Phar 722Pharmacy Practice III

Vitamins-

Cyancobalamin (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.

N

Co

N

N N

CH3

H2NOCH2C

HNOCH2CH2C

CH3

CH3

CH2CH2CONH2

H3CCH2CONH2

H2NOCH2CH2C

H3C

H2NOCH2C

H3C

N

O

OHO

HH

H

CH2OH

H

NCH3

CH3

P

O

H2C

O-

O

R

R = -CN, -OH, -CH3,

N

NN

N

NH2

O

OHOH

HH

HH

O

Adenosyl

CH3CH3CH2CH2CONH2

A B

CD

Benzimidazole

No methylene carbon

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.

– In the alkaline intestine, the R protein-cobalamin complex dissociates.

R + B12 R B12

R B12R + 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).

– 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.

B12 + IF B12 IF

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

5,10-CH2-THF

dUMP dTMP

DHF (Dihydrofolate)

THFDHF Reductase

DHFReductase

DHFDHF Reductase

Folic Acid (standard monoglutamate dosageform)

Ser

Gly

5-CH3-THF

DietaryFolates

FADH2

FAD

Homocysteine Met

B12coenzyme

[CH3]

This is where folic acid and cobalamincome together.

If cobalamin is deficient, the methyl group cannotbe removed from 5-CH3-THF. Also note that homocysteine levels will increase. Thisis 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 5-

methyl 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.

H3C CH

C

H

O

SCoA

Propionyl CoA

BiotinH3C CH

C

C

O

SCoA

O

O-

D-Methylmalonyl CoA

H3C CH

C

O

O-

O

SCoA

L-Methylmalonyl CoA

H2C CH

C

H

O

O-

Succinyl CoA

CoAS

O

Cobalamin

Methylmalonyl-CoA mutase

Methylmalonyl-CoA epimerase

CO2

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.