At the end of this posting, I've included some more conversions of cellular folate levels into intracellular folate levels, for cells cultured in media containing various concentrations of extracellular folic acid.
This article [http://carcin.oxfordjournals.org/cgi/content/full/26/5/981 (pubmed: http://www.ncbi.nlm.nih.gov/pubmed/15695236?dopt=Abstract) (Stempak et al., 2005)] is great and shows the way the S-adenosylmethionine and SAM-e/SAH ratio can vary in response to different intracellular folate levels and that DNA methylation can be really, really chaotic. The article shows the way the extents of CpG island methylation, such as in the promoters of genes (researchers sometimes express this in terms of the percentage of some segment of DNA that's methylated), can vary in an unpredictable way. They found, for example, that cells can have very high SAM-e/SAH ratios and still have "undermethylated" DNA. But this doesn't mean that methylfolate and methylcobalamin have no utility in influencing DNA methylation. Some of these cell lines are sort of "out there" in terms of crazy gene expression patterns. But it's very cell-type and cell-context-specific (DNA methylation). I'm sure there are hundreds of proteins that interact directly or directly with the DNA methyltransferase enzymes and influence their site-specific activities, etc.
The SAM-e and SAH levels have never been very reliable in humans as "markers" of folate status, either. They should theoretically correlate predictably with folate status, but the SAM-e levels in the blood or in other cells tend to be increased in B12 deficiency. And there can be significant effects from increasing extracellular folate that become independent of SAM-e and SAH levels and the SAM-e/SAH ratio (Brown et al., 2006: http://www.ncbi.nlm.nih.gov/pubmed/16469322). Using DNA methylation patterns to evaluate the effects of folate supplementation seems really suspect to me, and it can lead to erroneous conclusions. That article (Stempak et al., 2005) is great because it shows that different cells accumulate different levels of intracellular folates and that it's not easy to establish rigid "rules" about the way cells "should" behave, essentially.
Here's another interesting study (Maureen van den Donk et al., 2007: (http://jn.nutrition.org/cgi/content/full/137/9/2114) (http://www.ncbi.nlm.nih.gov/pubmed/17709451?dopt=Abstract)] in humans, too, that shows that colonocytes can actually show increased DNA uracil (uracil misincorporation, produced by an elevated dUMP/dTMP ratio due to folate depletion) in people taking 5 mg/d of folic acid and a small amount of B12. The authors make some good points and note that unmetabolized folic acid can constitute a major percentage of serum folate above intake levels of about 400 ug at a time.
But one thing that could explain their results is this strange phenomenon in which there's a biphasic degree of uracil misincorporation, as the extracellular folate level increases. When the cells are extremely deficient and depleted (0 nM extracellular folate in Mashiyama et al., 2004), there can actually be less uracil misincorporation into DNA [Mashiyama et al., 2004: (http://www.ncbi.nlm.nih.gov/pubmed/15183762)]. Mashiyama et al. (2004) suggested that the cells don't have enough thymidine, under those conditions, to divide, to initiate DNA replication, and the low growth rate allows the DNA replication that does occur to have access to more thymidine than more rapidly-dividing (and more folate-repleted) cells would have access to. At an extracellular folate level of 20 nM, the level of uracil misincorporation was found to be drastically increased over the level that had occurred at 0 nM extracellular folate (Mashiyama et al., 2004). At that level, there's thought to be enough thymidine for cell division to occur but not enough to minimize significant uracil misincorporation. Then, past another point, the levels start to decrease below controls and were not minimal until concentrations as high as 3,000 nM extracellular folate.
Given the very low intracellular folate levels in some of these cells that were cultured in 2.3 uM extracellular folate (see below), I'll bet the colonocytes used by van den Donk et al. (2007) had extremely low intracellular folate levels and had been exposed to extremely low extracellular folate levels in vivo. That biphasic phenomenon could reasonably be expected to have occurred. The authors referred to their doses as high-dose supplementation, but, when it comes to increasing the intracellular folate levels in extrahepatic cells, 5 mg/d folic acid is not a high dose. A dose of 5 mg/d of folic acid has, in some studies, not been shown to elevate the serum folate concentration much at all. Even an increase of 100 nM in the serum folate might only elevate the intracellular folate levels in colonocytes enough to increase thymidine-dependent DNA replication and increase the uracil DNA content in the newly-replicated DNA. These intracellular concentrations, converted from the data of Stempak et al. (2005) and Madeleine Kane et al. (1987) [(http://cancerres.aacrjournals.org/cgi/reprint/47/24_Part_1/6444.pdf) (http://www.ncbi.nlm.nih.gov/pubmed/2445472)] could conceivably be much lower in vivo, in human colonocytes. I used these conversion factors [I'll put a summary statement at the bottom of this long posting, soon, to highlight the factors I chose: (http://hardcorephysiologyfun.blogspot.com/2008/12/cell-biology-conversion-factors-for-ngg.html)].
In different cell types (from Stempak et al., 2005) grown in a medium containing 2.3 uM extracellular folate, the intracellular total folates were:
(22.44 ng intracellular folates/5 x 10^6 cells) x (1 nmol/459.44 ng) x (1.9 x 10^8 cells/mL intracellular water) x (1000 mL water/1 L water) = 1856 nM = 1.856 uM in NIH/3T3 cells [murine (mouse-derived) embryonic fibroblasts]
(22.87 ng/5 x 10^6 cells) = 1.892 uM for CHO-K1 cells (chinese hamster ovary cells)
(19.72 ng/5 x 10^6 cells) = 1.631 uM for HCT116 cells (human colonic epithelial carcinoma cells)
213.20 ng folates/5 x 10^6 cells) = 17634 nM = ~17.63 uM in Caco-2 cells (human epithelial colorectal carcinoma cells)
These are the data on cellular folate content (converted by me into estimates of intracellular total folates) from Kane et al. (1987) in cultured, epithelial cell-derived, human nasopharyngeal carcinoma cells (KB cells), grown in either 2.3 uM extracellular folate or 4 nM extracellular folate:
In 2.3 uM extracellular folic acid:
(60 pmol/10^6 cells) x (1 nmol/1000 pmol) x (1.9 x 10^8 cells/mL intracellular water) x (1000 mL/1 L water) = 11,400 nM = 11.4 uM
When the cells were placed in a medium of 4 nM extracellular folate, the concentration of intracellular total folates gradually declined to 5.2 pmol/10^6 cells (980 nM), then to 4 pmol/10^6 cells (760 nM), and then to 1 pmol/10^6 cells (190 nM).
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