Folic acid, chemically known as pteroylglutamic acid (PGA), is a member of the B-complex vitamin family and plays a central role in one-carbon metabolism, which underpins DNA synthesis, cell division, and multiple biosynthetic pathways.

It exists in nature in biologically active forms collectively called folates, while the synthetic, pharmaceutical form is folic acid.

1. Chemical Nature and Structure

Folic acid is a yellow crystalline compound that is only slightly soluble in water.

• Stable in neutral and alkaline conditions
• Unstable in acidic environments
• Heat-labile during prolonged cooking, especially in aqueous conditions

Structural components:

Folic acid is composed of three key moieties:

1. A pteridine ring derivative
2. Para-aminobenzoic acid (PABA)
3. Glutamic acid

Polyglutamate forms:

Naturally occurring folates often contain multiple glutamate residues (polyglutamates), which influence their absorption and cellular retention.

A key distinction:

Natural folates = polyglutamate forms (less stable, must be processed for absorption)

Synthetic folic acid = monoglutamate form (more stable, better absorbed)

2. Absorption, Transport and Storage of Folic Acid

Folic acid is absorbed mainly in the jejunum (proximal small intestine).

• Dietary folate (polyglutamate form) is first converted into monoglutamate form by intestinal enzymes (folate conjugases).
• It is then absorbed by active transport at low concentrations and by passive diffusion at higher concentrations.

Inside intestinal cells:

• Folic acid is reduced to tetrahydrofolate (THF).
• It is then converted mainly into N⁵-methyl-THF, which is the major circulating form.

👉 Transport:

Folate circulates in plasma mainly as N⁵-methyl-THF, loosely bound to albumin.

👉 Storage:

It is stored mainly in the liver. However, body stores are limited, so deficiency can develop within 2–4 months of inadequate intake.

3. Occurrence of Folic Acid

This vitamin is very widely distributed in nature. It is named folic acid because it occurs especially in the foliage of plants; it has also been called folacin. The modern tendency is to refer to it by its chemical name, i.e., pteroylglutamic acid (PGA), while biologically active forms are referred to as folates.

Its chief dietary sources are liver, kidney, beef, cauliflower, green leafy vegetables, legumes, and whole grains.

👉 Folate is easily destroyed during cooking, especially by overcooking and boiling, which can significantly reduce its content in food.

Folic acid is also synthesized by intestinal bacteria, but this contribution is generally not sufficient to meet human requirements. It is stored primarily in the liver.

4. Biochemical Role of Folic Acid

Folic acid is not active as such but is first reduced to its active tetrahydro form, tetrahydrofolate (THF).

This reaction is catalyzed by the enzyme dihydrofolate reductase (DHFR) and requires NADPH as a cofactor.

THF is chemically 5,6,7,8-tetrahydrofolate.

5. Important Concept: One-Carbon Metabolism

THF acts as a carrier of one-carbon units, which are essential for biosynthetic reactions.

Sources of one-carbon units include:

• Serine (major source)
• Glycine
• Histidine
• Methionine

   One-Carbon Substituted Forms of THF

One Carbon Group	Mode of Attachment to THF	Name of resulting compound
–CHO (Formyl)	To N⁵	N⁵-formyl-THF (folinic acid or leucovorin)
–CHO (Formyl)	To N¹⁰	N¹⁰-formyl-THF
–CH=NH (Formimino)	To N⁵	N⁵-formimino-THF
–CH₃ (Methyl)	To N⁵	N⁵-methyl-THF
–CH₂OH (Hydroxymethyl)	To N⁵ or N¹⁰	Hydroxymethyl-THF
–CH₂– (Methylene)	Between N⁵ & N¹⁰	N⁵,N¹⁰-methylene-THF
=CH– (Methenyl)	Between N⁵ & N¹⁰	N⁵,N¹⁰-methenyl-THF

These one-carbon groups are utilized in the synthesis of compounds like serine, purines, thymine (dTMP), methionine, histidine, and choline. Thus, folate coenzymes are involved in the metabolism of nucleic acids, amino acids, and phospholipids.

Examples of Reactions

• N⁵-methyl-THF + Homocysteine → Methionine + THF
  This reaction requires vitamin B₁₂ (cobalamin) as a cofactor (via methionine synthase).
• Glycine + N⁵,N¹⁰-methylene-THF → Serine + THF
• N¹⁰-formyl-THF serves as a donor of formyl groups in purine nucleotide synthesis and in the formation of formylmethionine-tRNA (fMet-tRNA) in prokaryotes (initiation of protein synthesis).

It can be seen that folate coenzymes are concerned with a large number of reactions in tissue cells. Their participation in the synthesis of purines and thymine makes them especially important in the growth and reproduction of cells. Therefore, folic acid deficiency is manifested by abnormalities in tissues with high mitotic rates, such as the cells of the hematopoietic system and the gastrointestinal epithelium.

6. Major Functions of Folate

1. Synthesis of Purines

THF derivatives donate carbon atoms required for formation of adenine and guanine, which are essential components of DNA and RNA.

2. Synthesis of Thymidine (Very Important Reaction)

N⁵,N¹⁰-methylene-THF is required for conversion of:
dUMP → dTMP

👉 This reaction is essential for DNA synthesis.
👉 Defect in this step leads to impaired cell division and megaloblastic anemia.

3. Amino Acid Metabolism

• Glycine ↔ Serine conversion
• Histidine metabolism (via FIGLU)

4. Methionine Formation and Methylation Reactions

N⁵-methyl-THF converts:

• Homocysteine → Methionine (requires vitamin B₁₂)

Methionine is further converted into S-adenosylmethionine (SAM).

👉 SAM is a universal methyl donor used in:

• DNA methylation
• Neurotransmitter synthesis
• Phospholipid synthesis

👉 Thus, folate plays an important role in gene regulation and brain function.

7. Important Clinical Concept: Methyl Trap Hypothesis

In vitamin B₁₂ deficiency:

• N⁵-methyl-THF cannot donate its methyl group
• THF is not regenerated
• Folate becomes trapped in inactive form

👉 This leads to functional folate deficiency, even if folate intake is adequate.

👉 Additional clarification:
The reaction involving N⁵-methyl-THF and homocysteine is the only reaction that regenerates free THF from methyl-THF, making it critically important.

9. Effects of Folic Acid Deficiency

1. In experimental animals (e.g., rats), deficiency may cause graying of hair and staining of fur and whiskers with porphyrins, along with anemia and leukopenia.
2. In humans, deficiency leads to macrocytic (megaloblastic) anemia, resembling pernicious anemia, except that the neurological manifestations of vitamin B₁₂ deficiency are absent. It is often accompanied by glossitis and gastrointestinal disturbances.

👉 Additional clinical features:

• Weakness and fatigue
• Diarrhea
• Increased homocysteine levels (risk factor for cardiovascular disease)

10. Causes of Folic Acid Deficiency

• Poor dietary intake
• Chronic alcoholism
• Malabsorption (e.g., celiac disease)
• Increased requirement (pregnancy, infancy, hemolytic anemia)
• Drugs:
• Methotrexate
• Phenytoin
• Sulfonamides
• Oral contraceptives (long-term use)

11. Urinary FIGLU Test for Folic Acid Deficiency in Humans

A dose of histidine (historically 15 g histidine monohydrochloride) is administered orally. Urine is then collected after 3 to 8 hours and analyzed for formiminoglutamic acid (FIGLU).
Normally, little or no FIGLU is excreted in urine. In folic acid deficiency, increased amounts are excreted, often exceeding ~2 mg/hour.

The test is based on the fact that FIGLU is an intermediate in histidine metabolism. During histidine breakdown, FIGLU normally transfers its formimino group to tetrahydrofolate (THF), forming glutamic acid and N⁵-formimino-THF.

In folate deficiency, THF is not available in adequate amounts, so this transfer reaction is impaired. As a result, FIGLU accumulates and is excreted unchanged in urine.

👉 Key biochemical point:

The FIGLU accumulation reflects a block in one-carbon transfer reactions due to

insufficient THF availability.

Clinical Note:

This test is now largely obsolete in clinical practice. It has been replaced by more reliable and convenient investigations, including:

• Serum folate levels
• Red blood cell (RBC) folate levels (more accurate for tissue stores)
• Plasma homocysteine levels (increased in folate deficiency)

👉 These modern tests provide better sensitivity, specificity, and clinical usefulness compared to the FIGLU test.

12. Clinical Uses of Folic Acid

• It is very useful in certain macrocytic anemias, such as those associated with sprue, pregnancy, infancy, malnutrition, and after gastric surgery.
• It is routinely used in pregnancy to prevent neural tube defects (e.g., spina bifida, anencephaly).

👉 Additional point:

It is also used in conditions with increased cell turnover, such as hemolytic anemia.

👉 Recommended prophylaxis in pregnancy:

Folic acid supplementation is advised before conception and during early pregnancy, as neural tube closure occurs very early in fetal development.

It can also correct the anemia of pernicious anemia, but it does not treat the underlying vitamin B₁₂ deficiency and may allow progression of neurological damage. Therefore, folic acid should not be given alone in suspected vitamin B₁₂ deficiency.

13. Folic Acid Antagonists

Drugs like aminopterin and methotrexate inhibit dihydrofolate reductase (DHFR). This inhibition blocks the formation of THF, thereby preventing the synthesis of purines and thymidine, leading to inhibition of DNA synthesis.

As a result, cell division is reduced, particularly in rapidly dividing cells such as malignant cells. These drugs are used in the treatment of certain leukemias, cancers, and autoimmune diseases.

However, resistance to these drugs may develop.

Leucovorin (Folinic Acid) Rescue (Important Addition)

Folinic acid (N⁵-formyl-THF) is used to:

• Protect normal cells during methotrexate therapy
• Bypass DHFR inhibition

👉 This is called leucovorin rescue therapy.

Trimethoprim and Sulfonamides

Another drug, trimethoprim, inhibits bacterial DHFR and is used as an antibacterial agent (often in combination with sulfamethoxazole).

👉 Important concept:

• Bacteria synthesize folic acid → targeted by sulfonamides
• Humans do not synthesize folic acid → dietary requirement

This explains the selective toxicity of these drugs.

14. Daily Requirement

👉 Recommended daily intake:

• Adults: ~400 µg/day
• Pregnancy: ~600 µg/day
• Lactation: ~500 µg/day

   Final Conceptual Summary

Folic acid is essential for:

• DNA synthesis (purines and thymidine)
• Cell division
• Amino acid metabolism
• Methylation reactions

👉 Therefore, deficiency mainly affects:

• Bone marrow (anemia)
• Intestinal mucosa
• Developing fetus