Folic Acid and Folate

Sample for Folic Acid and Folate

1. Fasting venous blood is needed to prepare the serum.
   • Some of the labs take a sample without fasting.
   • Protect the blood from light.
2. If RBCs folate is needed, then take blood in EDTA.
3. The sample is stable for 24 hours at 4 °C.
   1. 6 to 8 weeks at -20 °C.
4. Also, perform hematocrit.
5. Avoid hemolysis.
6. Avoid repeated freezing and thawing.

Precautions for Folic Acid and Folate

1. Certain drugs decrease folic acids like Alcohol, PAS, Ampicillin, Antimalarial, Erythromycin, Methotrexate, Oral contraceptives, Chloramphenicol, Penicillins Phenobarbital, Aminopterin, Tetracyclines, and Phenytoin.
2. Avoid administration of radionuclides for at least 24 hours.
3. Hemolysis gives a false elevated level.
4. Iron deficiency anemia gives a false increase.
5. Draw blood before the administration of the injection of B12.

Purpose of the test (Indications) for folic acid

1. It is advised in megaloblastic anemia.
2. This test is advised in :
   1. To rule out the folic acid deficiency.
   2. In iron-deficiency anemia.
   3. In Hypersegmented neutrophils.
3. It is advised in pregnancy.

Definition and structure of folic acid and Folate

1. Folic acid and Folate are used for a family of compounds related to Pteroic acid.
   1. Folic acid or pteroylglutamic acid is the parent compound of the folate complex.
2. Folacin is the generic term for folic acid and related substances that have the activity of folic acid.
3. Folic acid or folates consists of:
   1. A pteridine is a base.
   2. It is attached to one molecule of p-aminobenzoic acid.
   3. And glutamic acid.

4. Pteridine and p-aminobenzoic acid are called Pteroyl.
5. Rest names are given according to the number of glutamic acids, such as pteroyl monoglutamate or pteroyl polyglutamate.
6. Folic acid /folates are a generic term for a family of compounds that function as coenzymes for processing one-carbon units.
7. Another definition:
   1. Pteroic acid, when combined with one molecule of L-glutamic acid, will form pteroylglutamic acid (Folic acid).
   2. It will reduce to dihydrofolic acid or tetrahydrofolate. These reduced forms are biologically active.
8. Folic acid and folates are forms of water-soluble vitamins like Vit. B 12.

Folic Acid and Folate facts:

1. Humans cannot synthesize p-aminobenzoic acid or attach glutamic acid to pteridine.
2. This is the reason that folates occur naturally in food like:
   2. Yeast.
   3. Liver.
   4. Leafy vegetables.
   5. While folic acid is a synthetic form of the vitamin.
3. Folate activity is around 95% in the RBCs.
   1. The activity in the serum is about 40% and is protein bound.
4. Vitamin B12 and Folate are linked by the transfer of the methyl group from the 5-methyltetrahydrofolate (5-MTHF).
   1. 1. In the case of cobalamin deficiency, 5-MTHF is metabolically inactive.

The requirement of Folic Acid and Folate:

1. A normal diet consists of 500 to 700 µg of Folate, and 50 µg is absorbed daily.
   1. Another source says that the normal dietary intake of Folate is 200 to 250 µg.
2. The minimum daily adult requirement is 100 to 150 µg.
   1. Another source says daily requirements are 60 to 280 µg to replace the losses.
3. The body storage is 10 to 12 mg and is sufficient for 4 months (another source= storage for one month).
   1. Another source says total body stores are 12 to 28 mg.

Synthesis and site of absorption of Folic Acid and Folate:

1. Its absorption site is the duodenum and jejunum.
2. The bacteria form folic acid in the intestine.
3. Folic acid is stored in the liver.
   1. A liver biopsy shows half of the body’s stores of folates.
4. 0.5  to 1% of the body’s stores are catabolized or excreted daily.
5. The intestinal enzyme cleaved folate derivatives in the diet to monoglutamyl Folate for absorption.
6. Enzyme folate reductase reduces it into tetrahydrofolate by using NADPH as a donor.

Sources of Folic Acid and Folate

1.  It is present in:
   1. Eggs and milk.
   2. Leafy vegetables (spinach, broccoli, and lettuce).
      1. Okara, and asparagus.
   3. Fruits (bananas, melons, lemons).
      1. Orange juices and tomato juices.
   4. Beans, yeast, mushrooms,  and meat (beef liver and kidney).
   5. Liver.
   6. Yeast.
   7. Orange juice.
2. Folic acid is added into:
   1. Cereals.
   2. Bread.
   3. Flour.
   4. Pasta.
   5. Bakery items.
   6. Cookies.

3. This is stable in acid solution and rapid absorption in an empty stomach.
   1. This is unstable in light.
   2. Supplemental folate absorption is 100% in comparison to dietary folates.

Functions of the Folic Acid and Folate:

1. Folic acid produces and maintains new cells.
2. It prevents changes in the DNA, which may lead to cancer.
3. Folic acid is needed for the normal function and maturation of RBCs and WBCs.
4. Folic acid is a potent growth promotor, and it depends upon the normal functioning of the intestinal mucosa for absorption.
5. Folic acid is also needed to synthesize purine and pyrimidines, which, in turn, are precursors of cell DNA.
6. Folic acid is needed to synthesize purines and pyrimidines, a precursor of cell DNA.
7. Folic acid is needed for the DNA.
8. Folate is also needed for methionine synthesis, histidine catabolism, and the metabolism of serine and glycine.
9. It is a hypothesis that folic acid is involved in congenital abnormalities.
10. Methylation of homocysteine to methionine.
    1. Methionine is converted to S-adenosylmethionine, a universal donor of a methyl group to:
       1. DNA.
       2. RNA.
       3. Hormones.
       4. Neurotransmitter. 
       5. Membrane lipids.
       6. Proteins.
    2. The elevation of homocysteine concentration has shown an increased risk for coronary artery disease and cerebrovascular disease.

Sources of Folic Acid and Folate:

1. Folates occur naturally in foods, and folic acid is the synthetic form.

2. Folic acid has been added to cold cereals, flour, bread, pasta, bakery items, cookies, and crackers, as required by federal law from 1998.
   1. Foods rich in folates are spinach, lettuce, broccoli, okra, asparagus, and rich fruits, including bananas, watermelon, lemon, orange juice, and tomato juices.
   2. Folic acid is present in eggs, milk, yeast, liver, fruits, leafy vegetables, and fruits.
   3. Folate is also present in beef liver and kidneys.
3. Folic acid is a more potent growth factor than Vit.B12.
4. Determination of serum folic acid and RBC folic acid is the best measure to rule out the folic acid deficiency.
5. Vit B12 is needed to incorporate folic acid into tissue cells.

Deficiency of Folic Acid and folate results from:

1. Megaloblastic anemia is the major manifestation of folate deficiency.
   1. Folic acid deficiency causes megaloblastic anemia and can not be differentiated from pernicious anemia in laboratory tests except the Schilling test without intrinsic factor (IF).
   2. Neurological symptoms are absent in folic acid deficiency.
   3. Pernicious anemia is due to a deficiency of vitamin B12 and not a folic acid deficiency.
   4. It is important to differentiate B12 from folic acid
2. The absence of intestinal bacteria (sterilized gut).
3. Poor intestinal absorption may be seen after surgery or sprue.
4. Insufficient dietary intake.
5. Excessive demands, as in pregnancy, liver diseases, or malignancies.
6. Treatment with antifolate drugs like methotrexate anticonvulsant therapy.
7. In alcoholics.

Toxic effects of an overdose of Folic Acid and Folate:

1. Can Precipitate neuropathy in patients having a deficiency of Vitamin B12.
2. It masks vitamin B12 deficiency.
3. This may delay the treatment of deficiency and allow progress to the neuropathy.

Normal Folic acid and folates:

Source 4

1. Adult (fasting) = 3 to 20 ng/mL,  (11 to 57 nmol/L)
2. Adult (serum) = 2 to 20 ng/mL,  (4.5 to 45.3 nmol/L)
3. Children (serum) = 5 to 21 ng/mL (11.3 to 47.6 nmol/L)
4. Infants = 14 to 51 ng/mL (31.7 to 115.5 nmol/L)
5. RBCs folate:
   1. Adults = 140 to 628 ng/mL (317 to 1422 nmol/L)
   2. Children = >160 ng/mL (>362 nmol/L)

Another source

• <60 years adults = 1.8 to 9.0 ng/mL  (4.1 to 20.4 nmol/L)
• >60 years adults = 1.2 to 12 ng/mL (1.2 to 12 nmol/L)

Dietary folate equivalent (DEF):

1. It is dietary daily requirements of the body:
   1. 0 to 6 months DEF     =   65 µg/day.
   2. 1 to 3 years DEF    = 150 µg/day.
   3. 4 to 8 years  DEF            = 200 µg/day.
   4. 9 to 13 years DEF    = 200 µg/day.
   5. 19 years to older DEF = 300 µg/day.
   6. 14 to 18 years DEF     =  300 µg/day.
   7. Pregnant women  DEF = 600 µg/day.

Source 1

Normal Folate

• 2 to 16 years = 5 to 21 ng/mL.
• >16 years = 3 to 20 ng/mL.
• To convert into SI unit x 2.265 = nmol/L

Source 2

Normal folic acid (Folate)

• 5 to 25 ng/mL (11 to 57 nmol/L)

Another source

• Adult fasting serum folate = 3  to 20 ng/mL (7 to 45 nmol/L).

Source From Medscape

• Adult = 2 to 20 ng/mL
• Children = 5 to 21 ng/mL
• Infants = 14 to 51 ng/mL

The folic acid used to treat:

1. Memory loss.
2. Age-related hearing loss.
3. Alzheimer disease.
4. It reduces the aging effect.
5. Sleep problems, nerve pain, depression, and muscle pain.
6. In a patient with methotrexate treatment.
7. Some people recommend preventing colon cancer and cervical cancer.

Lab diagnosis of folic acid/folate deficiency:

1. Serum measurement of the folic acid diagnoses folic acid deficiency.
   1. RIA assay is simple than the bacterial method.
2. RBC folate level may be advised.
3. Draw-backs of serum folate assay:
   1. The small laboratory can not perform this test.
   2. Serum folate levels fall below normal limits 3 to 4 weeks after the dietary or absorption-induced deficiency begins.
   3. RBC folate levels become abnormal about 3 months later than serum folate.
   4. Anemia develops 5 months after the onset of deficiency of folates.
   5. A diet full of folate may increase the serum folate level. While RBC folate will be low.
   6. Serum folate levels may be low in severe liver and kidney diseases.
4. A therapeutic trial of the folate may be advised.

Increased level of folic acid seen in:

1. Pernicious anemia.
2. Massive blood transfusion in recent times.
3. Vegetarian food contains more folic acid.

Decreased level of folic acid/Folate is seen in the following:

1. Malnutrition. Dietary deficiency is the most common cause.
   1. Dietary deficiency is most common in alcoholics.
   2. It is postulated that alcohol inhibits folate absorption and interferes with folate metabolism.
2. Malabsorption syndrome, e.g., Sprue, Celiac disease.
   1. This is the second most common cause and is due to primary small bowel disease.
3. Pregnancy.
   1. This may be seen in  10% to 25% of pregnant ladies to have some degree of folic acid deficiency.
   2. In pregnancy, folic acid deficiency is due to diet and increased fetal demands.
   3. More severe deficiency (<5%) may be seen in the third trimester.
4. Hemolytic anemia.
5. Megaloblastic anemia.
6. Liver diseases.
7. Malignancies.
8. Chronic renal disease.
9. Vit.B12 deficiency.
10. Malignant tumor-like metastatic carcinoma, acute leukemias.
11. Myelofibrosis.
12. Crohn’s disease.
13. Ulcerative colitis.
14. Intestinal resection and jejunal bypass procedure.
15. In chronic alcoholics.
16. In the case of anorexia nervosa.
17. Drugs like:
    1. Anticonvulsant drugs like phenytoin show in roughly 30% of the cases. It is also seen in primidone.
    2. Cytotoxic drugs like methotrexate exert an antitumor effect by interfering with folate metabolism.
    3. Colchicine, para-aminosalicylic acid (PAS), and neomycin interfere with folic acid absorption in some of the patients.
18. Diet, pregnancy, and anticonvulsant drugs show normal Schilling test.

Drugs that act as an antagonist to folic acid are:

1. 1. Anticonvulsant.
   2. Methotrexate and aminopterin.
   3. Antimalarial.
   4. Heavy use of antacids.
   5. Oral contraceptives.

Questions and answers:

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