Transcript Phar 722 Pharmacy Practice III

Phar 722
Pharmacy Practice III
VitaminsCyancobalamin (B12)
Spring 2006
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
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.
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.
R
H2NOCH2CH2C
CH3
H3 C
CH2CONH2
H2NOCH2C
CH2CH2CONH2
B
A
H3 C
N
H3C
N
Co
No methylene carbon
N
N
D
CH3
C
H2NOCH2C
CH3
HNOCH2CH2C
CH3
CH3
CH2CH2CONH2
CH3
N
H2C
Benzimidazole
O
CH2OH
H
N
CH3
O
H
NH2
H
O
P
H
O
OH
N
N
OO
N
R = -CN, -OH, -CH3,
O
H
H
H
H
OH
OH
Adenosyl
N
Cobalamin Uptake & Metabolism-1
• R proteins
– Dietary cobalamins must be freed from the
animal tissue in the person’s diet. This requires
a functioning stomach. The cobalamins combine
with a protein called R-factor.
R + B12
R
B12
– In the alkaline intestine, the R protein-cobalamin
complex dissociates.
R
B12
R + B12
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).
B12 + IF
B12
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.
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.
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.
Cobalamin’s 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.
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
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)
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.
Cobalamin’s 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.
Methyl Trap Hypothesis
dUMP
dTMP
DHF (Dihydrofolate)
5,10-CH2-THF
Dietary
Folates
FADH2
Gly
FAD
Ser
5-CH3-THF
DHF
Reductase
THF
DHF
DHF Reductase
This is where folic acid and cobalamin
come together.
B12
coenzyme
Homocysteine
[CH3]
DHF Reductase
Folic Acid (standard
monoglutamate dosage
form)
Met
If cobalamin is deficient, the methyl group cannot
be removed from 5-CH3-THF.
Also note that homocysteine levels will increase. This
is why cyanocobalamin and folic acid are recommended,
along with pyridoxine for homocysteinemia.
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 5methyl THF), megaloblastic anemia develops.
• Folic acid supplements correct the anemia, but
the B12 deficiency continues.
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.
O
O
C
H3 C
SCoA
C
SCoA
C
O-
Biotin
CH
H3 C
H
CH
CO2
Propionyl CoA
O
D-Methylmalonyl CoA
MethylmalonylCoA epimerase
O
O
C
H2 C
CoAS
O-
C
O-
Cobalamin
CH
H3 C
H
O
Succinyl CoA
MethylmalonylCoA mutase
CH
SCoA
O
L-Methylmalonyl CoA
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.
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.
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).
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
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
Food Sources
•
•
•
•
•
Liver
Kidney
Red meat
Dairy products
Clams and mussels high concentrations.