracellular total cobalamins are useful, they don't really help to define the level of plasma B12 level that will maximize the kinetics of the folate cycle and still be "safe" for the kidneys (or other tissues).
One can't easily draw conclusions about extracellular vs. intracellular cobalamin levels (or extracellular Cbl vs. methionine synthase or methylmalonyl-CoA mutase activities) from data in cell culture studies, though. The cell culture studies show that transcobalamin-bound B12 is 10-2,000 times as potent as free CNB12 or OHB12 in supporting cell growth (in being transported into the cells and coenzymated, etc.). In this article (http://www.jbc.org/cgi/content/abstract/256/20/10329 pubmed: http://www.ncbi.nlm.nih.gov/pubmed/6974730?dopt=Abstract) (Fujii et al., 1981), a concentration of free H2OCbl (aquacobalamin) of 4 nM was required to produce maximal or "optimal" cell growth, and only 0.002 nM TC-bound H2OCbl was required to produce the same effect. This article (Gary McLean et al., 1997(http://bloodjournal.hematologylibrary.org/cgi/content/abstract/89/1/235) and (pubmed: http://www.ncbi.nlm.nih.gov/pubmed/8978297?dopt=Abstract)] shows that about 2.3 nM of cobalamin bound to holotranscobalamin II was required for maximal cell proliferation. In contrast, 250 nM of extracellular, free CNCbl was necessary to maximize cell proliferation in their cell cultures.
But those results don't show that there would be no additional effect from serum Cbl concentrations in excess of the maximal molar concentrations of serum transcobalamin+holotranscobalamin binding sites. The serum transcobalamin level is 1 nM and may be as little as 10 percent saturated, and the holotranscobalamin concentration, of which up to 75 percent may be saturated, was estimated to be 0.4 nM (http://www.ncbi.nlm.nih.gov/pubmed/16760376?dopt=Abstract). Thus, the maximal concentration of TC-bound B12 in the serum would be something less than 1.4 nM. But then why would the 34-35 nM serum B12 concentrations (or concentrations somewhere between 0.5 and 35 nM, given that the serum B12 levels in that study were probably "overkill") produce such dramatically greater reductions in tHcy?
It may be that there's some benefit from albumin-bound B12 (B12 binds to albumin at concentrations higher than those that produce transcobalamin saturation), and there's actually research suggesting that albumin can transport B12 into some tissues in humans with genetic transcobalamin II deficiency. Here's one article showing that a human with no transcobalamin II binding proteins (due to a genetic mutation, apparently) was able to maintain functional B12 status and a serum B12 of 3,000 ng/mL (= 3,000 pg/mL, or ~2.2 nM) (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) by taking 2 mg/d of oral OHB12. The person's serum B12 was apparently mostly bound to albumin, as the authors found, implying that albumin can transport B12 into cells. A tiny fraction was bound to "R-protein," which is transcobalamin I. I guess R-proteins are also called haptocorrins. It's worth noting that the person began taking the 2 mg/d at an age of 2 and 1/2 years, and so the dose-response relationship is not the response that would occur in an adult. The authors also noted that OHB12 may bind relatively loosely to albumin, although I've seen research suggesting that cobinamide, a B12 analogue, binds tightly to albumin. Here's another one showing binding of B12 to albumin in the blood of a person with transcobalamin II deficiency [(Hall et al., 1982): http://www.ncbi.nlm.nih.gov/pubmed/7073991]. Here's one showing binding of B12 to a 70-kDa protein in a person with transcobalamin I deficiency: [Carmel, 1982 http://www.ncbi.nlm.nih.gov/pubmed/7053761].
It's interesting that a lot about albumin transport is not known, and multiple receptors can transport albumin into endothelial cells, for example. But that article shows that a relatively mild increase in serum B12 (not nearly up to the 35 nM range, but from, say, 2 to 3 or 4 nM) could reasonably be expected to improve homocysteine reduction strategies. So the cell culture studies showing that free Cbl (and, by implication, serum Cbl in excess of the TC saturation point) is only 1/1000th or 1/2000th of the potency of TC-bound B12 may not be perfectly relevant and may ignore the transport capacity of albumin. Albumin could also sequester free Cbl, as implied by Fujii et al. (1981), but the extent to which free or albumin-bound Cbl will be available for transport (and capable of being transported) into cells in vivo remains to be established. The articles on human transcobalamin II deficiency suggest that small increases of serum B12 might produce functional responses, in terms of enhancements of intracellular methionine synthase activity, that are not drastically less-pronounced than those produced by a small increase in the TC-bound fraction.
Also, the intrinsic-factor-mediated absorption of the different forms of vitamin B12 is saturable, but there's an intrinsic-factor-independent mode of absorption that allows 1-5 percent of doses between 0.1 and 10 mg (100-10,000 ug) to be absorbed (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=%22intrinsic+factor+independent%22). Here's one article that suggests a mechanism: (http://www.ncbi.nlm.nih.gov/pubmed/10053217). It seems as though dosages in many studies are still being chosen with the assumption that there will be no dose-dependence of the serum B12 responses to oral methylcobalamin or other forms. But there is a percentage that's absorbed as the dosage increases past the dose at which intrinsic-factor-mediated transport becomes saturated (less than 100 ug or something).
So the point of this is that there's a rationale for some of these slightly-higher doses of methylcobalamin, such as in the range of the 3-6 mg/d doses used to treat sleep disorders (a search on methylcobalamin and sleep would probably turn the articles up, and I have them but am not up for linking to them right now).
But there's still the issue of the free B12 accumulation in the kidneys, and I don't know what level would be safe in that area.
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