Bence Szabó Gál

Bence Szabó Gál

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The glucosamine salt of methylfolate is most stable when we examine how long the unopened package remains stable at room temperature, but is most unstable when looking at how long it remains stable when opened (e.g. after opening a product) or when looking at how much degradation product is formed in it.

Although the common (amorphous) calcium salts of methylfolate are only marginally better or equal, the crystalline forms of calcium salts are better in this respect, at least the so-called C-crystal calcium salt certainly is. In terms of absorption, glucosamine salt (Quatrefolic) is a bit better than amorphous (Extrafolate-S) and I-crystalline calcium salt (Metafolin), but significantly lags behind the C-type calcium salt of methylfoalte (Magnafolate). This latter one is the best form of methylfolate in all respects (longest shelf life under all conditions, purity, safety of degradation products, utilization, etc.) However, all the above do not matter much, it is just nitpicking, because all four of the above methylfolate salts are suitable. Supplementing with more than 800 mcg of vitamin B9 per day can be risky and is completely unnecessary. Although presumably the methylfolate form is significantly safer, perhaps even at doses above 800 mcg, current evidence does not rule out the possibility that it is as risky as folic acid supplementation. Presumably, B9 can only pose any risk if taken at doses above 800 mcg in combination with B2, B6 and B12 deficiency, but it is better not to exceed 800 mcg in pregnancy, and 400 mcg is not advisable in other cases. In other words, methylfolate supplementation between 200-400 mcg seems to be optimal in general, while during pregnancy, supplementation between 400-800 mcg is typical, but above 800 mcg is risky.  

Forms of vitamin B9 in foods 

The form of vitamin B9 in foods is mixed. They contain mainly the already active methyl folate,[15] but also other forms of folate, which are readily converted to dehydrofolate (DHF), which is then converted to methyl folate with varying efficacy from individual to individual. The point is that vitamin B9 from natural foods can basically be considered methyl folate, since it is mainly present in this form and is the part that is not, is in a form that is more easily converted than folic acid. Methyl folate is released into the bloodstream in its unchanged form, while folic acid must first be converted by the liver. Folic acid is absent or almost absent in natural foods. 

Folic acid and methylfolate can be used in food supplements. The latter is available in 4 forms (taking into account only salts containing only natural isomers). 3 forms of calcium salts and 1 form of glucosamine salt, but I’ll go into details on the comparison of methylfolate forms later... 

What about folic acid? 

Unlike the natural form of B9, methylfolate, folic acid must first be converted to DHF and then finally to methylfolate (5-MTHF), but it can be absorbed in its unchanged form without conversion, as can methylfolate. The transformation can get blocked at several points: [1-15] 

It is not at all converted to DHF, in which case it is absorbed in its unchanged form (UMFA - Unmetabolized Folic Acid) and enters the bloodstream. (The conversion is carried out by the enzyme DHFR) 

Although it is converted to DHF, but not to methylfolate (this is usually due to underactivity of the MTHFR enzyme, but may also be due to deficiencies of B6 and magnesium, which are required for other enzymes in the methylfolate pathway). 

Underactivation of DHFR and MTHFR is also common (polymorphism). Several studies indicate that the polymorphism responsible for the underactive MTHFR enzyme is not important if vitamin B2 intake is adequate.[5] Vitamin B6 may be the solution in the case of polymorphisms affecting the DHFR enzyme, at least even low-dose B6 supplementation prevents or reduces the elevated UMFA levels caused by high-dose folic acid supplementation. [1,2,3] 

Supplementation of vitamin B9 has been found to be problematic in several studies when higher than lifetime levels were used. It is very difficult to get more than 400 mcg from natural foods, while 800 mcg is almost impossible to achieve on a daily basis. From this perspective, it is not so surprising that supplementation above 800 mcg has been found to increase the risk of various problems,[4, 7, 10-12 ] while up to 400 mcg (or up to 800 mcg in pregnant women) has been found to be beneficial.[10,12] A correlation between an increase in UMFA levels, or unchanged levels of folic acid in the blood, and an increase in risk has been found in several studies, [8] but the appearance and increase in UMFA is also an indicator of excessive folic acid intake, and is associated with high blood levels of methylfolate + a possible indicator of vitamin B6 deficiency, so this correlation does not yet make it very likely that unchanged folic acid (UMFA) in the bloodstream from folic acid supplementation is the cause of the problems observed with high-dose B9 supplementation. In one study, supplementing 1 mg of folic acid per day was associated with an increased risk of colorectal cancer. However, the risk was not correlated with UMFA levels, but specifically with methylfolate levels. In case of those with the highest levels of methylfolate the risk was increased by almost 50%, while in case of those with the highest UMFA levels the risk was reduced by up to 40%. That is, UMFA surprisingly appeared to be a protective factor and only methylfolate a risk factor. (12) 

Summary I. 

Vitamin B9 should preferably only be supplemented in the form of methylfolate, not folic acid. Supplementation of between 200-400 mcg per day seems optimal for adults, and between 400-800 mcg per day for infants. Long-term daily supplementation above 800 mcg may be counterproductive, although perhaps only in the case of B12 deficiency and perhaps only in the case of folic acid; however, little is known yet to determine this... If you are supplementing B9, supplement B12 as well, or if your B9 intake is partly from folic acid or only from food, then watch your B2 and B6 intake. 

On the differences between the 4 types of methylfolate salts 

I only include methylfolate salts containing only natural methylfolate. All four forms are similarly good, there are only some differences in their stability and the degradation products, as well as their utilization, which aspects don't really matter, but since there is no substantial difference in their effectiveness, these are usually used as a basis for ranking one against the other in marketing materials... 

Methylfolate has a salt formed with glucosamine and a salt formed with calcium (and will soon have a salt formed with sodium...). Glucosamine salt does not have a crystalline structure, only an amorphous form. Calcium salt has both amorphous and two types of crystalline forms. Crystalline forms are generally cleaner, more stable and more effective than amorphous forms. 

 Evolution of methylfolate base materials: 

  • The so-called I-crystalline form of the calcium salt of methylfolate was the first to appear under the name Metafolin 

  • An amorphous version of the calcium salt of methylfolate was later released under the brand name Extrafolate-S 

  • Quatrefolic is the amorphous version of the glucosamine salt of methylfolate (of which there is no other) 

  • And the newest version of the calcium salt of methylfolate, known as magnefolate, is the so-called C crystal structure. 

In a recently published pre-clinical study, C-crystalline calcium salt was found to be significantly more bioavailable than glucosamine salt and I-crystalline calcium salt, the latter being slightly better than the former. [13] The glucosamine form was the least stable, although it did not contain the amorphous calcium form. However, in an unpublished ("in-house") study, a 60-day open storage test compared all four forms to see how much degradation occurred.[14] Here again, glucosamine salt degraded by far the fastest, with many degradation products, and C-crystalline calcium salt was the most stable, with very little degradation product. 

In terms of the shelf-life of raw materials in unopened packaging at room temperature, glucosamine salt is more stable than amorphous or I-crystalline calcium salt, but even in this respect it is still inferior to C-crystalline calcium salt. 

Summary II. 

All methylfolate salts are good, but if you're a perfectionist to the point of being overly maximalist, the C-crystalline calcium salt (Magnafolate) is the best, followed by the I-crystalline (Metafolin), while the amorphous calcium salt (Extrafolate-S) and glucosamine salt (Quatrefolic) are slightly less good. 

  1. Tam, C., O’Connor, D., & Koren, G. (2012). Circulating unmetabolized folic Acid: relationship to folate status and effect of supplementation. Obstetrics and gynecology international, 2012, 485179. https://doi.org/10.1155/2012/485179 

  2. Murphy MSQ, Muldoon KA, Sheyholislami H, et al. Impact of high-dose folic acid supplementation in pregnancy on biomarkers of folate status and 1-carbon metabolism: An ancillary study of the Folic Acid Clinical Trial (FACT). Am J Clin Nutr. 2021;113(5):1361-1371. doi:10.1093/ajcn/nqaa407 

  3. Obeid R, Kirsch SH, Dilmann S, et al. Folic acid causes higher prevalence of detectable unmetabolized folic acid in serum than B-complex: a randomized trial. Eur J Nutr. 2016;55(3):1021-1028. doi:10.1007/s00394-015-0916-z 

  4. Paniz, Clovis et al. “A Daily Dose of 5 mg Folic Acid for 90 Days Is Associated with Increased Serum Unmetabolized Folic Acid and Reduced Natural Killer Cell Cytotoxicity in Healthy Brazilian Adults.” The Journal of nutrition vol. 147,9 (2017): 1677-1685. doi:10.3945/jn.117.247445 

  5. EFSA: Opinion on Pyridoxal 5’-phosphate as a source for vitamin B6 added fornutritional purposes in food supplements  

  6. Kelly P, McPartlin J, Goggins M, Weir DG, Scott JM. Unmetabolized folic acid in serum: acute studies in subjects consuming fortified food and supplements. Am J Clin Nutr. 1997;65(6):1790-1795. doi:10.1093/ajcn/65.6.1790 

  7. Huang, Xiangyuan; Ye, Ying; Li, Yun; Zhang, Ying; Zhang, Yi; Jiang, Yuan; Chen, Xiaotian; Wang, Liuhui; Yan, Weili (2020). Maternal folate levels during pregnancy and childrens neuropsychological development at 2 years of age. European Journal of Clinical Nutrition, (), –. doi:10.1038/s41430-020-0612-9 

  8. Raghavan R, Selhub J, Paul L, et al. A prospective birth cohort study on cord blood folate subtypes and risk of autism spectrum disorder. Am J Clin Nutr. 2020;112(5):1304-1317. doi:10.1093/ajcn/nqaa208 

  9. Pentieva K, Selhub J, Paul L, et al. Evidence from a Randomized Trial That Exposure to Supplemental Folic Acid at Recommended Levels during Pregnancy Does Not Lead to Increased Unmetabolized Folic Acid Concentrations in Maternal or Cord Blood. J Nutr. 2016;146(3):494-500. doi:10.3945/jn.115.223644 

  10. Maruvada P, Stover PJ, Mason JB, et al. Knowledge gaps in understanding the metabolic and clinical effects of excess folates/folic acid: a summary, and perspectives, from an NIH workshop. Am J Clin Nutr. 2020;112(5):1390-1403. doi:10.1093/ajcn/nqaa259 

  11. Troen, Aron M.; Mitchell, Breeana; Sorensen, Bess; Wener, Mark H.; Johnston, Abbey; Wood, Brent; Selhub, Jacob; McTiernan, Anne; Yasui, Yutaka; Oral, Evrim; Potter, John D.; Ulrich, Cornelia M. (2006). Unmetabolized Folic Acid in Plasma Is Associated with Reduced Natural Killer Cell Cytotoxicity among Postmenopausal Women. The Journal of Nutrition, 136(1), 189–194. doi:10.1093/jn/136.1.189 

  12. Rees, Judy R et al. “Unmetabolized Folic Acid, Tetrahydrofolate, and Colorectal Adenoma Risk.” Cancer prevention research (Philadelphia, Pa.) vol. 10,8 (2017): 451-458. doi:10.1158/1940-6207.CAPR-16-0278 

  13. Lian Z, Chen H, Liu K, Jia Q, Qiu F, Cheng Y. Improved Stability of a Stable Crystal Form C of 6S-5-Methyltetrahydrofolate Calcium Salt, Method Development and Validation of an LC–MS/MS Method for Rat Pharmacokinetic Comparison. Molecules. 2021; 26(19):6011. https://doi.org/10.3390/molecules26196011 

  14. https://methyl-life.com/pages/methylfolate-types 

  15. EFSA NDA Panel (EFSA Panel on Nutrition, Novel Foods and Food Allergens), Turck, D, Castenmiller, J, De Henauw, S, Hirsch-Ernst, KI, Kearney, J, Maciuk, A, Mangelsdorf, I, McArdle, HJ, Naska, A, Pelaez, C, Pentieva, K, Siani, A, Thies, F, Tsabouri, S, Vinceti, M, Cubadda, F, Engel, K-H, Frenzel, T, Heinonen, M, Marchelli, R, Neuhäuser-Berthold, M, Poulsen, M, Sanz, Y, Schlatter, JR, van Loveren, H, Bernasconi, G, Germini, A and Knutsen, HK, 2020. Scientific Opinion on calcium l-methylfolate as a source of folate added for nutritional purposes to infant and follow-on formula, baby food and processed cereal-based food. EFSA Journal 2020;18(1):5947, 17 pp. https://doi.org/10.2903/j.efsa.2020.5947 

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