5. HISTORICAL BACKGROUND
Vitamin B12 structure was
demonstrated by Hodgkin, who
was awarded a Nobel Prize for
her crystallographic work.
Castle demonstrated that
gastric juice contains an
“intrinsic factor” (IF)
In 1926 Minot and Murphy
using the then new technique
of reticulocyte assessment,
showed that the manifestations
could be reversed by eating
prodigious amounts of liver.
In 1880, Ehrlich identified
megaloblasts
Neuropathy was noted by Osler
and Gardner in 1877
First description of pernicious
anemia : puzzling illness with
anemia, debilitation, languor,
and, finally, torpor and death
by Addison in 1849.
7. STRUCTURE
• Cobalt atom at the center of a corrin ring.
• In nature, vitamin B12 exists as 2 forms
• 2-deoxyadenosyl (ado) Cobalamin
• Located in mitochondria
• Co-factor for the enzyme methyl-malonyl CoA mutase.
• Methyl Cobalamin
• Found in human plasma and in cell cytoplasm.
• Co-factor for methionine synthase.
8. DIETARY SOURCES AND REQUIREMENT
• Cobalamin is synthesized solely by microorganisms.
• The only source for humans is food of animal origin, e.g., meat, fish, and dairy products.
• Vegetables, fruits, and other foods of nonanimal origin are free from cobalamin unless they are
contaminated by bacteria.
• Daily requirements are also about 1–3 μg (avg 2.4 mcg ) in adult ,0.4 mcg in infancy and 1.8 mcg
in preadolescence.
• Body stores are of the order of 2–3 mg, sufficient for 3–4 years if supplies are completely cut off.
9. ABSORBTION
• Two mechanisms
Passive
Occurs equally through buccal, duodenal, and ileal mucosa
Rapid but extremely inefficient, with <1% of an oral dose being absorbed.
Active : normal physiological phenomenon
Occurs through the ileum
Efficient and mediated by gastric intrinsic factor (IF).
• Dietary cobalamin is released from protein complexes by enzymes in the stomach, duodenum,
and jejunum combines rapidly with a salivary glycoprotein aka haptocorrins (HCs) In the
intestine, the haptocorrin is digested by pancreatic trypsin and the cobalamin is transferred to IF.
10. TRANSPORT
• The IF-cobalamin complex passes to the ileum, where IF attaches to a specific receptor (cubilin)
on the microvillus membrane of the enterocytes cobalamin-IF complex enters the ileal cell,
where IF is destroyed appears in portal blood attached to TC II after a delay of about 6 hr.
• TC II is synthesized by liver and by other tissues, including macrophages, ileum, and vascular
endothelium. It normally carries only 20–60 ng of cobalamin per liter of plasma and readily gives
up cobalamin to marrow, placenta, and other tissues, which it enters by receptor-mediated
endocytosis
• Between 0.5 and 5 μg of cobalamin enter the bile each day.
13. STRUCTURE , SOURCE AND REQUIREMENT
• Folic (pteroylglutamic) acid is a yellow, crystalline, water-soluble substance.
• Partly or completely reduced to di- or tetrahydrofolate (THF) derivatives.
• Usually contain a single carbon unit
• 70–90% of natural folates - folate-polyglutamates.
• Dietary source : The highest concentrations are found in liver, yeast, spinach, other greens, and nuts (>100
μg/100 g).
• Folate is easily destroyed by heating.
• Total body folate in the adult is ~10 mg, with the liver containing the largest store.
• The recommended dietary folate equivalent intake is 400 mcg daily for adults ,600 mcg for pregnant women
and 500 mcg for lactating women.
14. ABSORBTION AND TRANSPORT
• Absorbed in upper small intestine.
• All dietary folates are converted to 5-methylTHF (5-MTHF) within the small intestinal mucosa
before entering portal plasma achieves peak level in the blood 1 hr after ingestion.
• 2/3rd bound to albumin and 1/3rd free form.
• About 60–90 μg of folate enters the bile each day and is excreted into the small intestine.
• In all body fluids (plasma, CSF, milk, bile), folate is largely, if not entirely, 5-MTHF in the mono
glutamate form.
15. BIOCHEMICAL FUNCTIONS
• As coenzymes in the transfer of single-carbon units.
• Purine and pyrimidine synthesis necessary for DNA and RNA replication.
• As a coenzyme for methionine synthesis, in which methylcobalamin is also involved and in which THF is
regenerated.
• Methionine, the other product of the methionine synthase reaction, is the precursor for S-
adenosylmethionine (SAM), the universal methyl donor involved in >100 methyltransferase reactions .
• During thymidylate synthesis, 5,10-methylene-THF is oxidized to DHF (dihydrofolate). The enzyme DHF
reductase converts this to THF.
• The drugs methotrexate, pyrimethamine, and (mainly in bacteria) trimethoprim inhibit DHF reductase
and so prevent formation of active THF from DHF.
16.
17.
18.
19. BIOCHEMICAL BASIS
• Defect in DNA synthesis that affects rapidly dividing cells in the bone marrow.
• Failure to convert deoxyuridine monophosphate (dUMP) to deoxythymidine
monophosphate (dTMP).
• Folate is needed as the coenzyme 5,10-methylene-THF for conversion of dUMP to dTMP;
the availability of which is reduced in either cobalamin or folate deficiency.
• Mis-incorporation of uracil into DNA as consequence of the block in conversion of dUMP
to dTMP resulting in DNA breakage arrest of hematopoietic precursor cell in various
stages of interphase.
20. COBALAMIN-FOLATE RELATIONS
• Only two reactions in the body are known to require cobalamin.
• Methylmalonyl CoA isomerization requires SAM.
• Methylation of homocysteine to methionine requires both methylcobalamin and 5-MTHF.
• In cobalamin deficiency, MTHF accumulates in plasma, and intracellular folate concentrations fall
due to failure of formation of THF : THF starvation or methylfolate trap.
24. CLINICAL FEATURES
• Asymptomatic : increased MCV
• LOA - usually marked , LOW
• Diarrhea, or constipation.
• Glossitis, angular cheilitis.
• Jaundice (unconjugated)- lemon yellow tinge, and reversible melanin skin hyperpigmentation
• Pallor, fatigue and other features of anemia – more common.
• Mucocutaneous bleed d/t thrombocytopenia and infections d/t leucopenia are less common.
25. CAUSES OF MACROCYTOSIS(MCV>97 fl)
MEGALOBLASTIC ANEMIA
Cobalamin or folate deficiency
Some metabolic disorders (e.g., thiamine-responsive
anemia)
Cytotoxic drugs (e.g., hydroxyurea, 5-fluorouracil)
27. GENERAL TISSUE EFFECTS OF VITAMIN B12
AND FOLATE DEFICIENCY
• Epithelial Surfaces
• After BM, the next most frequently affected tissues are the epithelial cell surfaces of the
mouth, stomach, and small intestine and the respiratory, urinary, and female genital tracts.
• Complications of Pregnancy
• Infertility is common in both men and women (with def. of both )
• Prematurity is common with folate deficiency.
• Recurrent fetal loss and neural tube defects ( with def. of both)
28. • Neural Tube Defects
• FA supplement at the time of conception and in the first 12 WOP reduce by ~70% the
incidence of NTDs (anencephaly, meningomyelocele, encephalocele, and spina bifida) in the
fetus.
• Malignancy
• Prophylactic FA in pregnancy has been found in some but not all to reduce the subsequent
incidence of ALL in childhood.
• Lower risk for colorectal cancer.
29. • Cardiovascular Disease
• Severe homocystinuria (blood levels ≥100 μmol/L) due to deficiency of one of three enzymes,
methionine synthase, MTHFR, or cystathionine synthase.
• Premature CVD, CAD,PAD and unexplained and recurrent venous thrombosis.
• Prospective RCT with supplements of folic acid, vitamin B12, and vitamin B6 against placebo over
a 5-year period
• No reduction of first event fatal or nonfatal MI.
• No reduction in the risk of recurrent cardiovascular disease after an acute MI
• 18% reduction in strokes but no significant prevention of death from any cause.
30. NEUROLOGICAL DYSFUNCTION
• CNS : Demyelination with subsequent axonal disruption and gliosis.
• PNS : Axonal degeneration without demyelination.
• The classic myelopathic syndrome is subacute combined degeneration, in which posterior and lateral
column damage predominates; dorsal, pyramidal, and spinocerebellar tracts are affected.
• The earliest changes appear in the cervical or thoracic spine and can be detected by MRI as
hyperintensity on T2WI.
• Larger, more heavily myelinated fibers tend to be affected most often.
• Unlike anemia, neurological dysfunction does not always reverse after cobalamin therapy. Residual
deficit persist in 6% of pts.
31. CLINICAL PRESENTATIONS
1) Young children : Developmental delay ,lethargy , cerebral atrophy, and seizure.
2) Ataxia ( sensory + cerebellar)
3) Pyramidal tract sign : Babinski sign and spasticity
4) Loss of DTR
5) Autonomic dysfunction : Bowel and bladder dysfunction , Impotence.
32. 6) Visual changes , Optic neuritis
7) Disturbance of smell or taste
8) Dementia
• Manifestations tend to be symmetrical
• Involvement ascends up the legs, and, eventually, hands are affected as well
33.
34. HEMATOGICAL FINDINGS
• Panmyelosis
• N/C disassociation : morphological hallmark.
• Giant band cells and metamyelocytes with large and often misshapen nuclei are typical.
• Neutrophils with characteristic hypersegmented nuclei appear in the blood early in the course.
• 6 lobes in ≥ 1 cell
• 5 lobes in ≥ 5 cells.
35. Cont..
• As megaloblastic anemia worsens, neutropenia and thrombocytopenia develop.
• Erythroid macrocytosis is an early change f/b gradual rise in overall MCH and then MCV before
the hemoglobin levels fall.
• Macro-ovalocytes are especially characteristic of megaloblastic anemia but are not specific.
38. BONE MARROW FINDINGS
• BM hyperplasia is intense but reticulocytosis does not occur.
• Giant and abnormally shaped metamyelocytes and enlarged hyper-polyploid megakaryocytes
are characteristic.
• Megaloblastoid : Refer to cells with both immature-appearing nuclei and defective
hemoglobinization and is usually seen in myelodysplasia.
39. • CHROMOSOME
• BM cells, transformed lymphocytes, and other proliferating cells in the body show a variety of
changes – includes
• Random breaks,
• Reduced contraction,
• Spreading of the centromere, and
• Exaggeration of secondary chromosomal constrictions and overprominent satellites.
40. • INEFFECTIVE HEMATOPOIESIS
• Increase in unconjugated bilirubin d/t death of nucleated RBCs in the marrow (ineffective
erythropoiesis).
• Raised urine urobilinogen,
• Reduced haptoglobins,
• Positive urine hemosiderin,
• Raised LDH.
41. CLINICAL DEF. Vs SUB-CLINICAL DEF.
CHARACTERISTICS CLINICAL DEFICIENCY SUB-CLINICAL DEFICIENCY
Biochemical abnormalities Often severe Usually mild
Clinical abnormalities Megaloblastic anemia is present
in >75% of cases
Neurologic or cognitive changes
are present in >50% of cases
Electrophysiologic (neurologic)
abnormalities are usually
present
Anemia is absent
Neurologic changes are absent
Mild electrophysiologic changes
are sometimes present
Cobalamin absorption status IF-related malabsorption causes
>90% of cases
Normal absorption is
uncommon (e.g., veganism)
IF-related malabsorption is
usually absent
Most persons have normal
absorption
42. CHARACTERISTICS CLINICAL DEFICIENCY SUB-CLINICAL DEFICIENCY
Diagnostic criteria Almost always one or more
clinical abnormalities
At least one abnormal
biochemical finding
No clinical signs of cobalamin
deficiency
Ideally, at least two abnormal
biochemical findings should be
demonstrated
Likelihood of progression of
deficiency
Very high because of the usual
presence of IF-related
malabsorption
Unknown, but probably small
Need for cobalamin therapy Urgent in all cases Unknown
Medical implications Clinical deficiency indicates that
medical management is needed
None known, but if SCCD is
found during medical evaluation
it must be evaluated medically
46. • INDICATIONS
Well-documented megaloblastic anemia or other hematologic abnormalities and
Neuropathy d/t to the deficiency.
47. VITAMIN B12 DOSE
• Replenishment of body stores should be complete with six 1000-μg IM injections of
hydroxocobalamin given at 3- to 7-day intervals.
• For maintenance therapy, 1000 μg hydroxocobalamin IM once every 3 months is satisfactory.
• When absorption is normal, oral cyanocobalamin can usually be used, and doses of 2 to 5 μg do
not substantially exceed IF capacity.
48. FOLATE DOSE
• 5 mg of FA daily.
• Duration : 4 months
• Long-term folic acid therapy is required when the underlying cause of the deficiency cannot be
corrected and the deficiency is likely to recur, e.g., in chronic dialysis or hemolytic anemias.
• Folinic Acid (5-Formyl-THF)
• Stable form of fully reduced folate.
• Given orally or parenterally to overcome the toxic effects of methotrexate or other DHF
reductase inhibitors, e.g., trimethoprim or cotrimoxazole.
49. REVERSAL OF PRESENTING SYMPTOMS
The patient often begins to feel better within a day, before anemia improves.
New reticulocytes appear in 2 to 3 days.
Although neutrophil hyper-segmentation persists for 2 weeks or more, WBC and platelet count rise in
the 1st week if they had been decreased.
Peak reticulocytosis occurs at 7 to 10 days.
The blood count becomes fully normal before 8 weeks have elapsed.
Neurologic improvement becomes noticeable in the first few weeks.
50. MONITORING THE RESPONSE
• Homocysteine and MMA levels begin to fall within a few days of therapy and can be used to
monitor response.
• Monitoring vitamin levels, whether cobalamin, holo-TC II, or serum folate, has little value because
plasma levels rise passively regardless of therapeutic effectiveness.
• Reticulocyte response can be assessed best at the end of the first week, and completeness of
hematologic response after 8 weeks.
• The extent of neurologic improvement cannot always be predicted.
• The likelihood of full reversal with cobalamin is inversely related to the duration of symptoms
before therapy was started and the extent of the original dysfunction. Whatever reversal can be
expected is usually completed by 6 months.
