Vitamin B12 and Folate

Question 1

1. What are some important dietary sources of folate?
2. What structural feature distinguishes dietary folates from the folate found in vitamin supplements?

Answer 1

1. Green, leafy vegetables (Broccoli, Spinach, Lettuce)

Dietary Folate: contains multiple gamma (y) glutamic acid residues

Vitamin/Supplemental Folate: contains one gamma (g) glutamic acid residue

Question 2

1. What enzyme converts folate to tetrahydrofolate (THF)?

Answer 2

Dihydrofolate Reductase

Question 3

How does the drug methotrexate work, and what is it used for?

Answer 3

Inhibits Dihydrofolate Reductase

Treats: Cancer, severe psoriasis, and rheumatoid arthritis

Question 4

What is the most oxidized form of THF?

What is the most reduced form?

Answer 4

Most Oxidized: N₁₀-formyl THF

Most Reduced: N₅-methyl THF

Question 5

Which reaction is the source of most of the carbon in the one-carbon pool? (The rxn. catalyed by…)

Answer 5

The reaction catalyzed by Serine Hydroxymethyltransferase

Serine + THF <-> Glycine + N₅,N₁₀-methylene THF + H₂O

Question 6

Describe how dietary folate is absorbed in the intestine and then released into the circulation. What is the major form of THF in the circulation?

Answer 6

Dietary Form = chiefly folate polyglutamate

***Polyglutamate is hydrolyzed in the intestinal lumen and folate enters the cell in the monoglutamate form

Some folate may enter the circulation directly, but most is first methylated and reduced, forming N₅-methyl THF

***Major form of THF in circulation –> N₅-methyl THF

Question 7

How is folate taken up from the circulation? What happens to folate inside the cell? Why is this modification important?

Answer 7

***Folate is taken up from the circulation via receptor-mediated endocytosis (receptors have a very high affinity for folate monoglutamates, particularly N₅-methyl THF)

***Inside the cell, folate is rapidly metabolized by the addition of polyglutamate

***This addition of charged glutamate residues is instrumental in maintaining folate within the cell

Question 8

Which reaction requiring folate derivatives appears to be of greatest clinical importance? (The reaction catalyzed by…)

Answer 8

The reaction catalyzed by Thymidylate Synthase

Converts

dUMP ——> dTMP

***Essential in DNA synthesis***

Question 9

What is the ultimate source of all vitamin B12 (i.e. cobalamin)?

What are some important dietary sources?

Answer 9

Ultimate source: Bacteria

Dietary sources: animal products –> meats, liver, dairy products, shellfish

Question 10

Describe how vitamin B12 is liberated from food and absorbed in the intestine.

Answer 10

1. Dietary B12 is released from food in the presence of gastric acid and pepsin
2. Vitamin B12 then binds to R proteins in the stomach
3. R proteins are digested by pancreatic proteases in the duodenum and free vitamin B12 is released
4. Intrinsic factor (from parietal cells of stomach) then binds vitamin B12
5. Intrinsic Factor/vitamin B12 complex is then taken up via receptor-mediated endocytosis in the ileum

Question 11

Describe how vitamin B12 is transported in the blood.

Answer 11

1. Vitamin B12 is liberated from the intrinsic factor/receptor complex and passes into the cytoplasm from where it is released into the circulation complexed with Transcobalamin (made by the Ileal mucosa)

Question 12

Where is Vitamin B12 mainly stored?

Answer 12

Liver (and in the kidneys)

Question 13

Describe uptake of B12 into cells?

What must Vitamin B12 be bound to to be taken up into cells?

Answer 13

a. Taken up via receptor-mediated endocytosis involving a specific TC receptor on the cell surface
b. TC/Vitamin B12 complex –> lysosome, where TC is degraded, freeing B12
c. Specific Lysosomal Transport System then actively moves Vitamin B12 from the lysosome into the cytoplasm

***Transcobalamin (TC)***

Question 14

What is the majority of vitamin B12 bound to in circulation?

Answer 14

Haptocorrins

(Circulating store of B12?)

Question 15

Describe B12 uptake in the liver.

What is the Enterohepatic Vitamin B12 cycle?

Answer 15

1. Haptocorrin/Vitamin B12 complexes are taken up by the hepatocytes and degreded, with the free B12 being excreted in the bile.
2. Vitamin B12 binds haptocorrin/R proteins again
3. Haptocorrin/B12 complexes are digested in the duodenum
4. B12 binds intrinsic factor and is reabsorbed in the ileum

These steps are the Enterohepatic Vitamin B12 Cycle (Same as Folate)

Question 16

What are the metabolically active forms of vitamin B12?

Answer 16

Adenosylcobalamin and Methylcobalamin

(converted to these within the cell via reduction and alkylation)

Question 17

Describe how Part 1 of the Schilling test are performed.

Answer 17

Part 1:

• Radioactive B12 is administered orally to a patient
• Non-radioactive B12 is given by IM injection to saturate circulating cobalamin-binding proteins
• 24 hour urine collection is started
• The radioactivity in the 24 hour urine collection is determined

***Normal individuals should excrete at least 7% of the administered radioactivity within the 24 hour period. Failure to do so indicates a defect in B12 absorption***

Question 18

Describe how Part 2 of the Schilling test are performed

Answer 18

Part II:

• Radioactive B12 is administered orally to the patient along with purified intrinsic factor
• Non-radioactive B12 is given by IM injection to saturate circulating cobalamin-binding proteins
• 24 hour urine collection is started

***Again, the expectation is at least 7% of the administered radioactivity recovered within the first 24 hours***

Question 19

Schilling Test

What does an abnormal Part 1 but normal Part 2 tell you?

How about abnormal Part 1 and Part 2?

Answer 19

Abnormal Part 1 and Normal Part 2: This will show up in patients with defects in Intrinsic Factor Production (i.e. Pernicious Anemia)

Abnormal Part I and Part II: This will show up in patients who have defects in Absorption

Question 20

What is the likely alternative to the Schilling Test that will be found in the U.S.?

Answer 20

Anti-Intrinsic Factor or Anti-Parietal Cell Antibody Test

Question 21

Describe the role of adenosylcobalamin in propionate metabolism.

How is this step affected by B12 deficiency?

Answer 21

Adenosylcobalamin is required by mutase, the enzyme that converts L-methylmalonyl-CoA into Succinyl-Coa in the last step of Propionyl-CoA (Propionate) metabolism

Deficiency: Inability to convert L-methylmalonyl-CoA to Succinyl-CoA leading to development of Methylmalonyl Acidemia

Question 22

Describe the role of methylcobalamin in homocysteine metabolism.

Answer 22

Methionine Synthase has an absolute requirement for methylcobalamin

Homocysteine is converted to methionine, regenerating THF from N₅-methyl THF

Question 23

What is meant by the ‘methyl trap hypothesis’?

Answer 23

The only way that THF can be regenerated from N₅-methyl THF is via the methionine synthase reaction

Methyl Trap Hypothesis

-A lack of Vitamin B12 in the diet will produce a functional folate deficiency as the body’s folate pool becomes “trapped” in the N₅-methyl THF form

***Have found success clinically with administration of methionine to patients with demyelintation from defects in B12 metabolism***

Question 24

Why does deficiency of either folate or vitamin B12 result in a megaloblastic anemia?

What step does this occur at?

Answer 24

***Folate or B12 Deficiency = Most common type of Megaloblastic Aemia***

***Deficiency in either compound will result in impaired DNA synthesis

***Step: the block in DNA synthesis due to these deficiencies appears to occur at the step catalyzed by Thymidylate Synthase, which converts dUMP to dTMP and has a requirement for N₅,N₁₀-methylene THF

(Note: dUTP is incorporated into DNA instead of dTTP –> DNA fragmentation and dell death)

Question 25

What circumstances might increase folate needs?

Under what circumstances may folate deficiency be seen in a patient?

Answer 25

Increased Folate Needs: Pregnancy***, Lactation, Periods of Growth, Chronic Hemolytic Anemia

***Deficiency can lead to neural tube defects

Folate Deficiency: Alcoholism, Old Age, Poverty, Celiac Disease/Tropical Sprue (and other conditions of impaired absorption)

Question 26

What circumstances might increase vitamin B12 needs?

Under what circumstances may vitamin B12 deficiency be seen in a patient?

Answer 26

Increased Vitamin B12 Needs: Pregnancy and Periods of Growth

Vitamin B12 Deficiency: Strict Vegans (who do not consume fortified foods or B12 supplements), Breast-fed infants of strict vegans, Pernicious Anemia, Celiac Disease/Tropical Sprue (other malabsorptive conditions), Impaired gastric acid secretion, Competing Instestinal Flora (blind loop syndrome), Ileitis/Extensive Resection of the Ileum, Certain Parasitic Infections (fish tapeworm)

Question 27

By what mechanism can nitrous oxide anesthesia provoke an acute megaloblastic anemia?

Answer 27

Note: Megaloblastic Anemia normally develops over weeks to months

***Nitrous Oxide (NO) rapidly destroys methylcobalamin (needed to convert homocysteine to methionine)

Presentation: 8 month old with fever, progressive lethargy, and athetoid movements (slow, involuntary, convulated, writhing movements)