Here's a paper with data on both cytosolic and mitochondrial total folates in young rats fed either folate-deficient or folate-supplemented diets (8 mg folic acid/kg diet) [Mesbaheddin Balaghi et al., 1993: (http://www.biochemj.org/bj/291/0145/2910145.pdf) (http://www.ncbi.nlm.nih.gov/pubmed/8471033)]. The weanling rats weighed 47 g but evidently gained some weight during the experiment. I'm just going to use the standard conversion factors for young rats, because the WHO document lists the food intake of a weanling rat as 10 g. Balaghi et al. (1993) showed that the food intakes for the rats had increased, during the four weeks of the experiment, from 7.5 to 17.8 g/d. But the body weights also increased, making them closer to the WHO approximation of 0.10 kg for a weanling rat. The way the authors explain the weight gain and increases in food intake, it's not possible to figure out what the precise folate intakes were, in mg/kg diet/d. I have to approximate, using standard conversions for weanling rats.
The 8 mg folic acid/kg diet converts to 800 ug/kg bw/d for the rats (http://hardcorephysiologyfun.blogspot.com/2008/12/equations-for-animal-food-intake-and.html). This scales, using the 7.14 conversion factor, to 112 ug/kg bw/d, or 7.84 mg/d, for a 70-kg human.
I'm going to use the values found by Balaghi et al. (1993) for the intracytosolic and intramitochondrial total folates at the end of week 4 of the experiment. The authors used a method of centrifuging the liver cells to obtain the mitochondrial fraction as a pellet and the cytosolic constituents (cytosolic protein+other cytosolic constituents+cytosolic folates) as the supernatant. So these nmol/g protein are going to be dry weight values, because that's what you'd get out of centrifugation [these articles show that it is necessary to convert from dry weight to wet weight following the separation of a mitochondrial pellet from tissue homogenates by centrifugation: Michail Tonkonogi et al., 2000: (http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2270128) and Ulla Glas and Gunter Bahr, 1966: (http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2106972)].
The intracytoplasmic total folate level in the rats fed the 800 ug/kg bw/d at that time point was a mean of 14.6 nmol/g dry weight liver, and the intramitochondrial total folate level was 15.7 nmol/g dry weight liver. The two pools do not equilibrate, and the authors mention that. It's been confirmed by more recent experiments. I'm going to have to do the conversions for the overall cells, but there are data on the percent of cytosolic volume that are mitochondria. Those values vary between cell types considerably, and I haven't looked for all of those conversion factors. One article found that the volume of the mitochondrial matrix fluid was between 15 and 20 percent of the total cytosolic volume for cells in the rat liver [Patricia Lund and David Wiggins, 1987: (http://www.ncbi.nlm.nih.gov/pubmed/3620602)]. I won't do those additional conversions here, to estimate the intramitochondrial vs. intracytoplasmic concentrations. These data for the mitochondrial and cytosolic pools are nice to see, though. In my conversion factors, the "cytosolic volume" is the total intracellular volume, and the mitochondrial fluid volumes are included within that. So I'm adding the two numbers together (http://hardcorephysiologyfun.blogspot.com/2008/12/cell-biology-conversion-factors-for-ngg.html). I'm first going to have to apply the 3.33 conversion factor for dry weight to wet weight [Balaghi et al. (1993) used male rats]:
(30.3 nmol/g dry weight liver) x (1 g dw liver/3.33 g ww liver) = 9.099 nmol/g ww
(9.099 nmol/g ww) x (2500) = ~22748 nM = ~22.7 uM intracellular total folates
That's about what other studies have shown, but I'll do these for some other articles. I'm not going to do the conversions for the folate-deficient rats (the authors don't say what their dietary folate intakes were), but the initial value is 1.5 nmol/g dw liver. So it's 22.7/20.2 (~1.12 uM) for the folate-deficient rats.
This article has some interesting data on the S-adenosylmethionine/S-adenosylhomocysteine ratios, but I won't go into all those data. The folate-replete rats have a ratio that is about 5 or 6 times higher than the ratio in the folate-deficient rats.
Here are my estimates from this article [Joanne Kotsopoulos et al., 2005: (http://carcin.oxfordjournals.org/cgi/content/full/26/9/1603) (http://www.ncbi.nlm.nih.gov/pubmed/15888495?dopt=Abstract)]. The rats are young rats, and so the 2 and 8 mg/kg diet regimens of folic acid convert to 200 or 800 ug/kg bw/d. These values scale to 28 and 112 ug/kg bw/d (~2.0 or 7.8 mg folic acid/d) for a 70-kg human. Here are the mean serum folate values for the three groups of rats (0, 2, and 8 mg folic acid/kg diet) after 4 weeks (this is the data from the 4 weeks before the interval during which all groups got the different doses for 22 weeks). To convert ng/mL to nM, multiply by 2.26:
0 mg/kg diet: 21.7 ng/mL = 49 nM
2 mg/kg diet: 64.7 ng/mL = 146 nM
8 mg/kg diet: 107.8 ng/mL = 244 nM
[I first have to do the conversions from ng total folates/g ww tissue to nmol/g ww.
Balaghi et al. (1993) found that 5-Me-THF, THF, and 10-CHO-THF accounted, together, for 96.4 percent and 89.1 percent of the intracytosolic and intramitochondrial total folates in the livers of rats fed 800 ug/kg bw/d. So I'm going to just average their molar masses [THF = 445.43 g/mol, 10-CHO-THF = 473.44 g/mol, and 5-Me-THF = 459.46 g/mol]. The average is 459.44 g/mol. To convert ng total folates/g ww tissue]:
(Y ng total folates/g ww) x (1/459.44 g total folates) = Z nmol total folates/g ww
These values are tissue folates for Kotsopoulos et al. (2005) after 22 weeks of different doses of folic acid:
Hepatic folate for rats given 0 mg/kg diet supplemental folate:
(2.7 ug total folates/g ww liver) x (1000 ng/1 ug) x (1/459.44 g total folates) = 5.877 nmol/g ww
(5.877 nmol/g ww) x 2500 = 14,693 nM = ~15 uM
Rats given 2 mg folate/kg bw/d: 7.3 ug/g ww = 39,722 nM = ~40 uM
Rats given 8 mg folate/kg bw/d: 9.3 ug/g ww = 50,605 nM = ~51 uM
Mammary tissue (I'm assuming it's roughly the same percent water as other cell types, but it's conceivable it's less for adipose tissue or mammary tissue overall, even if the target cell types for folates are epithelial cells in the mammary tissues):
Rats given 0 mg folate/kg diet: (69.2 ng total folates/g ww) x (1/459.44) x 2500 = 377 nM
Rats given 2 mg folate/kg diet: 110.3 ng/g ww = 600 nM
Rats given 8 mg folate/kg diet: 146.4 ng/g ww = 797 nM
Those data are pretty shocking and show that dietary folate can be generous by usual standards, when it comes to the scaled human doses, and still produce very low levels of intracellular total folates in mammary tissues. Those levels wouldn't support basic functions of the folate cycle, given that the folate-cofactor-dependent enzymes are thought to not be able to sustain activities below an intracellular concentration of about 5 uM (cited in my paper--I don't feel like linking to the reference). The intracellular values for the liver are not that far off other values. They're actually double the levels in the other study I went through, but there's considerable variation in human studies also. The liver values are more than adequate, when you just look at the crude, overall values (that don't tell you about the ratios of the different folate-derived coenzymes). But the values for the mammary tissue are shockingly low.
Those data are not consistent with the idea that extrahepatic tissues can efficiently regulate their intracellular folate levels in response to minor increases in serum folate or dosages of supplemental folate. Those data also have implications for breast cancer prevention strategies, even though it's just one study. It's a great animal study, though.
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