Dr. Amy Saenger discusses the limited utility of testing for erythrocyte folate in the age of folic acid supplementation. She also offers alternative choices for the diagnosis of folate and vitamin B12 deficiency.
Presenter: Amy K. Saenger, PhD
- Director of the Cardiovascular Laboratory in the Department of Laboratory Medicine & Pathology
- Consultant in the Division of Clinical Core Laboratory Services at Mayo Clinic
Welcome to Mayo Medical Laboratories' Hot Topics. These presentations provide short discussions of current topics and may be helpful to you in your practice.
Our presenter for this program is Amy K. Saenger, PhD, Director of the Central Clinical Laboratory in the Department of Laboratory Medicine & Pathology at Mayo Clinic in Rochester, Minnesota. Dr. Saenger will discuss the limited utility of testing for erythrocyte folate in the age of folic acid supplementation. She will also offer alternative choices for the diagnosis of folate and vitamin B12 deficiency.
Folate is a water-soluble B vitamin that is essential for adequate health. Folate, along with vitamin B12, is essential for DNA synthesis, repair, and methylation. Folate coenzymes are also integral in reactions involving one-carbon metabolism, and although there are various forms of methylated folate in the body, the primary form is 5-methyltetrahydrofolate. 5-methylTHF is also a cosubstrate required by methionine synthase for the conversion and demethylation of homocysteine to methionine *required to re-methylate homocysteine to form methionine, a reaction catalyzed by the enzyme methionine synthase. Homocysteine may also be re-methylated to methionine by other folate independent enzymes such as betaine homocysteine methyltransferase (BHMT) but generally to a much lower extent. Therefore, in the setting of folate deficiency there will be an accumulation of homocysteine. Folate and vitamin B12 (or cobalamin) metabolism are linked by the reaction that transfers a methyl group from 5-methyltetrahydrofolate to B12. Homocysteine, along with methylmalonic acid, will also be elevated in the setting of B12 deficiency, and a combination of MMA and homocysteine has been proposed by some to distinguish between B12 and folate deficiency.
* Bold section denotes correction made in August 2013.
Folate deficiency manifests clinically in a variety of ways; notably it is strongly linked to an increased risk of neural tube defects and several observational and controlled trials have demonstrated that neural tube defects are significantly reduced with periconceptual folic acid supplementation. Individuals with folate deficiency may present clinically with unexplained, nonspecific neurological symptoms including dementia, weakness, and headaches. Folate and vitamin B12 deficiencies are both associated with a reduction in hemoglobin and megaloblastic changes in the bone marrow or other tissues. Megaloblastic anemia is the primary manifestation of folate deficiency, where erythrocytes become abnormally large and nucleated due to the lack of folate necessary for DNA synthesis and cell division.
Recognition of the significant relationship between folate and neural tube defects, cancer, and cardiovascular disease led to FDA-mandated fortification of breads, cereals, flours, pasta, and other grain products. Complete fortification was fully implemented in the United States in 1998, with the primary goal of reduction of neural tube defects. Overall, fortification has been successful and the prevalence of low serum folate among women of childbearing age declined from 20.6% in 1988-1994 to 0.8% in 2005-2006. The incidence of neural tube defects was reduced by 36% from 1995 to 2006 in the United States (10.8 to 6.9 per 10,000 pregnancies).
Selected Food Sources of Folate and Folic Acid
The term "folate" is utilized when referring to the naturally occurring forms of folate, while "folic acid”" refers to forms used in food and drink supplementation. Folate is found naturally in raw leafy vegetables, citrus fruits, dried beans and peas. Widespread fortification of foods with folic acid makes it difficult to not have adequate folate status; breakfast cereals and pasta are routinely consumed food products in the United States and have some of the highest fortification concentrations, as noted by the starred items in this table.
Causes of Folate Deficiency
There are a variety of clinical conditions that contribute to folate or folic acid deficiency. A deficiency of folate may occur in situations where there is an increased demand for folate, such as during pregnancy, lactation, infancy, malignancy (where there is increased cell turnover), infection (due to an immunoproliferative response), or chronic hemolytic anemia (due to increased hematopoiesis). Clinical conditions associated with malabsorption, such as celiac disease, also place patients at a higher risk for folate deficiency. Metabolism of folate may also be impaired due to various folic acid antagonist drugs, and patients with severe liver or kidney disease may have increased excretion of folate. Furthermore, despite widespread fortification of foods with folic acid, deficiency still occurs in situations where there is inadequate ingestion or poor nutrition. High-risk patient populations include people with alcoholism, patients with psychiatric morbidities, and the elderly.
Laboratory Diagnosis of Folate Deficiency
Suspicion of folate deficiency may originate if patient history reveals any clinical conditions previously mentioned and/or from results from a routine complete blood count, where a low hemoglobin and high MCV are observed. Use of the MCV alone is a nonspecific indicator when used by itself, as patients with concurrent iron and folate deficiency will not have the characteristic macrocytosis seen in folate deficiency alone. Laboratory diagnosis of folate deficiency further includes measurement of serum folate, and less often red blood cell (RBC) folate, also called erythrocyte folate. In blood 95% of folate is within the erythrocytes. Folate is taken up only by the developing erythrocyte; therefore RBC folate has historically been regarded as the better indicator of long-term folate storage. Serum folate concentrations reflect recent dietary intake of folate, but measurements need to be conducted after the patient fasts. Thus, theoretically, while RBC folate is less susceptible to rapid changes in dietary intake, analytically, the assays are plagued with imprecision issues. As mentioned previously, homocysteine and methylmalonic acid may also be utilized to distinguish between folate and B12 deficiency.
Folate and Vitamin B12 Deficiency
One critical point to remember prior to initiation of folic acid supplementation is to rule out cobalamin (vitamin B12) deficiency. Unlike dietary folate, synthetic folic acid (pteroylglutamic acid) is reduced directly to tetrahydrofolate without the need for cobalamin as a cofactor. For this reason, the administration of supplemental amounts of folic acid to a B12-deficient patient may correct the megaloblastic hematologic changes but still allow neurologic disease to progress. This damage is often irreversible.
Quantitation of Folate: Methods
There are a number of methods which can be utilized to quantitate serum or red cell folate. These include microbiologic assays, competitive protein-binding assays, or chromatography. A majority of laboratories, including Mayo, use a protein-binding assay with chemiluminescent detection for both serum and red blood cell folate, where the red cell hemolysate is prepared by lysing with ascorbic acid, which releases the intracellular folate, again primarily in the 5-tetrahydrofolate form.
Differences Between Folate Immunoassays
There are notable differences between results obtained for red cell and serum folate assays between manufacturers, which is not an uncommon occurrence for any unstandardized assay on the market. Bias between test methods and platforms may be as low as 10% and as high as 70%, despite similar claims of traceability, and occurs throughout the analytical range of the assays. Therefore, there is currently no one standard for folate measurement and values can vary significantly from one laboratory to the next because of method differences.
Analytical Imprecision: RBC Folate (CAP Surveys)
Analytical imprecision is notably worse with red cell folate assays compared to serum folate assays. This has been well documented in the literature and noticeable in proficiency testing surveys, where the variability even within the same peer group may be up to 50%. Manual sample preparation and variability among procedures to completely lyse the red blood cells are likely responsible for much of this variation.
Equivalence of Serum and RBC Folate
In addition to the documented analytical variables and assay quality issues, over the last decade there has been a growing body of literature demonstrating the equivalence of red cell and serum folate testing, in an effort to improve test utilization and reduce unnecessary spending.
Modern Folate Deficiency is Rare
Results from our laboratory and other studies suggest that American populations have indeed attained adequate concentrations of folate and that modern folate deficiency has essentially been eliminated. This graph shows results from Mayo Clinic patients (in Rochester, MN only) who had serum folate ordered over the last two years. Of the 24,177 serum folates performed, only one-half of a percent were less than 3.0 ng/mL, which is the deficient concentration as defined by NHANES and the CDC. Thus, a huge majority of samples are being tested that are normal; there are not many other routine laboratory tests that have this type of distribution, normally a more Gaussian distribution of results can be observed.
Mayo RBC/Serum Folate Orders 1999-2009
We further undertook a 10-year retrospective analysis of red blood cell and serum folate results to examine ordering patterns and evaluate the clinical utility of RBC folate in our patient population. Results were retrieved from all serum and RBC folate tests from the laboratory information system at Mayo ordered on inpatients and outpatients between 1999-2009. Data for patients who had simultaneous orders for serum and red cell folate were analyzed and chart reviews were conducted on those patients with normal serum folate but low RBC folate; these are the individuals who have the potential for misdiagnosis if screened with a serum folate alone. Abnormal values were defined two-fold: first by our current reference ranges (
True folate deficiency in the current era of FDA-mandated folic acid supplementation is exceedingly rare. There is no evidence to support routine ordering of RBC or serum folate, but serum folate concentrations provide equivalent clinical information to RBC folate in the assessment and diagnosis of folate deficiency. Based on these statistics, and because serum folate provides equivocal results to RBC folate in almost all clinical scenarios, routine ordering of RBC folate is no longer warranted. Furthermore, investigation of megaloblastic anemia should preferentially be initiated with vitamin B12 testing instead of folate due to the low incidence of modern folate deficiency. In the absence of B12 deficiency, it is more cost effective to simply supplement with folic acid rather than routinely test and monitor a patient's folate status, similar to other nutritional deficiencies such as vitamin D.