Vitamin B12- Chemical structure, Forms of B12, Sources, absorption, storage, transportation, metabolic role, deficiency, megaloblastic anemia and neurological changes, laboratory diagnosis and treatment

1. Vitamin B12 – Chemistry,
functions and clinical significance
Professor(Dr.) Namrata Chhabra
Biochemistry for medics- Lecture notes
www.namrata.co 1Namrata Chhabra

2. Synonyms
• Cyanocobalamine
• Anti Pernicious anemia factor
• Extrinsic factor of Castle
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3. Structure
• Cobalamin is analogous to heme in its
structure having as its base a tetrapyrrole ring.
• Instead of iron as a metal cofactor for heme,
cobalamin has cobalt in a coordination state
of six with
o a benzimidazole group nitrogen coordinated
to one axial position,
o the four equatorial positions coordinated by
the nitrogens of the four pyrrole groups and
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4. Structure of Vitamin B12
oThe sixth position occupied by either a deoxyadenosine group, a methyl group or a
CN– group in the commercially available form in vitamin tablets.
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5. Forms of Cobalamin
• Cobalamin (vitamin B12) exists in a number of different
chemical forms.
• All have a cobalt atom at the center of a corrin ring.
• In nature, the vitamin is mainly in the 2-deoxyadenosyl
(ado) form, which is located in mitochondria
• The other major natural cobalamin is
methylcobalamin, the form in human plasma and in
cell cytoplasm.
• There are also minor amounts of hydroxocobalamin to
which methyl- and adenosyl cobalamin are rapidly
converted by exposure to light.
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6. Dietary Sources
• Cobalamin is synthesized solely by
microorganisms.
• Ruminants obtain cobalamin from the foregut,
but the only source for humans is food of animal
origin, e.g. meat, fish, and dairy products.
• Vegetables, fruits, and other foods of non-animal
origin are free from cobalamin unless they are
contaminated by bacteria.
• Strict vegetarians are at risk of developing B12
deficiency.
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7. Sources of vitamin B12
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8. Requirements of vitamin B12
• A normal Western diet contains between
5 and 30 μg of cobalamin daily.
• Adult daily losses (mainly in the urine
and feces) are between 1 and 3 μg
(~0.1% of body stores) and, as the body
does not have the ability to degrade
cobalamin, daily requirements are also
about 1 to 3 μg.
• Body stores are of the order of 2 to 3
mg, sufficient for 3 to 4 years if supplies
are completely cut off.
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9. Absorption
• Two mechanisms exist for cobalamin absorption.
• Passive absorption-occurring equally through
buccal, duodenal and ileal mucosa; it is rapid but
extremely inefficient, <1 percent of an oral dose
being absorbed by this process.
• Active absorption-The normal physiologic
mechanism is active; it occurs through the ileum
and is efficient for small (a few micrograms) oral
doses of cobalamin and is mediated by gastric
intrinsic factor (IF).
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10. Absorption
• Dietary cobalamin is released from protein
complexes by enzymes in the stomach,
duodenum, and jejunum
• It combines rapidly with a salivary glycoprotein
that belongs to the family of cobalamin-binding
proteins known as haptocorrins (HCs).
• In the intestine, the haptocorrins are digested by
pancreatic trypsin and the cobalamin transferred
to intrinsic factor(IF).
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11. Absorption of Vitamin B12 and the role of Intrinsic factor
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12. Absorption and the role of Intrinsic
factor
• Intrinsic factor (IF) is produced in the gastric
parietal cells of the fundus and body of the
stomach, its secretion parallels that of
hydrochloric acid.
• The IF-cobalamin complex passes to the ileum,
where IF attaches to a specific receptor (Cubulin)
on the microvillus membrane of the enterocytes.
• Cubulin with its ligand IF-cobalamin complex is
endocytosed.
• The cobalamin-IF complex enters the ileal cell
where IF is destroyed.
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13. Intrinsic factor deficiency
• In the absence of the intrinsic factor
inadequate amounts of cobalamin are
absorbed (the dietary requirement is
approximately 200 ng/day) resulting in
Megaloblastic anemia.
• When the root cause of the resultant
Megaloblastic anemia is absence of or
inadequate amounts of intrinsic factor the
condition is called pernicious anemia.
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14. Transportation of Cobalamin
• Three plasma transport proteins have been
identified.
• Transcobalamine I and III (differing only in
carbohydrate structure) are secreted by white
blood cells.
• Although approximately 90 percent of plasma
vitamin B12 circulates bind to these proteins,
only transcobalamine II is capable of
transporting vitamin B12 into cells.
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15. Storage of Cobalamin
• The liver contains 2000 to 5000 mcg of stored
vitamin B12.
• Since daily losses are 1 to 3 mcg/day, the body
usually has sufficient stores of vitamin B12 so
that vitamin B12 deficiency develops more
than 3 years after vitamin B12 absorption
ceases.
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16. Metabolic Role of Cobalamin
1)Cobalamin plays a vital role in the
catabolism of odd-chain fatty acids,
threonine, methionine, and the branched-
chain amino acids (leucine, isoleucine, and
valine).
• The degradation of each of these compounds
produces the same metabolite, Propionyl-
CoA.
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17. Fate of Propionyl CoA
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18. Fate of Propionyl CoA in B12
deficiency
• In cases of cobalamin deficiency these reactions
of utilization of propionyl co A are compromised
leading to an accumulation of methylmalonyl-
CoA in serum, which has been suggested as a
possible source of neurologic defects seen in
cobalamin deficiency by decreasing lipid
synthesis.
• Excess methylmalonyl-CoA in B12 deficiency gets
excreted in urine causing methylmalonic aciduria
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19. 2.Role of cobalamin in DNA synthesis and the
biochemical basis of Megaloblastic anemia
• The cause of megaloblastic anemia seen in
strict vegetarians is attributed to the effects of
cobalamin deficiency on DNA synthesis,
specifically the thymidylate synthesize
reaction which converts dUMP→ dTMP.
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20. Reaction catalyzed by Thymidylate
synthase
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21. Implications of Inadequate
Thymidylate synthesis
• Inadequate dTMP restricts DNA but not RNA
synthesis leading to the appearance of large
erythroid cells with small nuclei containing a
high ratio of RNA to DNA.
• These cells are removed from the circulation,
thus stimulating erythrogenesis and giving rise
to anemia with an elevated presence of
megaloblasts.
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22. 3. Role of cobalamin in methionine
metabolism
• Cobalamin is required for the conversion of
homocysteine into methionine.
• Cobalamin must first undergo methyl transfer
to form methyl cobalamin.
• It receives the methyl group from N5-
methyltetrahydrofolate thus regenerating
tetrahydrofolate to participate in other one-
carbon transfers in purine metabolism or
pyrimidine remodeling.
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23. Folate trap
• In cobalamin deficiency, the methionine synthase reaction
cannot occur, N5-methyltetrahydrofolate accumulates and
the other C-1 donor forms of tetrahydrofolate cannot be
formed.
• The methionine synthesis from homocysteine ceases
allowing the “trapping” of the folate pool as N5-
methyltetrahydrofolate, diminishing levels of N5, N10-
methylenetetrahydrofolate
• N5,N10-methylenetetrahydrofolate, is required for the
methylation of dUMP to dTMP, thus in it’s deficiency ,the
thymidylate synthase reaction is slowed and dTMP levels
drops and hence DNA synthesis is also slowed down due to
non availability of deoxy ribonucleotides
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24. Roles of cobalamin and folic acid in
methionine metabolism
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25. Vitamin B12 deficiency
Causes of Vitamin B12 Deficiency
• Dietary deficiency (rare)
• Decreased production of intrinsic factor
• Pernicious anemia
• Gastrectomy
• Pancreatic insufficiency
• Fish tapeworm (rare)
• Helicobacter pylori infection
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26. Vitamin B12 deficiency
• Crohn’s disease
• Surgical resection
• Decreased ileal absorption of vitamin B12
• Transcobalamine II deficiency (rare)
• Competition for vitamin B12 in gut Blind loop
syndrome
• The most common cause of vitamin B12
deficiency is associated with pernicious anemia
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27. Vitamin B12 deficiency
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28. Megaloblastic anemia
Clinical Findings
• The hallmark of symptomatic vitamin B12 deficiency is
megaloblastic anemia.
• However, subclinical cobalamin deficiency is an increasingly
recognized condition, especially in those with predisposing
conditions such as ileal disease or gastric surgery.
• In advanced cases, the anemia may be severe, with hematocrit as
low as 10 to 15 percent, and may be accompanied by leucopenia
and thrombocytopenia.
• The megaloblastic state also produces changes in mucosal cells,
leading to glossitis, as well as other vague gastrointestinal
disturbances such as anorexia and diarrhea.
• Patients are usually pale and may be mildly icteric
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29. Peripheral blood smear in
Megaloblastic anemia
Blood film in vitamin B12deficiency showing macrocytic red
cells and a hyper segmented neutrophil.
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30. Neurological changes in B12
deficiency
• Vitamin B12 deficiency also leads to a complex
neurologic syndrome.
• Peripheral nerves are usually affected first,
and patients complain initially of paresthesias.
• The posterior columns next become impaired,
and patients complain of difficulty with
balance.
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31. Neurological changes in B12
deficiency
• In more advanced cases, cerebral function
may be altered as well, and on occasion
dementia and other neuropsychiatric changes
may precede hematologic changes.
• Neurologic examination may reveal decreased
vibration and position sense but is more
commonly normal in early stages of the
disease.
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33. Laboratory Findings
• The MCV is usually strikingly elevated, between 110
and 140 fL.
• The peripheral blood smear is usually strikingly
abnormal, with anisocytosis and poikilocytosis. A
characteristic finding is the macro-ovalocyte, but
numerous other abnormal shapes are usually seen. The
neutrophils are hyper segmented.
• The reticulocyte count is reduced.
• Because vitamin B12 deficiency affects all
hematopoietic cell lines, in severe cases the white
blood cell count and the platelet count are reduced,
and pancytopenia is present.
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34. Laboratory Findings
• Bone marrow morphology is characteristically
abnormal.
• Marked erythroid hyperplasia is present as a response
to defective red blood cell production (ineffective
erythropoiesis).
• Megaloblastic changes in the erythroid series include
abnormally large cell size and asynchronous
maturation of the nucleus and cytoplasm—i.e.
cytoplasmic maturation continues while impaired DNA
synthesis causes retarded nuclear development.
• In the myeloid series, giant metamyelocytes are
characteristically seen.
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35. Laboratory Findings
• Other laboratory abnormalities include
elevated serum lactate dehydrogenase (LDH)
and a modest increase in indirect bilirubin.
• These two findings are a reflection of
intramedullary destruction of developing
abnormal erythroid cells and are similar to
those observed in peripheral hemolytic
anemias
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36. Laboratory Findings
• Serum cobalamin level: The diagnosis of vitamin
B12 deficiency is made by finding an abnormally
low vitamin B12 (cobalamin) serum level.
• The normal vitamin B12 level is > 240 pg/ml,
• Most patients with overt vitamin B12 deficiency
can have serum levels < 170 pg/ml, with
symptomatic patients usually having levels < 100
pg/ml.
• A level of 170 to 240 pg/ml is borderline.
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37. Laboratory Findings
Estimation of serum methylmalonic acid levels
• When the serum level of vitamin B12 is
borderline, the diagnosis is best confirmed by
finding an elevated level of serum methylmalonic
acid (> 1000 nmol/L
• However, elevated levels of serum methylmalonic
acid can also be due to renal insufficiency.
• The Schilling test is now rarely used.
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38. Essentials of diagnosis
• Essentials of diagnosis are macrocytic anemia.
• Macro-ovalocytes and hyper segmented
neutrophils on peripheral blood smear, and
• serum vitamin B12 level less than 100 pg/ml.
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39. Differential Diagnosis
• Vitamin B12 deficiency should be differentiated
from folic acid deficiency, the other common
cause of megaloblastic anemia, in which red
blood cell folate is low while vitamin B12 levels
are normal.
• The distinction between vitamin B12 deficiency
and myelodysplasia (the other common cause of
macrocytic anemia with abnormal morphology) is
based on the characteristic morphology and the
low vitamin B12 level
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40. Pernicious Anemia
• Pernicious anemia is a chronic illness caused
by impaired absorption of vitamin B12
because of a lack of intrinsic factor (IF) in
gastric secretions.
• The disease was named pernicious anemia
because it was fatal before treatment became
available
• The term pernicious is no longer appropriate,
but it is retained for historical reasons.
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41. Pernicious Anemia
• Pernicious anemia occurs as a relatively
common adult form of anemia that is
associated with gastric atrophy and a loss of IF
production and
• as a rare congenital autosomal recessive form
in which IF production is lacking without
gastric atrophy.
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42. Clinical manifestations in Pernicious
anemia
• General findings: Weight loss of 10 to 15
pounds occurs in about 50 percent of
patients and probably is due to anorexia,
which is observed in most patients.
• Anemia: The anemia often is well tolerated in
pernicious anemia, and many patients are
ambulatory with hematocrit levels in the mid
teens.
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43. Clinical manifestations in Pernicious
anemia
Gastrointestinal findings:
• Approximately 50 percent of patients have a smooth tongue with
loss of papillae. The tongue may be painful and beefy red. These
symptoms may be associated with changes in taste and loss of
appetite.
• Patients may report either constipation or having several semisolid
bowel movements daily. This has been attributed to megaloblastic
changes of the cells of the intestinal mucosa.
• Nonspecific gastrointestinal symptoms include anorexia, nausea,
vomiting, heartburn, flatulence, and a sense of fullness.
• Rarely, patients present with severe abdominal pain associated with
abdominal rigidity; this has been attributed to spinal cord
pathology.
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44. Clinical manifestations in Pernicious
anemia
Nervous system:
• Neurological symptoms can be elicited in most
patients with pernicious anemia, and the most
common symptoms are paresthesias, weakness,
clumsiness, and an unsteady gait.
• These neurological symptoms are due to myelin
degeneration and loss of nerve fibers in the
dorsal and lateral columns of the spinal cord and
cerebral cortex.
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45. Clinical manifestations in Pernicious
anemia
Genitourinary system:
• Urinary retention and
• Impaired micturition may occur because of
spinal cord damage.
• This can predispose patients to urinary tract
infections.
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46. Laboratory Studies
• The peripheral smear shows oval macrocytes,
hyper segmented granulocytes, and
anisopoikilocytosis.
• In severe anemia, red blood cell inclusions
may include Howell-Jolly bodies, Cabot rings,
and punctate basophilia.
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47. Laboratory Studies
• Gastric secretions: Total gastric secretions are
decreased to about 10 percent of the
reference range.
• Most patients with pernicious anemia are
achlorhydric, even with histamine stimulation.
• IF is either absent or markedly decreased.
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48. Laboratory Studies
• Serum Cbl levels: The serum Cbl is low in
patients with pernicious anemia; however, it
may be within the reference range in certain
patients with other forms of Cbl deficiency.
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49. Laboratory Studies
• Schilling test: The Schilling test measures Cbl
absorption by increasing urine radioactivity after
an oral dose of radioactive Cbl.
• The test is useful in demonstrating that the
anemia is caused by an absence of IF and is not
secondary to other causes of Cbl deficiency.
• It is used to identify patients with classic
pernicious anemia, even after they have been
treated with vitamin B12
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50. Laboratory Studies
• Serum: The indirect bilirubin may be elevated
because pernicious anemia is a hemolytic
disorder associated with increased turnover
of bilirubin.
• The serum lactic dehydrogenase usually is
markedly increased
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51. Histological Findings
• The bone marrow biopsy and aspirate usually
are hyper cellular and show trilineage
differentiation.
• Erythroid precursors are large and often oval.
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52. Complications
• If patients are not treated early in the disease,
neurological complications can become
permanent.
• Severe anemia can cause congestive heart
failure or precipitate coronary insufficiency.
• The incidence of gastric adenocarcinoma is 2-
to 3-fold greater in patients with pernicious
anemia than in the general population of the
same age.
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53. Prognosis
• Early recognition and treatment of pernicious
anemia provides a normal, and usually
uncomplicated, lifespan.
• Delayed treatment permits progression of the
anemia and neurological complications. The
mental and
• neurological damage can become irreversible
without therapy.
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54. Treatment of vitamin B12 deficiency
• The indications for starting cobalamin therapy
are :
• A well-documented Megaloblastic anemia
• or other hematological abnormalities
• or neuropathy due to the deficiency.
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55. Treatment of vitamin B12 deficiency
• Patients with pernicious anemia have historically been
treated with parenteral therapy.
• Intramuscular injections of 100 mcg of vitamin B12 are
adequate for each dose.
• Replacement is usually given daily for the first week,
weekly for the first month, and then monthly for life.
• It is a lifelong disorder, and if patients discontinue their
monthly therapy the vitamin deficiency will recur.
• Oral cobalamin may be used instead of parenteral
therapy and can provide equivalent results. The dose is
1000 mcg/day and must be continued indefinitely.
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56. Further reading
• A case oriented approach towards
Biochemistry- By Namrata Chhabra
http://www.jaypeedigital.com/(X(1)S(vclizd4r0zr
y5eoz45exstdx))/Book/BookDetail?isbn=9789
350901885
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