L18 One Carbon Metabolism

Question 1

Overview:

1. What is one carbon metabolism?
2. What amino acids are the carbon donors?
3. What is the carrier molecule?
4. What are th two types of reactions modified folate participates in?

Answer 1

1. One carbon metabolism is a network of integrated metabolic pathways act together to continually
   supply single-carbon units needed for various biochemical reactions.
2. The amino acids serine, glycine, and histidine can serve as initial donors of one-carbon units. Serine is the major donor of one-carbon units in the
   body.
3. The donated carbon unit is first bound to a carrier molecule (a modified form of folate)
4. a. Folate can directly donate its one-carbon unit to nucleic acid intermediates in the synthesis of nucleotides (purines or thymidine).
5. b. Folate can also donate its one-carbon unit to a second type of onecarbon carrier molecule, homocysteine. This carrier is further modified and eventually donates its one-carbon methyl group for methylation reactions.

Question 2

Reaction Pathway Overview:

Answer 2

Folate undergoes a series of reductions and accepts C, some of the intermediates are needed for purine and dTMP synthesis:
folate→dihydrofolate(DHF)→terahydrofolate(THF)
THF→N¹⁰formyl-THF (used in purine synth) →
→ serine donates C→
→ N⁵-N¹⁰-methyleneTHF (used in dTMP synth) →N⁵-methylTHF

Then Vitamin B₁₂ dependent Homocysteine Methyltransferase:
Transfers C from N⁵-methylTHF to methionine, recycling THF and creatin SAMe:
Methionine→SAdenosylmethionine(SAMe) donates CH₃ to the target accepter

SAMe goes through a series of reactions to produce Homocysteine which can be recycled to methione or THF via Homocystein Methyltransferase
SAMe→S Adenosylhomocysteine(SAH) →
→ Homocysteine → methionine or THF

Question 3

1. What is folate?
2. What are it’s components?
3. What is the source of folate?
4. What is the most common cause of folate deficiency?
5. How does heat affect folate?

Answer 3

1. The group of compounds consisting of folic acid and its derivatives are collectively referred to as folates.
2. Folic acid, also called pteroyl glutamate or folyl monoglutamate, is composed of three parts: a pteridine ring, p-aminobenzoic acid (PABA), and one glutamic acid residue.
3. Fruits and vegetables are the main source of
   folate in the human diet.
4. Insufficient dietary intake is the most common cause
   of folate deficiency.
5. Folate is heat-labile and easily destroyed by
   cooking (50-90% of the vitamin depleted).

Question 4

1. What form is dietary folate usually in?
2. How is it named?
3. How is folate inititally modified?

Answer 4

1. Dietary folate (mainly from fruits and vegetables) is present largely as conjugates, in which folate is bound to multiple glutamic acids residues.
2. Conjugates are named according to the length of the glutamate chain (pteroyl + (mono-, di-, tri-, etc) glutamate).
3. In humans, glutamic acids can be removed from folate by the action of deconjugating enzymes (conjugases), neccessary for absorption.

Question 5

1. Where is folate deconjugated?
2. How is it absorbed?
3. What is the primary form of folate in circulation?

Answer 5

1. A brush-border conjugase on the enterocyte surface systematically removes single glutamate residues from the end of the glutamate chain, ultimately yielding folyl monoglutamate (aka folic acid or pteroyl glutamate).
2. Folyl monoglutamate is actively transported into the enterocyte via a reduced folate carrier protein. Within the enterocyte, folic acid is metabolized to N5-methyl
   tetrahydrofolate (N5-methyl-THF) and then released into the circulation.
3. N5-methyl-THF (a modified form of folate with one glutamic acid residue) is the primary form of folate in the circulation.

Question 6

How is folated taken up into tissue from circulation?
4 steps:

Answer 6

Receptor-mediated Endocytosis of Folate:

1. GPI-anchored folate receptors (on
   the cell plasma membrane) bind to N5-
   methyl-THF in the circulation with high
   affinity.
2. Folate undergoes receptormediated
   endocytosis and is subsequently
   released into the lumen of the acidified
   vesicle within the cytoplasm.
3. Folate is then moved from the
   lumen of the vesicle into the cytoplasm of
   the cell via a vesicular membrane folate
   transporter.
4. The vesicles containing folate
   receptors are recycled to the cell surface,
   where the receptors can once again take up
   folate.

Question 7

Metabolic Pathway of Folate

Answer 7

Folic acid first undergoes two
NADPH-dependent reductions
(via the enzyme dihydrofolate
reductase) to form the active
form of folate, tetrahydrofolate
(THF).

Binding to THF allows onecarbon
compounds to be
recognized and manipulated by
biosynthetic enzymes.

Question 8

How does folate contribute to nucleotide synthesis?

Answer 8

N¹⁰-formyl-THF and N⁵N¹⁰-methylene-THF are important carriers of one carbon units for the synthesis of purines (adenine and guanine) and dTMP (thymidine).

After the removal of its one-carbon unit, folate itself can be metabolized back to either DHF or THF. Conversion of DHF to THF is dependent on the activity of the enzyme dihydrofolate reductase.

• *The N⁵N¹⁰-methylene** form of THF can also be metabolized by the enzyme methylene tetrahydrofolate reductase (MTHR) to form N⁵-methyl-THF. (this form is the carbon donor)
• *The reaction catalyzed by MTHR is irreversible.**

The drug methotrexate is a competitive reversible inhibitor of dihydrofolate reductase, and is used in chemotherapy regimens for a variety of cancers.

Question 9

What is methotrexate?

Answer 9

Methotrexate, a structural analog of folic acid, inhibits the enzyme dihydrofolate reductase, reducing the ability of the cell to recycle folate. The inhibition of folate recycling causes an associated decline in the synthesis of dTMP and purines, which are most needed by rapidly-dividing cells synthesizing new DNA (erythroblasts/cancer).

Question 10

How can decreased folate metabolism contribute to Megablastic Anemia?

Answer 10

Asynchrony of cell growth as well as the decreased proliferation and death of erythropoietic cells can result in a condition called megaloblastic anemia. (cells get too big from continued protein synthesis w/out cell division)

Rapidly-dividing cells are very sensitive to folate deficiency related declines in nucleotide synthesis. The decreased availability of nucleotides slows DNA synthesis, leading to an asynchrony between DNA synthesis and protein production, and resulting in large cells.

Erythroid cells rapidly divide in the course of their development, and are the first to exhibit pathology when folate levels are abnormally low or folate
metabolism is compromised.

In addition, when folate levels are low, dTTP is scarce but dUTP is plentiful and can become inappropriately incorporated into DNA. This results in an over-activation of dUTP-directed DNA repair mechanisms which introduce
DNA strand breaks and can results in higher levels of cell death.

Question 11

What are the characteristics of Megaloblastic Anemia in a peripheral blood smear?

Answer 11

Oval macrocytes: These are large, oval, fully
hemoglobinized erythrocytes.

Marked anisocytosis (unequal size) and
poikilocytosis (varied shape); high RDW

Low or normal reticulocyte count (CR) due to
decreased erythropoiesis. (note: RC count is elevated w/ anemia but not w/ megaloblastic anemia = way to distuingish)

Large hypersegmented neutrophils: Greater
than 5 lobes in the nuclei is abnormal, and is
likely due to cell division/DNA synthesis defects.

Thrombocytopenia (decreased platelet number)
and leukopenia (decreased white cell number)

Question 12

What are the characteristics of a bone marrow smear with Megaloblastic Anemia?

Can peripheral blood smears or bone marrow smears distinguish between a Vit B12 deficiency and folate deficiency?

Answer 12

Megaloblastic anemia displays
megaloblastic changes in all stages of red cell development. Decreased DNA synthesis leads to hypoproliferation and the premature destruction of
megaloblasts within the bone marrow(ineffective erythropoiesis).This is the cause of the observed anemia.

Development of cells from other lineages, including white blood cells, can also be affected. Increased destruction of platelet precursors (including megakaryocytes) leads to thrombocytopenia (too few platelets).

Increased destruction of
granulocytes (white blood cell
precursors) leads to leukopenia
(decreased white cell count).

_Note: These morphology changes
cannot be used to distinguish a
folate deficiency from a vitamin B12
deficiency as the cause. This will be
addressed in the workshop._

Question 13

What is the difference in morphology between cells with microcytic and megaloblastic (macrocytic) anemia?

Answer 13

Macrocytic has large cells from continued protein production w/out cellular division, caused by folate or vitamin B12 deficiency.

Microcytic anema has smaller cells from a lack of protein (hemoglobin) production, in example, iron deficiency.

Question 14

What is the role of folate in methylation?

Answer 14

N⁵-methyl-THF can donate its methyl group to a second type of onecarbon carrier, homocysteine.

Vitamin B12 (also known as cobalamin) functions as a cofactor for the enzyme homocysteine methyltransferase.

Homocysteine methyltransferase takes the donated methyl group from N5-methyl-THF and binds it to vitamin B12, which is a cofactor bound to the enzyme. This forms methylcobalamin. Then the enzyme transfers the methyl group off of vitamin B12 and onto homocysteine, forming methionine.

The products of this reaction are methionine (used to ultimately provide one carbon units for methylation reactions) and THF, which can be recycled for use in folate-dependent reactions.

Question 15

What are the two reasons Homocysteine methyltransferase is vitally important in the cell?

Answer 15

1. Metabolizes N5-methyl-THF into THF, a form of folate that can again be used as a one-carbon carrier.

Note: N5-methyl-THF cannot be used to supply one-carbon units for nucleotide synthesis and MUST be metabolized back to THF to restore its bioavailability.

2. Facilitates the transfer of the one-carbon (CH3) group from N5-methyl-THF to homocysteine, forming methionine. This step is essential for the synthesis of the terminal methyl donor, S-adenosyl
   methionine (SAMe).

Question 16

1. How are S-adenosylmethionine (SAMe) methyly groups transferred and what molecules can the methyl group of (SAMe) be transferred to?
2. What are some methylation reactions that use SAMe as the methyl donor?
3. What is the only enzyme substrate used more than SAMe?
4. How is SAMe synthesized?

Answer 16

1. Most cells contain numerous SAMe-dependent methyltransferases that can transfer the SAMe methyl group to the oxygen, nitrogen or sulfur
   atoms of other molecules.
2. S-adenosylmethionine is utilized as a methyl donor in over 100 different methylation reactions in the cell. Examples include:
   - Epinephrine biosynthesis
   - Synthesis of phosphatidlycholine
   - Carnitine synthesis
   - DNA methylation (cytosine bases)
   - Histone methylation (lysine residues)
3. Following ATP, S-adenosylmethionine is the second most widely used enzyme substrate. 99% of all methyl transfers in the cell use SAMe as a methyl donor, making it the major methyl donor.
4. To synthesize SAMe, the adenosine residue of ATP is covalently bound to the sulfur atom of methionine.

Question 17

Compare folate and vitamin B12 sotarge, daily needs, cause of deficiency, and time until deficiency is present before megaloblastic anemia manifests

Answer 17

The daily need for folate is much larger than that of vitamin B12, resulting in a greater need for daily intake of sufficient dietary folate.

Question 18

Where is most of the bod’s folate stored and how is it recycled?

How does alcohol interfere with the recycling?

Answer 18

About half of the body’s folate
storesare found in theliver.

10% of this pool is released and

• *recycled** via the **enterohepatic
  circulation. **

Alcohol interferes with enterohepatic
recycling of folate in two ways:
• Decreases expression of
conjugase enzymes
• Decreases expression of
reduced folate carrier protein

Decreased absorption of folate
into the portal blood accelerates
folate loss from the body.

These decreases can also affect
the uptake of new dietary folate.

Question 19

How does alcohol abuse effect kindey folate recycling?

Why does alcohol abuse accelerate the development of folate deficiency?

Answer 19

The non-hepatic pool of folate circulates in the
blood to other tissues. Circulating folate is filtered and then reabsorbed by the renal tubular cells of the kidney (only ~1% of total body folate pool is normally excreted in urine).

Alcohol abuse decreases expression of the folate receptor and carrier protein in the kidney, thus increasing urinary excretion of circulating folate.

Alcoholic individuals lose more of their folate on a daily basis. Individuals with poor diets who are not alcoholic are able to maintain appropriate recycling of folate stores (even if they’re not taking in enough dietary folate). So, folate deficiency (and megaloblastic anemia) will take longer to develop in individuals who do not consume excessive alcohol (4-5 months versus 4-5 weeks for an alcoholic).

Question 20

What is the “folate trap” and what is it caused by?

What are the consequnces?

How can a folate deficiency be confused with a
Vitamin B12 deficiency?

Answer 20

When Vitamin B12 is deficient, folate cannot be properly recycled, and becomes “trapped” as N5-methyl-THF. This phenomena is known as the “folate trap”.

-N5-methyl-THF cannot be used for DNA synthesis. It also cannot be recycled to THF without the activity of homocysteine methyltransferase, a vitamin B12 dependent enzyme.
-Deficiencies in vitamin B12 can also impair
production of methionineand the
subsequent synthesis of the methyl donor,
S-adenosyl methionine.

A patient may appear to have a
megaloblastic anemia due to an “apparent”
folate deficiency, but folate is present just
not biologically available.The realcause of
the anemia is a vitamin B12 deficiency that
prevents appropriate recycling of folate.

Question 21

What are the consequences of Vitamin B12 Deficiency?

Answer 21

When vitamin B12 is deficient, the activity of homocysteine methyltransferase is impaired.

As a result, N5-methyl-THF and homocysteine accumulate in the cell.

N5-methyl-THF cannot be used for nucleotide synthesis reactions, and without vitamin B12, cannot be recycled to THF.

This decrease in bioavailable folate (as a result of B12 deficiency) is referred to as the “folate trap”.

Question 22

How does vitamin B12 deficiency impare methylation?

What are the consequences?

Answer 22

A vitamin B12 deficiency also results in impaired methylation of homocysteine, thus impairing the production of S-adenosylmethionine.

Decreased S-adenosylmethionine synthesis reduces the methylation of a variety of macromolecules, including DNA and cellular proteins.

Impaired methylation may affect myelin stability and is a probable cause of the peripheral neuropathy associated with a vitamin B12 deficiency. Symptoms may include numbness or tingling of the hands and feet,
weakness, loss of dexterity, impaired memory, and personality changes.

Neuropathy may be present with or without accompanying megaloblastic anemia.

It is not known why individuals with folate deficiency do not generally develop the peripheral neuropathy that is seen with B12 deficiency. Is the methylation theory incorrect? Is the neuropathy related to another B12
dependent enzyme, methylmalonyl CoA mutase?

Question 23

What is the transsulfuration pathway?

What vitamin is required?

How is it regulated?

Answer 23

In addition to participating in methionine synthesis, homocysteine can also be directed to the transsulfuration pathway to form cysteine.

Homocysteine is first converted to cystathione by the rate-limiting enzyme cystathione synthase, which transfers a sulfur atom from homocysteine to
serine.

Note that vitamin B₆ (pyridoxal phosphate) is required for both enzymes in the transsulfuration pathway.

Cystathione synthase is allosterically activated by SAMe to prevent excess methionine from being synthesized (diverts homocysteine out of methylation pathway):

increased SAMe → increased cystathione synthase activity →
Decreased: homocysteine, methionine
Increased: cysteine

Question 24

What is Homocysteinemia and what are the risks?

What are the causes?

Answer 24

Elevations in circulating homocysteine levels are a clinical concern, as moderate hyperhomocysteinemia is a risk factor for atherosclerosis and
venous thromboembolism.

The most common form of genetic hyperhomocysteinemia is a mutation in the enzyme that produces N5-methyl-THF (methylene tetrahydrofolate reductase, MTHR).

Approximately 5-14% of the general population is estimated to harbor a mutation in MTHR. Homozygosity for this variant is a relatively common cause of mildly elevated plasma homocysteine levels.

Remember: Increased blood levels of homocysteine may also reflect a folate or vitamin B12 deficiency, not always a mutation in MTHR.

Question 25

What is Homocystinuria?

What gene is involved?

What are the symptoms?

Treatment?

Answer 25

Homocystinuria is an autosomal recessive disease affecting methionine metabolism that results in increased urinary homocysteine (homocystinuria).

Cystathione synthase gene mutations give rise to a much more severe elevation in plasma homocysteine.

This disorder can cause intellectual disability, osteoporosis, chest deformities, ocular abnormalities (including dislocation of the lens of the eye), and premature atherosclerosis. Patients generally have a long thin build and spidery fingers (somewhat reminiscent of Marfan syndrome, but with tight rather than loose joints).

I f the diagnosis is made soon after birth
(some states test for this deficiency), a
low methionine diet can help prevent
some intellectual disability as well as
other symptoms. Around 50% of
individuals with this deficiency will also
respond well to treatment with high dose
vitamin B6.