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Folate, also called vitamin B9, is an essential nutrient produced by microorganisms and plants. Folate functions as a coenzyme necessary for the synthesis of thymidine, which is required for DNA synthesis.  The major nutritional source of folate is leafy green vegetables, citrus fruits, and beans. The daily requirement is about 400 ug. Naturally occurring folate is a water-soluble molecule that exists as tetrahydrofolate and methyl tetrahydrofolate. 

Folic acid is the synthetic form of folate that is found in supplements, fortified foods and medications. Folic acid must be reduced by the enzyme dihydrofolate reductase to dihydrofolate and then to tetrahydrofolate. The latter is converted to the biologically active 5-methyl tetrahydrofolate (5-MTHF). 

Both folate and folic acid are most efficiently absorbed in the proximal jejunum by the proton-coupled folate transporter. Folate is released by enterocytes into the portal circulation for storage in the liver. 

In 1996 the FDA mandated folate fortification of cereals and grains in the USA to reduce the risk of neural tube defects in pregnancy, resulting in a significant decrease in the incidence of folate deficiency. Mandatory fortification of grain products added about 0.1 mg folate to the daily intake of an adult.

The prevalence of samples with low serum folate levels has decreased significantly over the past two decades as a consequence of food fortification.


%Folate Deficient

% Folate  >20 ng/mL

Median (ng/mL)














Although folate deficiency is now uncommon in the United States, evaluation of folate status is still indicated in a patient with megaloblastic anemia associated with poor nutrition, debilitation, alcoholism or malabsorption.

Two tests were traditionally used in the evaluation of folate status – serum folate and red cell folate. The rationale for measuring red cell folate was that it more accurately reflected tissue folate stores than serum folate, since red cells acquired folate when they were produced, and the cellular concentration of folate did not change during the red cell lifespan. A single folate-rich meal could correct serum folate concentration in a deficient patient, whereas a persistently low red cell folate would indicate chronically depleted stores. However, a study of folate concentrations in the liver (tissue folate stores) showed that they correlated equally well with serum and red cell folate. Additionally the red cell folate assay hd several disadvantages, including a lack of specificity and sensitivity. Red cell folate levels were decreased in up to 63% of patients with vitamin B12 deficiency, and normal levels occurred in a significant number of individuals with documented folate deficiency. The red cell folate assay was more difficult to perform than the serum folate assay, resulting in poorer precision and reliability, and increased cost. 

For the small proportion of cases (approx. 5%) where serum folate is normal, but there is a high clinical suspicion of folate deficiency, further testing can be performed. Some authorities have suggested that plasma homocysteine is a satisfactory substitute for the problematic red cell folate assay in this situation. An elevation of plasma homocysteine is a sensitive and early indicator of functional folic acid deficiency.


Differentiation of Folate & B12 Deficiency        


Serum Folate

Serum  B12








Vitamin B12 










HC=homocysteine, MMA=methylmalonic acid

A normal homocysteine level makes the diagnosis of folate deficiency extremely unlikely. Of course, elevation of homocysteine is not specific for folate deficiency, and can accompany other disorders, including vitamin B12 deficiency (methylmalonic acid is also elevated in this disorder), pyridoxine deficiency, renal failure, and various enzyme defects in the homocysteine metabolic pathway. In view of the above findings, measurement of red cell folate is no longer recommended. 

The low prevalence of folate deficiency calls into question the common practice of simultaneously ordering folate and vitamin B12 . Folate deficiency, in the absence of megaloblastic anemia, is now rare in the United States. Even in patients with megaloblastic anemia, there are often clinical indications that would lead to ordering only one of the two tests. For example, a vegetarian is much more likely to have B12 deficiency, while a patient with poor nutrition is more likely to have folate deficiency. Vitamin B12 levels are often ordered during the workup of peripheral neuropathies, but folate levels are not indicated. Routinely ordering of both tests not only doubles the laboratory’s work, but also increases health care costs.   

Causes of a falsely elevated serum folate level include a recent folate-rich meal, and even slight hemolysis because red cell folate is 30 times higher than serum folate. A falsely low serum folate level may be seen in the absence of megaloblastosis in patients with recent poor dietary intake, alcoholism, normal pregnancy, and in patients receiving anticonvulsant therapy.

Reference range for serum folate is >3.4 ng/mL. 

Specimen requirement for serum folate assay is one red top tube of blood, and for plasma homocysteine is one lavender-top (EDTA) tube. Fasting samples are preferred for both assays because recent food intake may increase the serum folate level. The specimen should be protected from light until processing. Hemolyzed specimens cannot be used. The following substances may interfere with folate determination: heparin, ascorbic acid, fluoride, methotrexate, or other folic acid antagonist.


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