Folic Acid Metabolism

M.G. Marinus Biochemistry and Molecular Pharmacology, NRB823, x63330, martin.marinus@umassmed.edu

Salient facts Folic acid is an essential dietary requirement for most people at about 400 ug/day. The federal government has mandated that grain-based products (bread, breakfast cereal, pasta, etc.) be supplemented with this vitamin. Other sources rich in folic acid are leafy vegetables (Latin folium = leaf), yeast and liver. There is a positive correlation between the incidence of neural tube defects (e.g., spina bifida, anencephaly) in newborns and insufficient folic acid intake by the mother. It has been estimated that more than 75% of such birth defects could be prevented by folic acid supplementation. For women intending to become pregnant, the daily dose should be at least 600 ug/day. The problem is compounded by the requirement that vitamin supplementation should be ongoing at the time of conception. Folic acid deficiency leads to anemia. Chronic alcoholism is the most common cause of deficiency. Intake of larger amounts of folic acid than the recommended level does not appear to be harmful; the excess is simply excreted in urine and bile. The Federal Drug Administration, however, recommends that ingestion of folic acid be kept below 1 mg/day. Increased ingestion of folic acid may reduce the risk of vascular disease in patients with elevated serum homocysteine. Certain drugs interfere with folic acid metabolism and can induce folic acid deficiency. The role of folates in metabolism is to donate one carbon units in various biosynthetic pathways.

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Folate metabolism
TS TMP dUMP

F
Human Bacteria

DHFR DHF DHFR THF THF.C1

Substrates Methionine Purines

F=folic acid; DHF=dihydrofolic acid; THF=tetrahydrofolic acid; TS=thymidylate synthase; DHFR=dihydrofolic acid reductase; dUMP=deoxyuridine monophosphate; TMP=thymidine monophosphate

Precursors

F represents folic acid in the oxidized form found in food supplements and multivitamin preparations. DHF and THF from natural sources are polyglutamated and need to be enzymatically converted to the monoglutamate for absorption from the small intestine. THF.C1 represents the various active forms of THF that participate in specific reactions. Form Methyl-THF Methylene-THF Formyl-THF Formimino-THF Methenyl-THF Reaction Methionine synthesis Thymidine monmophosphate synthesis Purine biosynthesis Interconversion intermediate Interconversion intermediate

During the formation of methionine and purines, a single carbon atom is donated and the THF remains unchanged. In contrast, the formation of TMP involves the donation of two hydrogens as well as the one carbon atom. Consequently, the reaction product is DHF which must be reduced to THF by DHFR to re-enter the metabolic cycles. Bacteria and certain lower eukaryotes are able to biosynthesize folic acid from scratch. Unlike humans, they cannot take up folates from their environment. What are the consequences to macromolecular synthesis if folate synthesis is blocked?

Medical Biochemistry

Folic acid metabolism

M.G. Marinus

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Mechanism of action of sulfonamides The first clue to the mechanism of antibacterial action of sulfonamides was that para-amino benzoic acid (PABA) reversed the growth inhibition caused by sulfonamides.

Cells per ml

Time
Reversal of sulfonamide (S) action by PABA (P)

Sulfonamides are analogues of para-amino benzoic acid (PABA), which is an integral part of folic acid. Note similarity of the drug to the natural enzyme substrate.

O NH2 S O R

NH2

O _ O

Sulfonamide

para-aminobenzoic acid

NH2 HN

H N _ O HN

O O _O N O H

N O

Dihydrofolic acid (Dihydropteroylglutamic acid)

Medical Biochemistry

Folic acid metabolism

M.G. Marinus

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Biosynthesis of dihydrofolic acid and its inhibition by sulfonamides This occurs in two steps. First the condensation of PABA and dihydropteridine to form dihydropteroic acid. This reaction is catalyzed by dihydropteroate synthase (DPS). In the second reaction, dihydrofolic acid synthetase catalyses the formation of dihydrofolic acid from dihydropteroic acid and glutamic acid. Sulfonamides block the action of DPS by virtue of their resemblance to the normal substrate.
NH2 HN O + OH H2N O N H N NH2 HN (Dihydropteridine) O + O NH2 S O R N H N

CH2OH

CH2OH

Dihydropteroate synthase

NH2 HN

H N

NH2 HN H N OH O

H N

N O

N O HN

O S O R

+ O _ O O _O NH2

(Enzyme

bound)

Dihydrofolic acid synthetase

H N _ O HN N O H

NH2 HN

O O _O

N O

Dihydrofolic acid
Medical Biochemistry Folic acid metabolism

M.G. Marinus

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Mechanism of action of DHFR inhibitors These compounds bind tightly, but not covalently, to the enzyme forming a stable complex. Note the similarity in structure of the natural substrate and drugs.
H N _ O HN N O H
O

NH2 HN

NH2

N N

N _ N O

O O
NH2

O O _O N H

N O

_O

CH3

Dihydrofolate
NH2 N NH2 O CH3 N

Methotrexate
NH2 N N NH2

CH3 CH3 O
Cl

Trimethoprim

Pyrimethamine

The Table below indicates the molar concentration of drugs required to produce a 50% decrease in activity of purified dihydrofolic acid reductase (DHFR) from the indicated source (After Hitchings and Elion).

DHFR from Drug Trimethoprim Pyrimethamine Methotrexate E. coli 5 x 10-9 2.5 x 10-6 1 x 10-10 Plasmodium berghii 7 x 10-8 5 x 10-10 4 x 10-10 H. sapiens 4 x 10-6 1 x 10-7 2 x 10-10

E. coli = Escherichia coli, a principal cause of urinary tract infections; P. berghii causes malaria. Methotrexate is used principally for cancer chemotherapy especially in the treatment of childhood leukemias.

Medical Biochemistry

Folic acid metabolism

M.G. Marinus

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Combining antifolates in therapy Sulfonamides or trimethoprim (TRP) are bacteriostatic at therapeutic concentrations but the combination is bacteriocidal. Sulfamethoxazole is used in the combination.

10 Cells per ml

Control Drug Sulfa or TRP

Sulfa plus TRP

5

10 Time (hr)

Effect of sulfamethoxazole (Sulfa) (1 g/ml) and/or trimethoprim (TRP, 0.1 g/ml) on Escherichia coli growth.

Study Questions In some patients taking sulfamethoxazole-trimethoprim, megaloblastic anemia, leukopenia and granulocytopenia occur as a result of folate deficiency. This can be treated by simultaneous administration with which chemical form of folic acid? What is the rationale for this therapy? With methotrexate, a similar toxicity can occur but folic acid, in any of its forms, cannot be administered simultaneously. Why not? When is folic acid treatment appropriate in this case and what chemical form should it take?
The answer to the first question is THF or THF.C1 and the actual compound used is 5-methyltetrahydrofolic acid (folinic acid) simply because it is the most chemically stable of the group. The folininc acid will reverse any ill effects in the cells of the patient but since the bacterium is unable to transport folates it cannot be rescued. (Organisms that can synthesize their own folate seem to lack transporters for them). Foe the second question, the answer is the same compound (folinic acid) but this time it cannot be given simultaneously with the methotrexate because it will reverse the agent's cytotoxic effects. The compounds are administered sequentially for several cycles; first the methotrexate then the folinic acid, etc.

Medical Biochemistry

Folic acid metabolism