Phar 722 Pharmacy Practice III

1
Phar 722Pharmacy Practice III

• Vitamins-
• Cyancobalamin (B12)
• Spring 2006

2
Cyanocobalamin (B12) Study Guide

• The applicable study guide items in the Vitamin
  Introduction
• History
• Descriptive knowledge of the cofactor forms
• Function of the cofactor including the specific
  type of reactions with examples
• Role of intrinsic factor
• Probable mechanism of action of the masking of
  pernicious anemia by folic acid
• Deficiency condition and how deficiencies may
  occur
• Dietary and commercial forms of the vitamin

3
Cyanocobalamin History

• 1926
• Whole liver used for therapeutic control of
  pernicious anemia.
• The prognosis for pernicious anemia before the
  discovery of vitamin B12 was equivalent to a
  diagnosis for diabetes before the discovery of
  insulin.
• 1946
• One mg of the active material was isolated from
  400 gm of whole liver.
• 1948
• About 3 - 6 µg of red crystalline B12 was found
  to be effective in the treatment of pernicious
  anemia.
• Fermentation procedures were developed to
  eliminate the dependency on liver.
• 1991
• The initial portion of the biosynthetic pathway
  is identical to the porphyrins up to the
  uroporphyrinogen III step. It then diverts into
  the corrin pathway with the methylene bridge
  between pyrroles III and IV lost as acetate.

4
Cobalamin Chemistry

• There are a family of cobalamins.
• The vitamin, cyanocobalamin, is an artifact from
  the original isolation.
• The early procedures called for the use of
  charcoal chromatographic columns.
• Apparently there was a small amount of CN- anion
  in some of these columns.
• Today, KCN is added to the isolates from the
  fermentation media.

5
No methylene carbon
Benzimidazole
6
Cobalamin Uptake Metabolism-1

• R proteins
• Dietary cobalamins must be freed from the animal
  tissue in the persons diet. This requires a
  functioning stomach. The cobalamins combine with
  a protein called R-factor.
• In the alkaline intestine, the R
  protein-cobalamin complex dissociates.

7
Cobalamin Uptake Metabolism-2

• Intrinsic Factor (IF)
• This is a low molecular weight mucoprotein
  produced in the stomach which is required for the
  absorption of the vitamin.
• Rarely is diet the reason for a deficiency of
  vitamin B12.
• Instead, the problem is due to malabsorption due
  to a lack of intrinsic factor.
• In the alkaline intestine, the cobalamin free of
  the R-protein now combines with intrinsic factor
  (IF).
• In the presence of calcium supplied by the
  pancreas, specific receptors in the intestinal
  mucosa take up the cobalamin-IF complex.
• Without IF, only about 1 percent of cobalamins
  are absorbed.
• If no intrinsic factor present, 500 1000 mcg
  oral B12 may overcome this problem.
• Otherwise, the vitamin must be administered
  parenterally.

8
Cobalamin Uptake Metabolism-3

• NOTE
• For maximum absorption of cobalamins, humans
  require a functioning stomach, pancreas and
  intestine.
• R proteins are only required for dietary
  cobalamin.
• Intrinsic factor is required for both dietary and
  oral dosage forms.
• Oral vitamin only requires intrinsic factor.
• Parenteral and nasal dosage forms do not require
  R proteins or intrinsic factor.
• Once absorbed cobalamin is converted to the
  cofactor by replacing the anion with
  adenosine-based derivatives.

9
Cobalamin Uptake Metabolism-4

• Storage and Circulation
• Depending on the source, it has been estimated
  that a patient who has lost the ability to form
  intrinsic factor still has a 3 - 6 year supply of
  the vitamin in the liver.
• Some sources say that if the liver stores are
  excellent, the patient may get along for the next
  20 years due to efficient enterohepatic
  circulation.
• This latter conclusion is based on a healthy
  intestinal environment.

10
Cobalamins Biochemical Functions

• Regenerate methionine by transfering the methyl
  group from 5-CH3-THF to homocysteine.
• Folic acid can mask a cobalamin deficiency
  thereby masking the anemia of Pernicious
  Anemia.
• The final reaction in the conversion of propionyl
  CoA to succinyl CoA.
• Commonly referred to as a mutase reaction.
• Considered to be the cause of the irreversible
  nerve damage in Pernicious Anemia.

11
Cobalamin Deficiency

• Pernicious Anemia
• This is rare in humans (2 of the population over
  60), but when it develops it is lethal.
• At one time a diagnosis of pernicious anemia was
  the same as being informed that the patient had
  lung cancer or diabetes mellitus.
• The damage is to deterioration of the myelin
  sheath surround nerve fibers.
• Symptoms
• A characteristic macrocytic (megaloblastic)
  anemia which may be masked by a megaloblastic
  anemia caused by folic acid deficiency.
• Degeneration of the myelin in the spinal cord
• Degeneration of the peripheral nerves affecting
  the reflexes and walking
• Deterioration of the patient's mental state

12
Causes of a Cobalamin Deficiency-1

• Loss of Intrinsic Factor
• The loss of the ability to make intrinsic factor
  usually parallels a decrease in gastric acid
  production.
• This may be caused by an autoimmune destruction
  of the parietal cells.
• Many times a patient with achlorhydria will be
  have an increased risk of developing pernicious
  anemia.
• Diseased Intestine.
• Various chronic inflammatory conditions such as
  the sprues will interfere with Vitamin B12
  uptake.
• Correction of the inflammatory condition usually
  will return internal vitamin concentrations to
  normal levels.
• Surgical Removal of the Stomach or Part of the
  Stomach
• Decreased production of intrinsic factor
• Gastric cancer.
• Bariatric surgery (gastric bypass)

13
Causes of a Cobalamin Deficiency-2

• Diet
• This is rare.
• There have been a few isolated reports
  particularly of children fed strict vegetarian
  diets heavy in grains.
• There have been reports of pernicious anemia in
  children breast fed by mothers who followed
  strict vegetarian diets.
• Drug - Vitamin Interactions
• Proton-pump inhibitors and H2 blockers can reduce
  absorption of the vitamin by decreasing gastric
  acid production.
• This can be treated easily with a vitamin
  supplement containing cyanocobalamin.

14
Cobalamins Relationship to Folic Acid

• Methyl Trap Hypothesis
• Folic acid supplements will mask the anemia
  portion of pernicious anemia.
• Folic acid and Vitamin B12 come together with
  5-methyl tetrahydrofolate in what has been called
  the Methyl Trap Hypothesis.
• Remember that 5-methyl THF is the one
  irreversibly formed cofactor.
• Once formed, it cannot be converted to any of the
  other cofactor forms.
• In the situation of a B12 deficiency, more of the
  5-methyl THF will be formed in attempt to
  overcome the B12 deficiency.
• Therefore, an excess of folic acid will partially
  compensate for a lack of adequate B12.
• In this case, erythrocyte production will
  continue, but irreversible nerve damage also will
  occur. By the time the latter becomes
  noticeable, severe injury may have been incurred.

15
Methyl Trap Hypothesis
16
Methyl Trap Anemia

• Methylation Reactions or One Carbon Metabolism
  (see previous slide)
• The methyl trap model shows how folic acid can
  mask a B12 deficiency.
• Once 5-methyl THF forms, it can only function as
  a source of methyl groups if B12 is present.
• Without replacing the folic acid (lost as
  5-methyl THF), megaloblastic anemia develops.
• Folic acid supplements correct the anemia, but
  the B12 deficiency continues.

17
(No Transcript)
18
Rearrangement Reaction

• Rearrangement reaction
• Conversion of methylmalonyl CoA into succinyl
  CoA. The latter is metabolized further in the
  Krebs Cycle.
• It is believe that this rearrangement is the
  cause of the nerve damage seen with pernicious
  anemia. Two mechanism are suggested
• The buildup of methylmalonyl CoA is a competitive
  inhibitor of malonyl CoA during fatty acid
  synthesis.
• Methylmalonyl CoA replaces malonyl CoA as a
  substrate in fatty acid synthesis producing fatty
  acids with methyl substituents.
• These are incorporated into the lipids components
  of the myelin sheath producing a non-functioning
  myelin sheath.

19
(No Transcript)
20
Hypervitaminosis B12

• The vitamin is considered nontoxic.
• There has been some concern that the presence of
  the CN anion in the commercial vitamin might
  cause problems with megadoses.
• Look up the molecular weight of cyanocobalamin
  (1355.4) and calculate the number of millimoles
  of CN- in a 1000 µg (1 mg 0.001 gm) dose. The
  MW of CN is 26.
• (The LD50 of KCN in rats is 10 mg/kg.)
• 1000 µg of cyanocobalamin contains 0.02 mg of CN.

21
Cobalamin Dosage Forms

• This is the one, true all natural vitamin.
• It is obtained from anaerobic bacteria.
• Indeed, all plants and animals obtain this
  vitamin from bacteria.
• The bacteria synthesize the corrin ring system
  following the same porphyrin route all organisms
  use to form heme or cytochromes.
• While it may be produced by our intestinal flora,
  this production is below the site of absorption
  and would not be able to form a complex with the
  required intrinsic factor.
• Stability
• Some problems with oxidizing and reducing agents
  and light.

22
Oral Versus Parenteral Administration

• As long as there is some intrinsic factor
  present, the vitamin will be administered orally
  since only a few micrograms need be absorbed.
• Otherwise standard practice has been
  administering IM injections weekly or monthly
  schedule.
• Recent studies have shown that oral or sublingual
  administration of 500- 2,000 µg cyanocobalamin is
  effective.
• The trial indicated that oral administration
  should be tried before parenteral.
• NOTE About 1 of orally administered
  cyanocobalamin is absorbed without intrinsic
  factor.
• Another alternative to IM injection is a gel for
  intranasal administration using a metered
  inhaler.
• It is not indicated for patients with active
  pernicious anemia.
• Rather it is indicated for patients who have
  dietary deficiencies (potentially strict
  vegetarians) or malabsorption due to structural
  or functional damage to the stomach where
  intrinsic factor is produced or the ileum where
  B12 is absorbed.
• These conditions can be caused by surgical
  removal of the stomach or ileum or chronic
  intestinal inflamatory disease (parasites,
  enteropathies, autoimmune, etc).

23
Cyanocobalamin DRIs-1

• AI
• Infants 0.4 - 0.5 µg/day
• EAR
• Children (1 - 13 years) 0.7 - 1.5 µg/day
• Adolescents (14 - 18 years) 2.0 µg/day
• Men Women (19 - 50 years) 2.0 µg/day
• Pregnancy 2.2 µg/day
• Lactation 2.4 µg/day

24
Cyanocobalamin DRIs-2

• RDA
• Children (1 - 13 years) 0.9 - 1.8 µg/day
• Adolescents (14 - 18 years) 2.4 µg/day
• Men Women (19 - 50 years) 2.4 µg/day
• Pregnancy 2.6 µg/day
• Lactation 2.8 µg/day
• UL
• None reported

25
Food Sources

• Liver
• Kidney
• Red meat
• Dairy products
• Clams and mussels high concentrations.