Folic acid- Structure, forms, absorption, transportation, storage, excretion, role in one-carbon metabolism, role in methionine synthesis, role in nucleotide biosynthesis, folate trap, folate antagonists, megaloblastic anemia

1. Folic acid- Chemistry, functions
and clinical significance
Namrata Chhabra
MHPE, MD, CMCL FAIMER FELLOW- REGIONAL INSTITUTION,
Principal-in-charge, Professor& Head, Department of Biochemistry,
SSR Medical College, Mauritius

2. Learning Objectives
• Structure of Folic acid
• Sources,
• Recommended Daily allowance,
• Absorption, transportation, storage and excretion,
• Functions and coenzyme role,
• Biological and clinical significance of folic acid
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3. Folic acid
• Folate and folic acid are forms of a water-soluble B vitamin.
• Folate occurs naturally in food, and folic acid is the synthetic form of this
vitamin.
• Synonyms:
• Liver lactobacillus Caseifactor,
• Vitamin M,
• Streptococcus lactis R (SLR) factor,
• Vitamin Bc,
• Fermentation residue factor, and
• Pteroyl glutamic acid (PGA)
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4. Structure of Folic acid
• A composite molecule
• Made up of three parts:
• A pteridine ring system (6-
methylpterin),
• Para-amino benzoic acid, and
• Glutamic acid.
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5. Chemical forms of folic acid
• Monoglutamate (Vitamin Bc): Having one glutamic acid.
• Triglutamate“fermentation factor”: Having three glutamic acid
residues.
• Heptaglutamate (vitamin Bc conjugate of yeast ): Having seven
glutamate residues.
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6. Role of structural components of folic acid
• The glutamic acid doesn't participate in the coenzyme functions of
folic acid.
• Folic acid in the interior of the cell may contain a “chain” of three to
eight glutamic acid residues, which serves as a negatively charged
“handle” to keep the coenzyme inside cells and/or bound to the
appropriate enzymes.
• The pteridine portion of the coenzyme and the p-amino benzoic acid
portion participate directly in the metabolic reactions of folate.
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7. Sources of folic acid
• Green leafy vegetables (such
as spinach, broccoli, and
lettuce), okra, asparagus,
• Fruits (such as bananas,
melons, and lemons)beans,
yeast, mushrooms,
• Meat (such as beef liver and
kidney),
• Orange juice, and tomato
juice.
• Many microorganisms
including those inhabiting the
intestinal tract can synthesize
folic acid.
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8. Recommended daily allowance (RDA)
• Since 1998, as required by federal law, folic acid has been added to
cold cereals, flour, breads, pasta, bakery items, cookies, and crackers.
• Daily requirements of 50–100 mcg/d are usually met in the diet.
• Total body stores of folate are approximately 5000 mcg, enough to
supply requirements for 2–3 months.
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9. Absorption of folic acid
• Absorption occurs along the whole length of mucosa of small
intestine.
• Polyglutamates ingested in diet are converted to mono-glutamates
• Dihydrofolates are reduced to tetrahydrofolates by folate reductase
• Tetrahydrofolates are then converted to methyl tetrahydrofolates,
which enter the portal blood and then carried to liver.
• Conjugases (γ-glutamyl carboxypeptidases) in the intestinal lumen
cleave off extra glutamic acid residues before absorption of folate in
the small intestine.
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10. Transportation, plasma and tissue levels
• Transportation-Folate is transported in blood as methyl
tetrahydrofolate bound to a specific protein.
• Plasma level: In normal individuals, it varies from 3 to 21 ng/ml.
• Tissue folate: About 70 mg in the whole body, of which about 1/3 (5
to 15 μg/g) is in the liver.
• RB cells folate: Folate is incorporated into the RB cells during
erythropoiesis and is retained there during their entire life span.
• Red cell folate is a reliable indicator of the folate status of the body.
• Average level is 300 ng/ml of whole blood (range 160–640 ng/ml).
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11. Excretion of folate
• Urine: 2 to 5 μg/day.
• Excretion is much increased after an oral dose of folate if the tissues
are saturated.
• Feces contain 20 per cent of the ingested folates that remains
unabsorbed and some unabsorbed synthesis of folate by bacterial
flora of intestine.
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12. Activation of folic acid
• Folic acid, before functioning as a
coenzyme, must be reduced first
to 7, 8-dihydrofolic acid(F.H2) and
then to 5, 6, 7, 8 tetrahydrofolate
(F.H4).
• Both the reactions are catalyzed by
Folic acid reductases enzyme,
which use NADPH as hydrogen
donor.
• This enzyme also requires vitamin
C (ascorbic acid) as a cofactor.
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13. Biologically active forms of folic acid
Active “coenzyme” form of the vitamin is the reduced tetrahydro
derivative, Tetrahydrofolate F.H4, obtained by addition
of four hydrogens to the pteridine moiety at 5, 6, 7 and 8
position.
The other forms occur only in small quantities.
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14. Functions of folic acid
• One carbon metabolism-Folates act as coenzymes in the transfer of
single-carbon units.
• Replication and Transcription- Folate is involved in purine and
pyrimidine synthesis required for DNA and RNA synthesis.
• Folate is also a coenzyme for methionine synthesis, in which
Methylcobalamine is also involved.
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15. One carbon compounds
• One carbon moiety (C1) may be either:
• Methyl (–CH3),
• Formyl (–CHO),
• Formate (H.COOH),
• Formimino group (–CH=NH) or
• Hydroxymethyl (–CH2OH).
• Most of them are metabolically “interconvertible”
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16. Sources of one carbon fragments
• One carbon fragments are produced by the metabolism of :
• Serine
• Glycine
• Tryptophan
• Histidine, and
• Choline
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17. Utilization of one carbon fragments
• Methylene-, methenyl-, and 10-formyl-
tetrahydrofolates are interconvertible.
• The methylene form donates its methyl
group during the biosynthesis of
thymidine nucleotides for DNA synthesis,
• the methenyl form donates its group as a
Formyl group during purine biosynthesis,
and
• the methyl form is the donor of the
methyl group to sulfur during methionine
formation.
• When one-carbon folates are not
required, the oxidation of formyl-
tetrahydrofolate to yield carbon dioxide
provides a means of maintaining a pool of
free folate.
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18. Role of folic acid in nucleotide synthesis
• Two different forms of folate are
required for different aspects of
nucleotide biosynthesis.
• N10-formyl THF provides the C-2 and
C-8 carbons for the de novo synthesis
of purine rings, and thus is critical for
DNA metabolism.
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19. Role of folic acid in nucleotide synthesis
• The Methylene
form, N5,N10-
methylene THF, is
required for the
production of dTMP
from dUMP.
• Methotrexate,
inhibits
dihydrofolate
reductase and has
been exploited as an
anti-cancer
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20. Role of folic acid in Trans Methylation
reactions and Folate trap
• In the absence of B12,
THF can be trapped in
the N5-methyl THF
form and thus be
removed from the THF
pool. This is referred to
as the “methyl trap,”
which can impact other
areas of 1-carbon
metabolism, such as
dTMP production.
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21. Summary of functions of folic acid
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22. Folate antagonists
• 1) Sulfanilamide is the simplest of the sulfa
drugs, used as antibacterial agents.
• Because of structural similarity to p-
aminobenzoic acid, sulfanilamide inhibits the
growth of bacteria by interfering with their
ability to use p-aminobenzoic acid to synthesize
folic acid.
• Because humans don't make folic acid,
sulfanilamide is not toxic to humans in the doses
that inhibit bacteria.
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23. Folate antagonists
2) Trimethoprim and
Pyrimethamine
• The dihydrofolate reductases
of some bacteria and
parasites differ from the
human enzyme;
• Inhibitors of these enzymes
can be used as antibacterial
drugs (e.g., trimethoprim)
and Antimalarial drugs (e.g.,
pyrimethamine).
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24. Folate antagonists used as anti cancer drugs
1) Methotrexate, an analog of 10-methyl-tetrahydrofolate, inhibits
dihydrofolate reductase and has been exploited as an anti-cancer
2) Aminopterin is also an inhibitor of DHFR enzyme and is used as an
anticancer drug.
3) Trimetrexate, Lometrexol and Pemetrexed are also some of the
upcoming folate antagonists used in cancer chemotherapy.
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25. Leucovorin (LV)
• Leucovorin (LV) is a form of folic acid that can help "rescue" or reverse
the toxic effects of methotrexate.
• LV is not a folate antagonist per se, but the folate derivative 5-
formyltetrahydrofolate.
• N5-formyl THF is normally administered 24 hours following
treatment with methotrexate;
• It can be converted to THF by these normal cells by bypassing the
block caused by methotrexate.
• Therefore, these normal cells can synthesize deoxy thymidine and
carry out DNA synthesis.
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26. Megaloblastic anemia due to folate deficiency
• Inadequate dietary intake.
• Alcoholics, anorectic patients, persons who do not eat fresh fruits and
vegetables, and those who overcook their food are candidates for folate
deficiency.
• Drugs such as phenytoin, trimethoprim-sulfamethoxazole, or Sulfasalazine
may interfere with folate absorption.
• Folic acid requirements are increased in pregnancy, hemolytic anemia, and
exfoliative skin disease, and in these cases the increased requirements (five
to ten times normal) may not be met by a normal diet.
• Patients with increased folate requirements should receive
supplementation with 1 mg/d of folic acid.
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27. Biochemical Basis of Megaloblastic Anemia
• The common feature of all megaloblastic anemias is a defect in DNA
synthesis that affects rapidly dividing cells in the bone marrow.
• In deficiencies of either folate or cobalamin, there is failure to convert
deoxy uridine monophosphate (dUMP) to deoxythymidine
monophosphate (dTMP), the precursor of dTTP.
• This is because folate is needed as the coenzyme 5, 10-methylene-
THF polyglutamate for conversion of dUMP to dTMP; the availability
of 5, 10-methylene-THF is reduced in either cobalamin or folate
deficiency.
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28. Clinical manifestations
• History:
• The patient's history is important because it may reveal the
underlying disorder.
• Very often, a patient presents with a history of excessive alcohol
intake with concurrent poor diet intake.
• Other times, patients may be pregnant or lactating;
• may take certain drugs, such as phenytoin, sulfonamides, or
methotrexate;
• may have chronic hemolytic anemia; or
• may have underlying malabsorption.
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29. Clinical manifestations
2.Oral lesions
• Some patients complain of a sore tongue or pain
upon swallowing.
• The tongue may appear swollen, beefy, red, or shiny,
usually around the edges and tips initially.
• Angular stomatitis also may be observed.
• These oral lesions typically occur at the time when
folate depletion is severe enough to cause
megaloblastic anemia, although, occasionally, lesions
may occur before the anemia
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30. Clinical manifestations
• GI symptoms
• Patients may present with GI symptoms, such as nausea, vomiting,
abdominal pain, and diarrhea, especially after meals.
• Anorexia also is common and, in combination with the above
symptoms, may lead to marked weight loss.
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31. Clinical manifestations
• 4. Hyperpigmentation
• Patients with folate deficiency may have darkening of the skin and
mucous membranes, particularly at the dorsal surfaces of the fingers,
toes, and creases of palms and soles.
• Distribution typically is patchy.
• The hyperpigmentation gradually should resolve after weeks or
months of folate treatment.
• A modest temperature elevation (<102°F) is common in patients who
are folate deficient, despite the absence of any infection.
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32. Laboratory Investigations
• Serum folate (reference range 2.5-20 ng/mL) and serum cobalamin
(reference range 200-900 pg/mL)
• Ruling out cobalamin deficiency is very important because folate treatment
will not improve neurologic abnormalities due to cobalamin deficiency.
• Additional follow-up tests include serum homocysteine (reference range 5-
16 mmol/L), which is elevated in B-12 and folate deficiency, and serum
methylmalonic acid (reference range 70-270 mmol/L), which is elevated in
B-12 deficiency only.
• Red blood cell folate levels (reference range >140 ng/mL) tend to reflect
chronic folate status rather than acute changes in folate that are reflected
in serum folate levels,
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33. Laboratory Investigations
• Hematological Findings
• Peripheral Blood film
• Oval macrocytes, usually with considerable anisocytosis and poikilocytosis,
are the main feature.
• The MCV is usually >100 fL unless a cause of microcytosis (e.g., iron
deficiency or thalassemia trait) is present.
• Some of the neutrophils are hyper segmented (more than five nuclear
lobes).
• There may be leucopenia due to a reduction in granulocytes and
lymphocytes, but this is usually >1.5 x 109/L;
• the platelet count may be moderately reduced, rarely to <40 x 109/L.
• The severity of all these changes parallels the degree of anemia.
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34. Laboratory Investigations
• Bone Marrow
• Bone marrow morphology is characteristically abnormal.
• Marked erythroid hyperplasia is present as a response to defective
red blood cell production (ineffective erythropoiesis).
• Megaloblastic changes in the erythroid series include abnormally
large cell size and asynchronous maturation of the nucleus and
cytoplasm—i.e., cytoplasmic maturation continues while impaired
DNA synthesis causes retarded nuclear development.
• In the myeloid series, giant metamyelocytes are characteristically
seen
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35. Treatment
• Oral doses of 5–15 mg folic acid daily are satisfactory.
• It is customary to continue therapy for about 4 months, when all folate-
deficient red cells will have been eliminated and replaced by new folate-
replete populations.
• Cobalamin deficiency must be excluded and, if present, corrected,
otherwise cobalamin neuropathy may develop, despite a response of the
anemia of cobalamin deficiency to folate therapy.
• Long-term folic acid therapy is required when the underlying cause of the
deficiency cannot be corrected, and the deficiency is likely to recur.
• In any patient receiving long-term folic acid therapy, it is important to
measure the serum cobalamin level at regular (e.g., once yearly) intervals
to exclude the coincidental development of cobalamin deficiency.
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36. Therapeutic significance of folate
supplementation
• Folic acid is used for preventing and treating low blood levels of folate (folate
deficiency) and high blood levels of homocysteine (Hyperhomocysteinemia).
• Women who are pregnant or might become pregnant take folic acid to prevent
miscarriage and "neural tube defects "such as spina bifida.
• Folic acid is also used for many other conditions including depression, stroke,
decline in memory and thinking skills in older people
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37. Summary
• A composite molecule, made up of three parts :a pteridine ring
system, para-amino benzoic acid, and glutamic acid.
• Daily requirement is 50–100 mcg/d.
• Total body stores of folate are approximately 5000 mcg.
• Participates in one carbon metabolism, nucleotide biosynthesis and
as a coenzyme for methionine synthesis.
• In the absence of B12, THF can be trapped in the N5-methyl THF
form.
• Folate antagonists act as antibiotics and anticancer drugs.
• Folate deficiency causes megaloblastic anemia.
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38. Thank you
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