Phar 722Pharmacy Practice III

Vitamins-Folic Acid

Spring 2006

Folic Acid Study Guide• The applicable study guide items in the Vitamin

Introduction• History• Structure of the vitamin and conversion to the

cofactor forms• Role of conjugase in processing the vitamin• Function of the cofactors including the specific

types of reactions• Deficiency conditions and how they may occur• Importance in pregnancy• Role in controlling excess blood homocysteine

levels and cardiovascular disease• Drug interactions• Dietary and commercial forms of the vitamin

What is Anemia?• Definition

– Below normal levels of red blood cells or hemoglobin, or both, which can be caused by many different conditions.

• Symptoms may include fatigue, weakness, headache, and dizziness.

• Appropriate blood tests will confirm the diagnosis of anemia and shed light on its etiology.

• Anemia is a symptom. Classification and cause are essential for proper treatment.

Simplified Classification of Anemias-1• Size of erythrocyte

– Normocytic• Deficiency of normal sized erythrocytes

– Iron deficiency– Hemorrhage– Hemolysis (Hemolytic Anemia)– Anemia of chronic disease (autoimmune diseases,

cancer, etc.)

– Microcytic• Inability to make adequate amount of hemoglobin

leading to small sized erythrocytes– Iron deficiency

Simplified Classification of Anemias-2

– Megaloblastic-Macrocytic• Larger than normal sized erythrocytes• May or may not include larger than normal

precursor cells (megaloblasts).– Folic Acid– Cobalamin (Cyanocobalamin)

Simplified Classification of Anemias-3

– Aplastic Anemia• Decreased ability of the bone marrow to make

new erythrocytes.• May include deficiency in platelets and immune

system cells.– Toxins– Drug therapy– Radiation

– Defective Hemoglobins• Sickling Disease (Sickle Cell Anemia)• Thalessemias

Folic Acid History• More patients are seen with folic acid deficiencies

than any other vitamins.

• 1931 – First recognized as a treatment for megaloblastic anemia.– It was named by Professor Snell (U of Texas).

• Folic is derived from folium, the Latin word for leaf because it was isolated in quantities sufficient enough to characterize from four tons of spinach.

• 1993– Initial recommendations that women contemplating

pregnancy should take 0.4 mg to 1 mg per day of folic acid in order to prevent neural tube defects (NTDs) such as spina bifida and anencephaly.

Folic Acid Chemistry and Nomenclature

• The synthetic vitamin is composed of the heterocyclic pteridine ring linked to a p-aminobenzoic acid which, in turn, is connected to a glutamic acid by a peptide-like bond. – In this form it is called folic acid

• The natural vitamin found in foods is made up of a family of polyglutamates all connected to the initial glutamic acid at the γ-carboxyl group. – From that point on the glutamic acids are linked by normal

peptide bonds between α-amino and α-carboxyl moieties. – The length of this polyglutamate chain varies with the

source of the vitamin, but lengths of 3, 5, and 7 amino acids are seen.

– The various forms found in food are called folate or folates.

Folic Acid Uptake• The dietary polyglutamates are cleaved to

the monoglutamate vitamin by a γ-L-glutamylcarboxy peptidase commonly called conjugase.

• Folic acid and tetrahydrofolate is absorbed as the monoglutamate.

• Conjugase is found in the brush border of the intestine. – Chronic inflammatory conditions in the intestine

lead to low conjugase activity which can result in significant decreased folic acid absorption.

• At least one drug, phenytoin, can inhibit folic acid uptake.

Folic Acid Metabolism• The absorbed vitamin must be converted to the several

cofactor forms. – The first step requires adding back a five to seven member

glutamate chain.– Next there is a two step reduction to the active cofactor

tetrahydrofolic acid. • There are two common abbreviations for the cofactor form, FH4 and

THF. – Dihydrofolate reductase is one of the most studied enzymes

because it is the site of cancer chemotherapy (methotrexate) in humans, anti-bacterial activity in bacteria and anti-parasite activity in malaria.

– Synthesis of FH4/THF occurs in a wide variety of tissues. • The fact that the dihydrofolate reductase inhibitor methotrexate is

active in so many tissues indicates that the enzyme is widely distributed.

• The liver contains about a three to six months supply of the vitamin, presumably in the polyglutamate form. – Folic acid deficiencies are seen in patients who are chronic

alcoholics. This may be caused by alcohol interfering with storage and processing of the vitamin in the liver.

As polyglutamates

Cofactor Forms-1• There are at least five forms, four of

which are “cofactors” existing in each of three different oxidation states.– 5,10-methylene THF (N5,N10-methylene

THF)• Methylation of dUMP forming dTMP• Synthesis of serine from glycine

– 10-formyl THF• Conversion aminoimidazole carboxamide

ribotide (AICAR) to Formamidoimidazole carbxamide ribotide (FAICAR) in purine biosynthesis

Cofactor Forms-2– 5,10-methenyl THF (N5,N10-methenyl THF)

• Synthesis of formylglycine ribotide (FGAR) from glycine amide ribotide (GAR) in purine biosynthesis

– 5-methyl THF (N5-methyl THF)• Methylation of homocysteine forming

methionine• IMPORTANT:

– This is the one cofactor form which is not interchangeable with any of the other cofactor structures.

– It is the predominant cofactor form of the vitamin. » This aspect of folic acid chemistry is important to

appreciate and will be discussed again during cyanocobalamin (Vitamin B12) deficiencies.

A Fifth but Important Form– 5-Formyl THF (folinic acid, citrovorum

factor)• This compound is not a cofactor, but it can be

converted to any of the active cofactor forms.– It is administered following methotrexate therapy to

return tetrahydrofolate activity to normal levels in benign tissues.

THF

ATP

HN

N

NH

CH

CH2

HNH2N

O

CH2

HN

R

HN

N

NH

CH

CH2

HNH2N

O HC N

CH2

RO

HCOOHH2O

His

Ser

Gly

HN

N

N+

CH

CH2

HNH2N

O HC N

CH2

R

Met

HN

N

N

CH

CH2

HNH2N

O HN

CH2

R

CHO

NADP+

HN

N

N

CH

CH2

HNH2N

O H2C N

CH2

R

FADH2

FAD

HN

N

N

CH

CH2

HNH2N

CH3O

CH2

HN

R

5

10

N5-Methyl-THF

5

N5,N10-Methylene-THF

NADPH + H

5

N5,N10-Methenyl-THF

10

10

5

10

5

N10-Formyl-THF

10

ADP + Pi 5

N5-Formyl-THF(Folinic Acid;citrovorum factor;Leucovorum

10

Homocysteine

Methylcobalamin

Cofactor forms arepolyglutamates.

NH

O

O

H3C

N

O

HOH

HH

H

CH2

H

OP-O

O-

O

NH

O

ON

O

HOH

HH

H

CH2

H

OP-O

O-

O

DHF

THF

,

Deoxyuridylic acidDeoxythymidylic acidN5,N10-Methylene THF

Dihydrofolatereductase

NADP+

NADPH + H+

N

N

N

NH2N

NH2

CH2

N

CH3

C

HN

O

CH2

CO2-

CH2

CO2-

Methotrexate

Site of methotrexate inhibition

Folic Acid Deficiency-1

• Overview– Folic acid is a very important vitamin for

biosynthetic reactions required for the biosynthesis of purines and pyrimidines and regeneration of methionine from homocysteine.

• The main deficiency is a characteristic megaloblastic anemia due to a shortage of nucleotides required for the production of erythrocyte precursors.

– Most deficiencies are caused by a medical condition other than diet and usually are the result of malabsorption.

• The main exception to the latter is inadequate diet during pregnancy.

Folate Deficiency Causes-2• Inadequate nutrition during periods of

increased requirements-1– Megaloblastic anemia of pregnancy– Neural tube defects (NTDs)

• spina bifida• Anencephaly

– Doses of 4 mg (6.7X the RDA for pregnant women) given to women with prior NTD-affected pregnancies caused a 70% reduction of NTD-affected pregnancies.

– Based on extrapolations from the few published studies, doses of 0.4 – 0.8 mg (400 – 800 μg) will result in an expected 50% reduction of NTDs.

– RDA (Pregnancy): 0.6 mg (600 μg)

Folate Deficiency Causes-3• Inadequate nutrition during periods of increased

requirements-2– Thus, the current recommendations call for women

contemplating pregnancy to take 0.4 – 0.8 mg daily of folic acid.

– The main difference between the new recommendations and the earlier ones is that women formerly began taking their folic acid supplements (usually included in a standard prenatal vitamin formulation) after the pregnancy was confirmed.

– This could be one to three months following conception. The damage from NTDs can occur early in the pregnancy. Women not planning on becoming pregnant do not need a folic acid supplement.

• Mutant mice models indicate that folic acid, in its cofactor forms, is required for methylation of DNA.

– This is one of the regulatory mechanisms for turning genes on and off.

Folate Deficiency Causes-4

• Inadequate nutrition during periods of increased requirements-3– Do we obtain enough folic acid from our

diets? • Grain-based products are being fortified with

folic acid along with niacin, thiamine and riboflavin.

• Earlier slide showed women were not obtaining enough folic acid from their diets.

Uses of Dietary Reference Intakes-9

Folate Deficiency Causes-4

• Alcoholism– This is considered the leading cause of

folic acid deficiency in the United States. – There are two ways that excessive alcohol

consumption can interfere with folic acid activity.

• Impairment of folic acid reduction to the active THF forms

• Interference with folic acid storage and release from the liver possibly caused by increased lipid in the liver.

Folate Deficiency Causes-5• Chronic inflammation of the intestinal

mucosa (malabsorption)– There are two mechanisms that explain

how an inflamed mucosa can lead to a folic acid deficiency.

• First, there can be reduced production of the required conjugase enzyme which removes the polyglutamate chain.

• Second, and some believe more important, an inflamed mucosa inhibits folate transport.

Folate Deficiency Causes-6• Drug-vitamin interactions

– Phenytoin• The mechanism is uncertain, but there seems to be some type of

inhibition of folic acid uptake or utilization.– Methotrexate

• This chemotherapeutic agent inhibits dihydrofolate reductase preventing the conversion of the vitamin to its functional cofactor forms.

• NOTE: The rationale for the development of methotrexate is of interest because it illustrates how chance favors the prepared mind. It was noted that administration of folic acid supplements hastened the death of cancer patients. This led to the development of folic acid antagonists, of which methotrexate is the standard in the United States. (Trimethoprim is selective for bacterial dihydrofolate reductase.)

• Uses– Cancers– Rheumatoid Arthritis– Psoriasis– Other autoimmune diseases

– Depending on the indication and dosing, folic acid or leucovorin (folinic acid) will be part of the drug regimen with methotrexate.

Folate Deficiency Causes-7

• Too much sunlight (still a hypothesis)– Increases ultraviolet light (sunlight)

destroys body stores of folate. Some authorities discount this hypothesis.

• This is based on a correlation that more problems are seen with fair skinned individuals living in sunny areas.

Folate Deficiency??• Elevated blood homocysteine levels

– The positive correlation between homocysteine levels and cardiovascular disease is weakening.

• There may be a positive correlation between homocysteine and osteoporosis.

– Elevated homocysteine can be corrected, at least partially, with folate supplements, along with pyridoxine and cyanocobalamin.

– Methionine metabolism review: • Loss of the methyl group from methionine produces

homocysteine. • Homocysteine is converted back to methionine by

methylation.• The source of the methyl group is N5-methyl THF.

– Homocysteine may be an indication of inadequate levels of pyridoxine, folic acid and possibly cyanocobalamin.

Hypervitaminosis Folate• This apparently is not a problem.

– Transport across the intestinal mucosa may be regulated by a feedback mechanism or the rate of hydrolysis of the polyglutamate chain or a combination of both.

– What is very important is that taking the vitamin in doses above 400 μg (800 μg in pregnant and lactating women) can mask the macrocytic anemia seen with pernicious anemia caused by a cyanocobalamin deficiency.

– There are other tests for cyanocobalamin deficiency besides examination of a blood smear.

– In 2005 there was a report that elevated intake of folic acid caused mental deterioration in older adults (65+ years).

• It may be early signs of pernicious anemia.

Folate Dosage Forms• Stability

– Very unstable to sunlight, even in the dry state.– Very heat labile such that it can be destroyed during

cooking.

• Folic Acid Solubility: 1.6 gm/ml

• Commercial Forms– Folic Acid– Sodium Folate

• Used in parenteral dosage forms.– Leucovorin Calcium (5-formyl THF, folinic acid, citrovorum

factor)• Used in orally and parenterally to

– Reverse the toxicity of methotrexate– Administer an active form of folic acid when the intestinal uptake

or conversion to the tetrahydrofolate active form is impeded.– Also co-administered with 5-flurouracil (5FU) which blocks the

methylation of dUMP to dTTP.

Folic Acid DRIs-1• With the setting of the new DRIs, a new unit was

introduced.– Dietary Folate Equivalent (DFE)– It acknowledges the differences in bioavailability of

synthetic folic acid in supplements and fortified foods versus naturally occurring folates.

• 1 mcg of food folate = 1 mcg of DFE• 1 mcg of folic acid taken with meals or as fortified food = 1.7

mcg of DFE• 1 mcg of folic acid supplement taken on an empty stomach =

2 mcg of DFE.

• The closest analogy to the DFE is the retinol equivalent (RE) that takes into account the differences in source of β-carotene.

Folic Acid DRIs-2• AI

– Infants 65 - 80 μg DFE/day

• EAR– Children (1 - 8 years) 120 - 160 μg DFE/day– Children (9 - 13 years) 250 μg DFE/day– Adolescents (14 - 18 years) 330 μg DFE/day– Adults (19 - 50+ years) 320 μg DFE/day– Pregnancy 520 μg DFE/day– Lactation 450 μg DFE/day

Folic Acid DRIs-3• RDA

– Children (1 - 8 years) 150 - 200 μg DFE/day– Children (9 - 13 years) 300 μg

DFE/day– Adolescents (14 - 18 years) 400 μg

DFE/day– Adults (15 - 50+ years) 400 μg

DFE/day– Pregnancy 600 μg DFE/day– Lactation 500 μg DFE/day

Folic Acid DRIs-4• UL

– Children (1 - 3 years) 300 μg DFE/day– Children (4 - 8 years) 400 μg DFE/day– Children (9 - 13 years 600 μg DFE/day– Adolescents (14 - 18 years) 800 μg DFE/day

• The UL is based on the amount of folate that could mask the blood picture of cyanocobalamin deficiency.

Food Sources• Deep green leafy vegetables• Liver• Kidney• Yeast

• Dietary forms are tetrahydrofolate polyglutamates.

• Note: – Even though the bacteria in the colon do produce

folic acid polyglutamates, this is well below the upper third of the small intestine where most folic acid absorption occurs.

– Therefore, it is doubtful if humans receive any of this vitamin from their intestinal flora.