Phar 722 Pharmacy Practice III

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Phar 722Pharmacy Practice III

• Vitamins-
• Folic Acid
• Spring 2006

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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

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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.

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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

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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)

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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

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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.

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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 ?-carboxyl group.
• From that point on the glutamic acids are linked
  by normal peptide bonds between a-amino and
  a-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.

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Folic Acid Uptake

• The dietary polyglutamates are cleaved to the
  monoglutamate vitamin by a ?-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.

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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.

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As polyglutamates
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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

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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.

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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.

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Cofactor forms are polyglutamates.
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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.

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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)

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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.

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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.

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Uses of Dietary Reference Intakes-9
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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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 ß-carotene.

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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

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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

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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.

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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.