Absorption, transport and metabolism of cyanocobalamin

Absorption, transport and
metabolism of cyanocobalamin
Domina Petric, MD

Absorption
I.
April 23, 2018
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
2

Digestion
The naturally occurring vitamin B12 in foods is
bound in coenzyme form to proteins.
The vitamin is released from such complexes on
heating, gastric acidification and/or proteolysis
(especially by the action of pepsin).
Impaired gastric parietal cell function, as in
achlorhydria, impairs vitamin B12 utilization.
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3

Protein binding in the gut
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4

R proteins
The binding of vitamin B12 to these
glycoproteins may be adventitious.
They are found in human gastric
juice, intestinal contents and several
other bodily fluids.
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5

R proteins
R proteins show structural and
immunologic similarities.
Their differencies in
electrophoretic mobility are due
to differing carbohydrate
contents.
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R proteins
• The R proteins in the intestine are not
necessary for the enteric absorption of
vitamin B12, as they are normally
digested proteolytically in the alkaline
conditions of the small intestine,
whereupon they release their ligands
to be bound by intrinsic factor (IF).
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R proteins
Because vitamin B12 binds preferentially to R
proteins rather than to IF under the acidic
conditions of the stomach, R proteins can
interfere with the absorption of vitamin B12.
Patients with pancreatic exocrine
insufficiency, and consequent deficiencies of
proteolytic activities in the intestinal lumen,
can achieve high concentrations of R proteins
that cause poor absorption of the vitamin.
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8

Intrinsic factor
The intrinsic factor (IF) is synthesized and
secreted by the gastric parietal cells in
response to histamine, gastrin,
pentagastrin and the presence of food.
Individuals with loss of gastric parietal cell
function may be unable to use dietary
vitamin B12, as these cells produce both IF
and acid, both of which are required for the
enteric absorption of the vitamin.
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Intrinsic factor
Geriatric patients, many
of whom are hypoacidic,
may be at risk of low
vitamin B12 status.
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Intrinsic factor
• IF binds the four cobalamins with comparable,
high affinities under alkaline conditions:
methylcobalamin, adenosylcobalamin,
cyanocobalamin and aquocobalamin.
• Intrinsic factor also binds a specific receptor in
the ileal mucosal brush border.
• Cobalamin binding appears to have an allosteric
effect on the ileal receptor-binding center of IF,
causing the protein complex to dimerize and
increasing its binding to the receptor.
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11

Intrinsic factor
Formation of the IF-vitamin B12
complex protects the vitamin
from catabolism by intestinal
bacteria and protects IF from
hydrolytic attack by pepsin
and chymotrypsin.
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12

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13
Mechanisms of absorption

Active transport
The carrier-mediated absorption of vitamin B12 is highly
efficient and quantitatively important at low doses (1-2 µg).
Such doses appear in the blood within 3-4
hours of consumption.
The active transport of vitamin B12 depends on the
interactions of the IF-vitamin B12 complex with a specific
receptor in the microvilli of the ileum.
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14

Active transport
The ileal IF receptor, a
glycoprotein, binds the IF-
vitamin B12 complex, but
little, if any, free IF or free
vitamin B12.
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15

Active transport
Receptor binding occurs at neutral
pH and depends on Ca2+, in the
presence of which it forms a stable
IF-vitamin B12-IF receptor complex.
The receptor is anchored to the brush
border membrane and effects the enteric
absorption of vitamin B12 through the
endocytotic internalization of the
receptor-bound complex.
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16

Active transport
• The absorption of vitamin B12 by the enterocyte
involves the cellular uptake of the dissociated
vitamin, with the release of the unbound
IF to the intestinal lumen.
• Upon entering the enterocyte, the vitamin is
bound to an intracellular protein that is
immunologically similar to IF, and eventually
transferred to the portal circulation bound to a
specific carrier protein, transcobalamin II (TCII).
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Active transport
Human patients who
lack IF have very low
abilities to absorb
vitamin B12, excreting
in the feces 80-100%
of oral doses (versus
the 30-60% fecal
excretion rates of
individuals with
adequate IF).
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Simple diffusion
Diffusion of the vitamin occurs with low efficiency
(1%) throughout the small intestine and becomes
significant only at higher doses.
Such doses appear in the blood within
minutes of consumption.
This passive mechanism is utilized in therapy for
pernicious anemia, in which patients are given high
doses (>500µg/day) of vitamin B12 per os.
The vitamin must be given an hour before
or after a meal.
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Transport
II.
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20

Transport proteins
On absorption from the intestine, vitamin
B12 is initially transported in the plasma,
most of which is bound as the
adenosylcobalamin and methylcobalamin to
an R protein called transcobalamin I (TCI).
Most of the remainder is bound to another
binding protein transcobalamin II (TCII)
synthesized in several tissues, including the
intestinal mucosa, liver, seminal vesicles,
fibroblasts, bone marrow and macrophages.
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21

Transcobalamin I (TCI)
• It is a 60-kDa α-glycoprotein.
• This is an R protein, also referred to as
haptocorrin.
• Vitamin B12 bound to this protein appears to
turn over very slowly (half-life is 9-10 days),
becoming available for cellular uptake only over
fairly long time frames.
• TCI occurs at very high concentrations in saliva,
breast milk, tears and other secretions.
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Transcobalamin II (TCII)
• It is smaller (38 kDa).
• TCII serves as the chief transport protein of the
vitamin, binding it stoichiometrically in a 1:1 molar
ratio.
• Only 10-25% of plasma vitamin B12 is bound to this
transporter.
• The rapid turnover (half-life is 90 min) of the
protein-ligand complex renders TCII the only
functional source of the vitamin for cellular uptake.
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23

Transcobalamin III (TCIII)
It is electrophoretically similar to
TCI, but antigenically similar to TCII.
Its metabolic role is less clear.
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24

Transport proteins
• The movement of vitamin B12 from the intestinal
mucosal cells into the plasma appears to depend
on the formation of the TCII-vitamin B12 complex
(the vitamin is shuttled from IF to TCII).
• This complex turns over rapidly: half-life is about 6 min.
• TCII is necessary for normal cellular maturation
of the hematopoietic system.
• Because cobalamin is lost within days from TCII, the
amount bound to that protein can be a useful
parameter of early-stage vitamin B12 deficiency.
April 23, 2018
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Transcobalamin receptor
• Membrane-bound receptor proteins for TCII occur
in all cells.
• The TCII receptor is structurally similar to TC.
• It is a 50-kDa glycoprotein with a single binding
site for the TCII-vitamin B12 complex.
• The binding is of high affinity and requires Ca2+.
• The cellular uptake of vitamin B12 might involve such
TCII receptors mediating the pinocytotic entrance of
the vitamin-TCII complex into the cell.
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Role of R proteins
Most recently absorbed vitamin B12 is transferred to
the plasma R protein TCI, which binds approximately
three-quarters of the circulating vitamin B12.
Owing to the specificity of the TCI for
methylcobalamin, that vitamer
predominates in the circulation of humans.
Congenital deficiency of this R protein results in low
concentrations of vitamin B12 in the plasma, but not in
detectable losses in function, as cobalamins bound to TCI
do not appear to be available for cellular uptake.
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Intracellular protein binding
• After its cellular uptake, the TCII-receptor
complex is degraded in the lysosome to yield the
free vitamin, which can be converted to
methylcobalamin in the cytosol.
Virtually all of the vitamin within the cell is bound
to two vitamin B12-dependent enzymes:
● Methionine synthetase (also called methyl-FH4
methyltransferase) in the cytosol
● Methylmalonyl-CoA mutase in mitochondria
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Distribution in tissues
• Vitamin B12 is the best stored of the
vitamins.
• Under conditions of non-limiting intake,
the vitamin accumulates to very
appreciable amounts in the body,
mainly in the liver (about 60% of the
total body store) and muscles (about
30% of the total).
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Total hepatic reserve is about 1,5 mg.
Mean total body stores of vitamin
B12 are in the range of 2-5 mg.
Pituitary gland, kidneys, heart, spleen
and brain also contain substantial
amounts: 20-30 µg of vitamin B12.
April 23, 2018
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The great storage and long biological half-life
(350-400 days) of the vitamin provide substantial
protection against periods of deprivation.
The low reserve of the human infant (25 µg)
is sufficient to meet physiological needs for
about a year.
April 23, 2018
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The predominant form in
plasma is methylcobalamin
(60-80% of the total), owing
to the presence of TCI that
selectively binds that vitamer.
April 23, 2018
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The vitamin B12 concentration of human milk
varies widely (130-320 pg/ml) and is particularly
great (10-fold that of mature milk) in colostrum.
Although those products contain TCII, most of
the vitamin (mainly methylcobalamin) is bound
to R proteins.
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Metabolism
III.
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34

Activation to coenzyme forms
• Vitamin B12 is delivered to cells in the
oxidized form, hydroxycob(III)alamin,
where it is reduced by thiol- and
reduced flavin-dependent reduction of
the cobalt center of the vitamin (to
Co+) to form cob(I)amin, also called
vitamin B12s.
April 23, 2018
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Activation to coenzyme forms
• The vitamin is active in metabolism only as
derivatives that have either a methyl group
(methylcobalamin) or 5′-deoxyadenosyl
group (adenosylcobalamin) attached
covalently to the cobalt atom.
• The conversion to these coenzyme forms
involves two different enzymatic steps.
April 23, 2018
36

Generation of methylcobalamin
This step is catalyzed by the cytosolic
enzyme 5-methyl-FH4: homocysteine
methyltransferase.
By producing methylcobalamin, it
renders the vitamin a carrier for the
single carbon unit in the regeneration
of methionine from homocysteine.
April 23, 2018
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Generation of adenosylcobalamin
The adenosylation of the vitamin occurs in the
mitochondria due to the action of vitamin B12 coenzyme
synthetase, which catalyzes the reaction of cob(II)amin
with a deoxyadenosyl moiety derived from ATP.
This step depends on the entry of hydroxycobalamin into
the mitochondria and its subsequent reduction in
sequential, one electron steps involving NADH and NADPH
linked aquacobalamin reductases to yield cob(II)alamin.
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Catabolism
• Vitamin B12 is excreted as the
intact cobalamin.
• Only the free cobalamins (not the
methylated or adenosylated
forms) in the plasma are available
for excretion.
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Excretion
• Vitamin B12 is excreted via both renal and
biliary routes at the daily rate of about 0,1-
0,2% of total body reserves.
• Glomerular filtration of the vitamin is
minimal: <0,25 µg/day.
• Urinary excretion of the vitamin after a
small oral dose can be used to assess vitamin
B12 status: this is called the Schilling test.
April 23, 2018
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Excretion
• The biliary excretion of the vitamin is substantial,
accounting for the secretion into the intestine of
0,5-5 µg/day.
• Most (65-75%) of this amount is reabsorbed in
the ileum by IF-mediated active transport.
• This enterohepatic circulation constitutes a highly
efficient means of conservation, with biliary
vitamin B12 contributing only a small amount
to the feces.
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41

Literature
• Combs GF. The Vitamins. Fundamental Aspects in
April 23, 2018 42

Absorption, transport and metabolism of cyanocobalamin

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