Role o Vitamin B12 & Folic Acid

1. Folic Acid and Vitamin B 12
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Dealing with the topic
• Introduction
• Types and basic biochemistry
• Dietary Sources
• Absorption, Transport, Metabolism & Excretion
• RDA
• Deficiency and Clinical Manifestations
• Laboratory Assessments of Status
• Toxicity

3. Types and forms:
• Folate (most oxidized form)
• Dihydrofolate (FH2)
• Tetrahydrofolate (FH4) (most
reduced form)
Basic Structure of Folate
1. Bicyclic Pteridine Ring
2. PABA( Para-amino-benzoic acid)
3. Polyglutamate Tail
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4. Salient Points:
• N5-Methyl-FH4 once formed, the step is irreversible.
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5. Folate from
food Sources
Folate Monoglutamates
(Absorbed)
Folate Conjugases
Duodenum
Folate N5-methyl FH4
Within Intestinal Cell
Liver
Entero-hepatic circulation(through bile)
Urine
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Small degradation

6. Vitamin B12 Structure
• Contains a CORRIN ring.
• Two of the four pyrrole rings are
joined directly
rather than joined by methylene
bridges(heme)
• Unusual feature- Cobalt coordinated
with Corrin
ring.
• In the body, Co reacts with the carbon
of a methyl group, forming
methylcobalamin, or with the 5’-
carbon of 5’-deoxyadenosine, forming
5’-deoxyadenosylcobalamin.
• The form of Vit B12 found in
supplements is Cyanocobalamin.
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7. Absorption and Transport of Vitamin B12
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RDA of FOLATE
• Age Male Female Pregnant Lactating
• 0-6months* 65µg DFE* 65µg DFE*
• 7-12months* 80µg DFE* 80µg DFE*
• 1-3 years 150µg DFE 150µg DFE
• 4-8 years 200µg DFE 200µg DFE
• 9-13 years 300µg DFE 300µg DFE
• 14-18 years 400µg DFE 400µg DFE 600µg DFE 600µg DFE
• 19+ years 400µg DFE 400µg DFE 600µg DFE 600µg DFE
*Adequate Intake (AI)

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• 1 DFE(Dietary Folate Equivalent) = 1 µg of Folic acid in dietary food.
• 1DFE = 0.6µg of Folic acid when consumed along with dietary food.
• 1 DFE= 0.5µg of Folic Acid when consumed in empty stomach.

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RDA OF VITAMIN B12
• Age Male Female Pregnant Lactating
• 0-6months* 0.4µg 0.4µg
• 7-12months* 0.5µg 0.5µg
• 1-3 years 0.9µg 0.9µg
• 4-8 years 1.2µg 1.2µg
• 9-13 years 1.8µg 1.8µg
• 14+ years 2.4µg 2.4µg 2.6µg 2.8µg
• *Adequate Intake (AI)

11. Dietary Sources of Folate
• Spinach
• Green Leafy Vegetables
• Liver
• Yeast
• Legumes
Natural Dietary Sources- Reduced Co-enzyme form
Vitamin Supplements/Fortified Foods- Oxidized form of Pteridine Ring 11

12. Dietary Sources of Vitamin B12
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One Carbon Pool
• The collection of one-carbon groups attached to FH4 is known as the
one-carbon pool.
• While attached to FH4, these one-carbon units can be oxidized and
reduced.
• The most oxidized form is N10-formyl FH4. The most reduced form is
N5-methyl-FH4. Once the methyl group is formed, it is not readily
reoxidized back to N5, N10 methylene FH4, and thus N5-methyl-FH4
will tend to accumulate in the cell.

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• Serine’s hydroxyl-methyl group is transferred to FH4 in a reversible
reaction, catalyzed by the enzyme serine
hydroxymethyltransferase.
• This reaction produces glycine and N5, N10-methylene-FH4.
• Because serine can be synthesized from 3-phosphoglycerate, an
intermediate of glycolysis, dietary carbohydrate can serve as a
source of carbon for the one-carbon pool.
• The glycine that is produced may be further degraded by donation
of a carbon to folate
Salient points of Serine as Carbon donor

15. • The chief carbon donator is SERINE.
• Methionine metabolism is dependent both
on the Vitamin B12 as well as Folate.
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16. A deficiency of folate results in the
accumulation of FIGLU, which is
excreted in the urine. A histidine
load test can be used for detecting folate
deficiencies. Patients were given a test dose
of histidine (a histidine load), and the
amount of FIGLU that appeared in the urine
was measured.
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19. What do you think the
structure of Methotrexate
resembles to?
Folic Acid. Binds to DHFR 1000 times more
strongly
Methyl Group
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20. Two main reactions of Vitamin B12
• The transfer of a methyl group
from N5-methyl FH4 to homocysteine to form methionine.
• The rearrangement of
the methyl group of L-methylmalonyl CoA to form succinyl CoA
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The Methyl-Trap Hypothesis
• The equilibrium lies in the direction of the N5-methyl FH4 form.
• This appears to be the most stable form of carbon attached to the
vitamin.
• However, in only one reaction can the methyl group be removed from
N5-methyl FH4, and that is the methionine synthase reaction, which
requires vitamin B12.
• Therefore if there is Vitamin B12 deficiency, then most folate forms in
body is “trapped” in N5-methyl FH4 form- Functional Folate
Deficiency.
• This is the Methyl-Trap Hypothesis.

22. How Folate, Vitamin B12 and SAM are related?
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Tests done
Serum Cobalamin
• normal range- 160-200ng/L to 1000ng/L.
• If megaloblastic anemia then levels are <100ng/L.
• Measured by ELISA.
Serum Methylmalonate and Homocysteine
• Advantage- These can pick up def in early stages even in absence of
hematological abnormalities or subnormal levels of serum cobalamin.
• Disadvantage- Serum MMA varies with RF.

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Serum Folate
• Measured by ELISA.
• Normal range : 2µg/L - 15µg/L
Red Cell Folate
• Valuable test for body folate stores.
• It is though affected by recent diets and traces of hemolysis.
• In normal adults, range is 160-640µg/L of packed red cells.
Histidine Load test generally not done now a days

25. Detection of cause of Vitamin B12 by Schilling
Test
RL oral Vit B12 + IM
Unlabelled VitB12
Measure the 24hrs-48hrs urine sample
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Actual RL Vit B12- Dietary
deficiency
Decreased RL B12- Absorption
problem
STAGE 1
NOTE: Unlabelled Vit B12 through IM is given only once

26. RL oral Vit B12 +
Intrinsic factor
Measure the 24hrs-48hrs urine sample
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Actual RL Vit B12-
Pernicious anemia
Decreased RL B12- No
Dietary Def., No IF def.
STAGE 2

27. RL oral Vit B12 +
Antibiotics
Actual RL Vit B12- Bacterial
Overgrowth
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Decreased RL B12- No dietary
Def, No IF def, No Bact.
Overgrowth
STAGE 3
Measure the 24hrs-48hrs urine sample

28. RL oral Vit B12 +
Pancreatic enzymes
Measure the 24hrs-48hrs urine sample
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Actual RL Vit B12- Pancreatic
Insufficiency like Chronic
Pancreatitis
STAGE 4

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Deficiency of Vitamin B12
• Pernicious anemia with atrophic gastritis is the most common cause of
its deficiency in the western countries, however, in India, alcoholism,
malnutrition and ileo-cecal tuberculosis are the common causes.
• Two most common manifestation
1. Neurological Manifestation(Caused By Hypomethylation of Nervous
system)
2. Hematological Manifestation(Due to adverse effects of Vitamin B12
on Folate Metabolism)

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Neurological symptoms
• Symmetric numbness and tingling of the hands and feet, diminishing
vibratory and position sense, and progression to a spastic gait
disturbance.
• The patient may become somnolent or may become extremely irritable
(“megaloblastic madness”).
• Blind Spots in Visual field followed by alterations in Gustatory and
Olfactory function.
• This is believed to be caused by hypomethylation within the nervous
system, brought about by an inability to recycle homocysteine to
methionine and from there to SAM.
• Ultimately both ascending and descending tracts may be affected and
get degenerated- SUBACUTE COMBINED DEGENERATION OF SPINAL
CORD
• The nervous system lacks the betaine pathway of methionine
regeneration and is dependent on the B12 system.

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Other neurological Symptoms
• Additional symptoms of vitamin B12 deficiency include difficulty
maintaining balance, depression, confusion, dementia, poor memory,
and soreness of the mouth or tongue.
• The neurological symptoms of vitamin B12 deficiency can occur
without anemia, so early diagnosis and intervention is important to
avoid irreversible damage.

32. Betaine Pathway of Methionine Regeneration
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33. Severe Combined Degeneration of Spinal Cord
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Hematological Manifestations
Megaloblastic Anemia
• The presence of red cells that are macrocytic and oval (macro-ovalocytes) is
highly characteristic.
• There is marked variation in the size (anisocytosis) and shape
(poikilocytosis) of red cells.
• Neutrophils are also larger than normal (macropolymorphonuclear) and
hypersegmented, having five or more nuclear lobules instead of the normal
three to four.
• NOTE: Whatever the mechanism, lack of folate is the proximate cause of
anemia in vitamin B12 deficiency, since the anemia improves with
administration of folic acid- Functional Folate Deficiency

35. Hyper-segmented Neutrophils
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36. Ineffective Erythropoeisis
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Other system affected by Vitamin B12 and
Folate
• Epithelial surfaces: After marrow most frequently affected. Mouth,
Stomach,small intestines, respiratory, urinary and female genital
tracts- Macrocytosis + Increased multinucleate and dying cells.
• Complications of Pregnancy: The gonads are also affected, and
infertility is common in both men and women with either deficiency.
Maternal folate deficiency has been implicated as a cause of
prematurity, and both can cause recurrent fetal loss and neural tube
defects.

38. CVS and Hyperhomocysteinemia
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Neural Tube Defects
• Failure of a portion of the neural tube to close, or reopening of a
region of the tube after successful closure, may lead to one of several
malformations.
• Most common –Defect in caudal part of spinal cord.
• Types of Spinal dysraphism- Spina bifida
occulta, Myelomeningocele, Meningocele, Anencephaly.
• Folate deficiency during the initial weeks of gestation has been
implicated as a risk factor; differences in rates of neural tube defects
between populations can be attributed in part to polymorphisms in
enzymes of folic acid metabolism.
• Folate deficiency may affect cell division during critical periods that
coincide with closure of the neural tube.

40. Myelomeningocele
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41. Anencephaly
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All that is left is small, vascular mass of disorganized
neural tissue (cerebrovasculosa), mixed with
choroid plexus.

42. Therapy for megaloblastic Anemia
Cobalamin deficiency
• Lifelong regular cobalamin injections. Surgery if tropical sprue(gut
replacement surgeries), fish tapeworm, intestinal stagnant loop.
• Replenishment of body store complete with six 1000µg injections of
hydroxocobalamin given at 3- to 7-day interval. Maintenance therapy-
1000µg/3months.
• Even in Pernicious Anemia, its documented that very large doses
(1000-2000)µg oral doses can lead to absorption of Vit B12 from
mucous membranes.
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43. Folate Deficiency
• Oral doses of 5-15mg folate daily are satisfactory. Its important to
continue therapy for around 4months by which time all folate
deficient red cells are replaced by folate repleted population.
• Always check if the megaloblastic anemia is due to vit b12 def or not
else folate will correct the anemia(As methyl trap is bypassed) but not
the neurological symptoms of Vit B12.
Pregnancy- Folic Acid 400µg daily, as supplement before and
throughout pregnancy. Previous cases of NTDs mother, are given 5mg
daily dose. Some studies say to give Zinc if folate supplements are to be
given during pregnancy.
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Hypervitaminosis
• B12 is commonly given in doses much higher than the RDA (2.4 mcg (micrograms)) without known
toxicity. Most B12 supplements will provide at least 2,000 mcg of B12 and some deliver as much as
5,000mcg. There has been no scientific evidence demonstrating any significant toxicty when given at
this level. Currently no tolerable upper limit of B12 has been set by The Food and Nutrition Board
indicating this lack of toxic effects
• Folate: Folate is not considered to be toxic and even high doses of Folic Acid are considered to be safe
and non-toxic. However, high intakes of Folic Acid can make it difficult to detect a Vitamin B12
deficiency because Folic Acid also reduces Vitamin B12 deficiency symptoms but without correcting the
neurological damage that also occurs. This is why most Folic Acid products also contain Vitamin B12.
• Adverse Effects may include:
• fever
• itching
• mental changes
• shortness of breath
• skin rash
• sleep disturbances
• wheezing