Vitamin Biomarkers: Serum Folate

Optimal Takeaways

Folate is a B vitamin with essential roles in red and white blood cell development, methionine and homocysteine metabolism, and other methylation reactions. Folate insufficiency leads to megaloblastic anemia, hypersegmented neutrophils, altered SAM metabolism, elevated homocysteine, birth defects, cardiovascular disease, cognitive impairment, and increased cancer risk. Elevated serum folate may be seen with pernicious anemia, vegetarianism, and insufficiency of vitamin B12, which is required for folate metabolism. Excess synthetic folic acid may impair folate and homocysteine metabolism.

Standard Range: 5.50 - 27.00 ng/mL (12.46 – 61.18 nmol/L)

The ODX Range: 15.00 – 27.00 ng/mL (33.99 – 61.18 nmol/L)

Low folate levels are associated with megaloblastic folate deficiency anemia, hemolytic anemia, malabsorption, e.g., celiac or Crohn’s disease, malnutrition, anorexia nervosa, alcoholism, pregnancy, cancer, and the use of certain medications, including antacids, H2 blockers, oral contraceptives, estrogen, methotrexate, penicillin, tetracyclines, antimalarials, anti-convulsants (Pagana 2021), triamterene, and metformin (Bailey 2015).

Low folate can also be associated with compromised methylation of protein and DNA; impaired synthesis of hormones, phosphatidylcholine, creatine, and carnitine; hepatic triglyceride accumulation, steatohepatitis, and non-alcoholic fatty liver disease; altered expression of genes associated with obesity, metabolic syndrome, and lipid metabolism (da Silva 2014); compromised methionine metabolism (Zheng 2019); elevated homocysteine and pre-eclampsia (WHO 2015); impaired red and white blood cell production (Pfeiffer 2012); leukemia, lymphoma, cardiovascular disease, cognitive dysfunction (Bailey 2015); depression (Bender 2017); impaired reproductive health and congenital disabilities, especially neural tube defects such as spina bifida (Naderi 2018). Other birth defects associated with compromised folate status include oral clefts, congenital heart defects, restricted growth, low birth weight, and premature birth (Chen 2019).

High folate levels can be seen with pernicious anemia, B12 deficiency, vegetarianism, and recent blood transfusions (Pagana 2021). Elevations in unmetabolized synthetic folic acid inhibit the re-methylation of homocysteine to methionine (da Silva 2014).

Overview

The term folate refers to a family of naturally occurring compounds that include 5-methyltetrahydrofolate, folinic acid, and various forms of tetrahydrofolate. Folic acid is a synthetic form not found naturally in food though it may be formed from the oxidation of folates in cooked or stored foods (Scaglione 2014). Folic acid is more shelf-stable and is currently the form used to fortify processed foods in many countries.

Folate, also known as vitamin B9, is required for the normal production and function of red and white blood cells and the synthesis of DNA precursors. Transport of folate into the cell requires vitamin B12. Therefore, serum folate can be elevated with a B12 deficiency, while intracellular folate will be low (Pagana 2021). A B12 deficiency can lead to cellular accumulation of 5-MTHF (the “methyl-folate trap”) as well as homocysteine despite adequate folate availability (Ohrvik 2011). Folate interacts closely with vitamins B2 and B6 as well and should be assessed along with folate (Bailey 2015).

Folate plays a vital role in methylation (1 carbon metabolism) and the metabolism of S-adenosylmethionine (SAM), homocysteine, phosphatidylcholine, and carnitine. Folate deficiency disrupts SAM metabolism and impairs protein modification, DNA methylation, hormone synthesis, and homocysteine metabolism, ultimately increasing serum homocysteine. Inadequate methylation of phosphatidylcholine contributes to steatohepatitis and non-alcoholic fatty liver disease (da Silva 2014). Folate also plays an essential role in mitochondrial metabolism and function (Zheng 2019).

Food fortification with folic acid, the synthetic form of folate, has reduced the incidence of neural tube defects. However, excess intake of the synthetic form can mask a B12 deficiency and may promote folate-sensitive cancers such as colorectal cancer (Naderi 2018). Excess synthetic folic acid intake above 200 ug/day increases unmetabolized folic acid in the blood. Elevated folic acid inhibits the MTHFR enzyme and re-methylation of homocysteine, resulting in a functional folate deficiency. The preferred form of supplemental folate is 5-MTHF which increases serum folate without increasing unmetabolized folic acid (da Silva 2014). The adverse effects of unmetabolized folic acid may be magnified with compromised vitamin B12 status, resulting in elevated homocysteine and methylmalonic acid and decreased holotranscobalamin (active B12). Researchers note that the tolerable upper intake limit of 1 mg/day of folic acid is too high, citing increased neurological effects associated with sustained folic acid doses of 0.5-1 mg/day in conjunction with B12 deficiency. Food fortification can lead to excess intake of folic acid, where mandatory fortification of flour may be as high as 80 ug/100 grams in the United States and 200 ug/100g in Chile. A combination of elevated folate and insufficient B12 in mothers may contribute to the incidence of insulin resistance and stunted growth in their offspring. The combination of folate and B12 deficiency is associated with neuropathy, cognitive decline, and depression (Sobczyńska-Malefora 2018). Folate supplementation should always be combined with vitamin B12 to avoid masking a B12 deficiency (Pizzorno 2016).

The 5-MTHF form does not mask B12 deficiency as synthetic folic acid does. 5-MTHF is better absorbed despite alterations in gastric pH. It reduces drug interactions that interfere with folate metabolism and overrides the metabolic defects associated with the MTHFR polymorphism. The association between high folate intake and cancer, especially breast cancer, is attributed to high doses of synthetic folic acid versus natural folate (Scaglione 2014). The potential for high-dose synthetic folic acid to promote cancer and other adverse effects has called into question its mandatory fortification in food (Ohrvik 2011).

Food sources contain natural folate, not synthetic folic acid, and include leafy green vegetables, legumes, egg yolks, and liver (folate is stored in the liver). Serum folate levels reflect recent intake, whereas red blood cell levels reflect long-term folate status. Some individuals may have impaired folate metabolism due to a polymorphism in the MTHFR gene. Homocysteine is a functional indicator of folate status; increased levels likely indicate insufficiency or impaired folate metabolism (WHO 2015).

The MTHFR polymorphism is associated with impaired riboflavin status, high blood pressure, and stroke (McNulty 2019). It is also associated with an increased risk of neural tube defects and cardiovascular disease. The methylated form of folate is required to re-methylate homocysteine to methionine, a B12-dependent process (Scaglione 2014).

Folate insufficiency is one of the most common nutrient deficiencies (Scaglione 2014) and is characterized by stages. The first stage is characterized by a decrease in serum folate followed by increasing homocysteine; decreasing red blood cell folate; alterations in rapidly dividing tissues, including bone marrow which results in large immature (megaloblastic) RBCs, hypersegmented neutrophils, chromosome breakage, and generation of lymphocyte micronuclei and Howell-Jolly bodies; anemia; and decreased oxygen-carrying capacity which contributes to shortness of breath, weakness, fatigue, and irritability (Bailey 2015). Some laboratory methods may underestimate circulating 5-MTHF despite it being the major circulating metabolite, resulting in potentially inaccurate measurement of folate status (WHO 2015)

Low serum folate below 7 ng/mL (15.86 nmol/L) was found to correlate with markers of nutritional deficiency, including low prealbumin, albumin, hemoglobin, and B12, compared to folate of 15 ng/mL (33.99 nmol/L). Biomarkers of nutrient deficiency were present even in subjects with a BMI above 25, demonstrating that malnutrition can occur in obesity. Folate below 7 ng/mL was also associated with gastrointestinal disorders such as IBD and celiac disease, sepsis, increased creatinine, and low MCV (possibly due to a concurrent iron deficiency). Serum folate below 3.4 ng/mL (7.7 nmol/L) was associated with a lower serum ALT level, possibly due to a concurrent vitamin B6 deficiency (Kozman 2020). Researchers confirm the need for revised conventional reference ranges for serum folate and that a reduction in neural tube defects was associated with a level of at least 13 ng/mL (29.46 nmol/L) or above (Singh 2015).

Folate insufficiency may also increase the risk of specific cancers, including head and neck, oral cavity, pharynx, esophagus, pancreas, bladder, and cervix. Combining folate insufficiency with increased alcohol intake may increase the risk of breast cancer (Pieroth 2018).

References

Bailey, Lynn B et al. “Biomarkers of Nutrition for Development-Folate Review.” The Journal of nutrition vol. 145,7 (2015): 1636S-1680S. doi:10.3945/jn.114.206599

Bender, Ansley et al. “The association of folate and depression: A meta-analysis.” Journal of psychiatric research vol. 95 (2017): 9-18. doi:10.1016/j.jpsychires.2017.07.019

Chen, Meng-Yu et al. “Defining the plasma folate concentration associated with the red blood cell folate concentration threshold for optimal neural tube defects prevention: a population-based, randomized trial of folic acid supplementation.” The American journal of clinical nutrition vol. 109,5 (2019): 1452-1461. doi:10.1093/ajcn/nqz027

da Silva, Robin P et al. “Novel insights on interactions between folate and lipid metabolism.” BioFactors (Oxford, England) vol. 40,3 (2014): 277-83. doi:10.1002/biof.1154

Devalia, Vinod et al. “Guidelines for the diagnosis and treatment of cobalamin and folate disorders.” British journal of haematology vol. 166,4 (2014): 496-513. doi:10.1111/bjh.12959

Ebara, Shuhei. “Nutritional role of folate.” Congenital anomalies vol. 57,5 (2017): 138-141. doi:10.1111/cga.12233

Froese, D Sean et al. “Vitamin B12 , folate, and the methionine remethylation cycle-biochemistry, pathways, and regulation.” Journal of inherited metabolic disease vol. 42,4 (2019): 673-685. doi:10.1002/jimd.12009

Kozman, Diana et al. “Serum Folate of Less than 7.0 ng/mL is a Marker of Malnutrition.” Laboratory medicine vol. 51,5 (2020): 507-511. doi:10.1093/labmed/lmz101

McNulty, Helene et al. “Addressing optimal folate and related B-vitamin status through the lifecycle: health impacts and challenges.” The Proceedings of the Nutrition Society vol. 78,3 (2019): 449-462. doi:10.1017/S0029665119000661

Naderi, Nassim, and James D House. “Recent Developments in Folate Nutrition.” Advances in food and nutrition research vol. 83 (2018): 195-213. doi:10.1016/bs.afnr.2017.12.006

Ohrvik, Veronica E, and Cornelia M Witthoft. “Human folate bioavailability.” Nutrients vol. 3,4 (2011): 475-90. doi:10.3390/nu3040475

Pagana, Kathleen Deska, et al. Mosby's Diagnostic and Laboratory Test Reference. 15th ed., Mosby, 2021.

Pfeiffer, Christine M et al. “Estimation of trends in serum and RBC folate in the U.S. population from pre- to postfortification using assay-adjusted data from the NHANES 1988-2010.” The Journal of nutrition vol. 142,5 (2012): 886-93. doi:10.3945/jn.111.156919

Pieroth, Renee et al. “Folate and Its Impact on Cancer Risk.” Current nutrition reports vol. 7,3 (2018): 70-84. doi:10.1007/s13668-018-0237-y

Pizzorno, Joseph E., Michael T. Murray, and Herb Joiner-Bey. The Clinician's handbook of natural medicine E-book. Elsevier Health Sciences, 2016.

Scaglione, Francesco, and Giscardo Panzavolta. “Folate, folic acid and 5-methyltetrahydrofolate are not the same thing.” Xenobiotica; the fate of foreign compounds in biological systems vol. 44,5 (2014): 480-8. doi:10.3109/00498254.2013.845705

Singh, Gurmukh et al. “Clinical utility of serum folate measurement in tertiary care patients: Argument for revising reference range for serum folate from 3.0 ng/mL to 13.0 ng/mL.” Practical laboratory medicine vol. 1 35-41. 26 Mar. 2015, doi:10.1016/j.plabm.2015.03.005

Sobczyńska-Malefora, Agata, and Dominic J Harrington. “Laboratory assessment of folate (vitamin B9) status.” Journal of clinical pathology vol. 71,11 (2018): 949-956. doi:10.1136/jclinpath-2018-205048

World Health Organization. Serum and red blood cell folate concentrations for assessing folate status in populations. No. WHO/NMH/NHD/EPG/15.01. World Health Organization, 2015. https://apps.who.int/iris/bitstream/handle/10665/162114/WHO_NMH_NHD_EPG_15.01.pdf

Zheng, Yuxiang, and Lewis C Cantley. “Toward a better understanding of folate metabolism in health and disease.” The Journal of experimental medicine vol. 216,2 (2019): 253-266. doi:10.1084/jem.20181965