Effects of Reduced Folates vs. Folic Acid in Cell Culture Studies

These articles show that reduced folates can be between 10 and 100 times as potent, depending on the variables that researchers have considered, as folic acid in producing certain effects on cultured cells.

Conversion factors:

1 nM serum folate = 2.265 ng/mL
Molar mass of folic acid: 441.4 g/mol
Molar mass of 5-formyltetrahydrofolate (5-CHO-THF): 473.44 g/mol
Molar mass of 5-methyltetrahydrofolate (MTHF): 459.46 g/mol

Balk et al. (1978) used cultured chicken fibroblasts and found that either L-5-methyltetrahydrofolate (MTHF) or L-5-formylTHF (L-leucovorin) produced a maximal rate of cell proliferation at an extracellular concentration of 10 ng/mL (21.1 nM 5-CHO-THF or 21.8 nM MTHF) [Balk et al., 1978: (http://cancerres.aacrjournals.org/cgi/reprint/38/11_Part_1/3966.pdf) (http://www.ncbi.nlm.nih.gov/pubmed/212184)]. In contrast, 1,000 ng/mL (2,266 nM, or 2.26 uM) of extracellular folic acid was required to maximize the rate of cell proliferation and make the rate comparable to the rate that 21-22 nM extracellular 5-CHO-THF and MTHF had produced. Here's an example of a conversion:

(10 ng 5-CHO-THF/mL) x (1000 mL/1 L) x (1 nmol/473.44 ng) = 21.1 nM

One thing that might have caused such a large difference might be that Balk et al. (1978) apparently used a fairly large extracellular cobalamin concentration of 1500-1700 pg/mL (1.12-1.27 nM). It's not clear, given the way the authors discussed the cobalamin concentration in the article, what the actual cobalamin concentration had been during the experiments. But that might have potentiated the effects of the reduced folates. The authors also discuss research showing that reduced folates are about 100 times as potent as folic acid in producing cell proliferation rates of cultured cells. The value of 2.26 uM extracellular folic acid, producing something approaching maximal cell proliferation, is comparable to the results in some other articles.

This article [Watkins and Cooper, 1983: (http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1152268)] is great and shows that racemic folinic acid (a reduced folate) is somewhere around ten times as potent as folic acid in producing cell proliferation (when one looks at the concentrations of the extracellular folic acid or folinic acid). The authors also show the intracellular (ICL) and extracellular (ECL) total folates and MTHF concentrations in uM, and I don't have to convert those:

Folic acid ECL 10,000 nM: 0.78 doublings/day; ICL folates 7,070 nM; ICL MTHF 2,340 nM
Folic acid ECL 1,000 nM: 0.83 doublings/day; ICL folates 3,690 nM; ICL MTHF 2,580 nM
Folic acid ECL 100 nM: 0.69 doublings/day; ICL folates 610 nM; ICL MTHF 500 nM
Folic acid ECL 10 nM: 0.34 doublings/day; ICL folates 450 nM; ICL MTHF 210 nM

D,L-folinic acid ECL 200 nM: 0.74 doublings/day; ICL folates 6,020 nM; ICL MTHF 2,900 nM
D,L-folinic acid ECL 20 nM: 0.71 doublings/day; ICL folates 3,150 nM; ICL MTHF 2,730 nM
D,L-folinic acid ECL 2 nM: 0.65 doublings/day; ICL folates 570 nM; ICL MTHF 510 nM
D,L-folinic acid ECL 0.2 nM: 0.38 doublings/day; ICL folates 490 nM; ICL MTHF 420 nM

The authors also found that the proliferation rate, when the extracellular folate level was 1 nM, was independent of the total intracellular concentration of reduced folates, other than MTHF, when the intracellular total folates were more than 1-1.5 uM. This doesn't mean that there's no more growth at intracellular folates above 1.5 uM. It means that the cells in the culture competed for the low folate supply, essentially, and reached a dynamic equilibrium in which some of them were able to maintain higher intracellular folate levels than others. So that relationship was specific to the low 1 nM extracellular folate level. It's interesting that the growth rate, under folate-depleted conditions, was dependent on the "total reduced folates other than MTHF" concentration intracellularly but was not dependent on the MTHF level intracellularly. This may not be very telling, though, given the folate-starved conditions that produced that relationship.

Again, the folate-free medium contained 1 nM folic acid but contained 1 uM cyanocobalamin. That would be expected to potentiate the effects of the reduced and ordinary folates, and that underscores the importance of cobalamin status to folate metabolism.