Phar 722 Pharmacy Practice III

1. Phar 722Pharmacy Practice III Vitamins- Folic Acid Spring 2006

2. 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

3. 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.

4. 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

5. 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)

6. 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

7. 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.

8. 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.

10. 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.

11. 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.

12. As polyglutamates

13. 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

14. 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.

15. 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.

16. Cofactor forms are polyglutamates.

18. 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.

19. 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)

20. 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.

21. 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.

22. Uses of Dietary Reference Intakes-9

23. 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.

24. 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.

25. 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.

26. 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.

27. 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.

28. 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.

29. 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.

30. 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.

31. 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

32. 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

33. 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.

34. 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.