Folate (Folic Acid)

Folate measurements are used in the diagnosis and management of megaloblastic anemia. The reference range of the plasma folate level varies by age, as follows ^[1] :

• Adults - 2-20 ng/mL, 2-20 μg/L, or 4.5-45.3 nmol/L
• Children - 5-21 ng/mL, 5-21 μg/L, or 11.3-47.6 nmol/L
• Infants - 14-51 ng/mL, 14-51 μg/L, or 31.7-115.5 nmol/L

The reference range of the red blood cell (RBC) folate level also varies by age, as follows ^[1] :

• Adults: 140-628 ng/mL or 317-1422 nmol/L
• Children: Over 160 ng/mL or over 362 nmol/L

However, since the United States instituted mandatory fortification of cereal grain products with folic acid, new adult reference values of 5.8-32.8 ng/mL (13.1-74.3 nmol/L) for serum folate and 153-515 ng/mL (347-1167 nmol/L) for erythrocyte folate have been proposed, demonstrating the importance of establishing regional and national reference values. ^[2]

Biochemical deficiency has been defined as a concentration of less than 3 ng/mL (< 6.7 nmol/L) for serum folate and less than 140 ng/ml (< 322 nmol/L) for erythrocyte folate. ^[3]

Circulating folate levels are strongly influenced by recent intake and are an unreliable index of tissue stores. The plasma folate level can be restored to normal with one folate-rich meal. Principal food sources of folate are liver, spinach, legumes, and orange juice, although in countries that fortify cereal with folic acid, this is often the major source. ^[3]

RBC folate provides information about folate status over the lifetime of RBCs, similar to hemoglobin A1C as used in blood glucose monitoring. Approximately 5% of people with normal serum folate levels may have evidence of folate deficiency if analyzed using RBC folate testing. ^[4]

In patents with normal or low-normal folate levels in whom folate deficiency is clinically suspected (based on risk factors such as malabsorption or other findings, including megaloblastic anemia), it is more common to use ancillary testing of methylmalonic acid (MMA) and homocysteine, rather than RBC folate analysis, to confirm folate deficiency. In folate deficiency, the plasma homocysteine level is elevated but MMA is normal, whereas both plasma homocysteine and MMA are elevated in vitamin-B12 deficiency. ^[5]

Causes of folate deficiency include the following ^[6] :

• Decreased folate intake
• Alcohol use
• Advanced age
• Poor dietary intake
• Overcooked food
• Malabsorption -  Alcoholism,  inflammatory bowel disease,  celiac disease, infiltrative disease,  short-bowel syndrome
• Increased folate demand -  Lactation, pregnancy,  exfoliative dermatitis, hemodialysis, chronic hemolysis,  leukemias,  hypothyroidism
• Medications - Methotrexate, trimethoprim, phenytoin, antimalarials, antacids, oral contraceptives

Researchers found higher rates of anemia and other blood abnormalities in people with low vitamin-B12 levels who also had high folate levels. ^[7] To address the concerns associated with this finding, the National Institutes of Health (NIH) conducted a study on vitamin-B12 and folic acid levels in the population, particularly as high folic acid levels may result from the fortification of grains in the United States and other Western nations, and concluded that high folic acid levels do not worsen the manifestations of vitamin-B12 deficiency. ^[8]

Most commercial laboratories offer the quantitative determination of folate in human serum, plasma, and RBCs using the ARCHITECT chemiluminescent microparticle immunoassay (CMIA, Abbott) ^[9] or electrochemiluminescence (ECL) technology with flexible assay protocols (Roche). Other analytic tools are also available, such as the Immulite immunoassay (Siemens), ^[10] the Quantaphase II Folate/Vitamin B12 radioassay kit (Bio-Rad), ^[11]  and the microbiologic method. ^[12]  Another analytic technique, using a competitive binding receptor assay (the access folate assay) is available at Mayo Clinic Laboratories. ^[13]

Specifics of the ARCHITECT and the Immulite systems are listed below.

ARCHITECT folate assay - serum folate level

Specifics for this assay include the following ^[9] :

• Specimen type - Venous blood
• Container - Serum (glass or plastic tube), serum separator tube (SST), lithium heparin plasma separator tube (PST); do not use plasma in ethylenediaminetetraacetic acid (EDTA) tubes for folate; protect specimen from light; hemolyzed specimens will give falsely elevated levels
• Collection method - Venipuncture
• Specimen volume - 3 mL
• Collection instructions - Test should be performed after an 8-hour fast; avoid vitamin-B12 injection before test
• Other instructions - Do not use specimens that have been heat inactivated, pooled, or hemolyzed or that have obvious bacterial contamination

The following lead to inaccurate results:

• The use of human anti-mouse antibodies (HAMA)
• Routine exposure to animals/animal serum products that have heterophilic antibodies in their serum and can react with reagent immunoglobulins
• The use of methotrexate, aminopterin, or folinic acid (leucovorin)

If testing is not being performed immediately, specimens may be stored at 2-8 ºC for 7 days or at -10 ºC for 30 days.

Immulite 2000 folic acid test - RBC folate level

Specifics for this assay include the following ^[10] :

• Specimen type - Venous blood; calculation of RBC folate requires simultaneous testing of the patient’s hematocrit and serum folate level
• Container - Fresh heparinized or EDTA whole blood (EDTA plasma should not be used)
• Collection method - Venipuncture
• Specimen volume - 100 μL fresh heparinized or EDTA whole blood for RBC folate, 50 μL serum/heparinized plasma for serum folate
• Collection instructions - Test should be performed after an 8-hour fast; avoid vitamin-B12 injection before test
• Other instructions - To clear a lipemic sample, ultracentrifuge use is recommended; ensure complete clot formation prior to centrifugation

If testing will not be performed immediately, specimens may be stored at 2-8 ºC for 8 hours or at -20 ºC for 6-8 weeks. 

The natural variety of vitamin B9 is known as folate. Although the terms folate and folic acid are used interchangeably, folic acid is synthetically made.

Folate itself is not biologically active. It is converted to its active form, tetrahydrofolic acid (THF), by the enzyme dihydrofolate reductase. Compounds such as serine, glycine, and histidine contribute one-carbon fragments to THF, which transfers them into intermediates used in amino acid, purine, and thymidine synthesis, with thymidine being the characteristic pyrimidine in DNA. ^[14]

THF is involved in the conversion of homocysteine to methionine; hence, elevated homocysteine levels are noted in folate deficiency. 

Folate is found in the following foods ^[15] :

• Meats - Liver, chicken, kidney, egg yolk
• Legumes - Dried beans, lentils, soya products, almonds, nuts
• Starches - Whole-grain breads, wheat flour, potatoes
• Fruit and vegetables - Spinach, beetroot, Brussels sprouts, broccoli, cabbage, asparagus, bananas, oranges, peaches

Normal folate requirements are about 200-400 µg/day. The requirements in pregnant and lactating individuals increase to 500-800 µg/day. Healthy individuals have about 500-20,000 µg of folate stored, mainly in the liver.

Folate testing is mainly indicated as part of the workup for megaloblastic anemia and hypersegmented neutrophils.

Screening for folate deficiency is performed by checking the serum folic acid level. Serum levels are sensitive to false elevation after a meal, so fasting levels are more reliable. RBC levels are not recommended as a screening test but rather as a diagnostic test when serum levels are low or low-normal. However, although RBC folate is more accurate, it is a costly and less available test. Alternatively, elevated serum homocysteine levels with normal MMA levels can be used to confirm folate deficiency. ^[4]

Pregnancy is not an indication for routine folate screening, although it is an indication for prophylactic folate supplementation.

Folate deficiency is treated with folic acid supplementation and management of the underlying cause.

The main presentation of folate deficiency is megaloblastic anemia without neurologic changes, in contrast to vitamin-B12 deficiency, in which neurologic changes may be observed.

Folic acid can partially reverse some of the hematologic abnormalities of vitamin-B12 deficiency but not the neurologic manifestations. Thus, megaloblastic anemia is not treated with folic acid until vitamin-B12 deficiency has been excluded.

Folate deficiency can also cause congenital neural tube defects, the incidence of which is reduced by the administration of folic acid supplements in pregnant women.

The fact that folate is required for the synthesis and repair of DNA makes it a target for many cancer medications. Methotrexate, for example, is a dihydrofolate reductase inhibitor that prevents the conversion of inactive folate to its active form, THF. 

In a case-control study that evaluated 750 patients with documented vascular disease and 800 control subjects, low levels of folate and vitamin B6 were associated with an increased risk for atherosclerosis, independent of conventional risk factors. The folate-linked risk was partially explained by increased serum homocysteine. ^[16]