Absorption, transport and metabolism of cyanocobalamin

Absorption, transport and metabolism of cyanocobalamin

1. Absorption, transport and
metabolism of cyanocobalamin
Domina Petric, MD

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

3. Digestion
The naturally occurring vitamin B12 in foods is
boundin coenzyme form to proteins.
The vitamin is releasedfrom such complexes on
heating, gastric acidificationand/or proteolysis
(especially by the action of pepsin).
Impairedgastric parietal cell function, as in
achlorhydria, impairs vitamin B12 utilization.
April 23, 2018
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
3

4. Protein binding in the gut
April 23, 2018
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
4

5. R proteins
The binding of vitamin B12 to these
glycoproteins may be adventitious.
They arefound in human gastric
juice, intestinal contentsand several
other bodily fluids.
April 23, 2018
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
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6. R proteins
R proteinsshow structural and
immunologicsimilarities.
Their differenciesin
electrophoreticmobility aredue
to differing carbohydrate
contents.
April 23, 2018
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
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7. R proteins
•The R proteins in the intestine are not
necessary for the enteric absorption of
vitaminB12, as they are normally
digested proteolyticallyin the alkaline
conditions of the small intestine,
whereupon they release their ligands
to be boundby intrinsic factor (IF).
April 23, 2018
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
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8. R proteins
BecausevitaminB12 binds preferentially to R
proteins ratherthan to IF under the acidic
conditions of thestomach, R proteinscan
interfere with the absorptionof vitamin B12.
Patients with pancreatic exocrine
insufficiency, and consequent deficiencies of
proteolytic activities in the intestinal lumen,
canachieve high concentrations of R proteins
thatcause poor absorption of the vitamin.
April 23, 2018
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
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9. Intrinsic factor
The intrinsic factor (IF) issynthesized and
secretedby the gastric parietalcells in
response to histamine, gastrin,
pentagastrinand the presence of food.
Individuals with loss of gastric parietal cell
function maybe unable to use dietary
vitamin B12, as thesecells produce both IF
and acid, both of whichare required for the
enteric absorption of thevitamin.
April 23, 2018
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
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10. Intrinsic factor
Geriatricpatients,many
of whom are hypoacidic,
may be at riskof low
vitamin B12 status.
April 23, 2018
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
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11. 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 receptorin
the ilealmucosal brush border.
•Cobalaminbinding appears to have an allosteric
effect onthe ilealreceptor-binding center of IF,
causingthe protein complex to dimerize and
increasingits binding to the receptor.
April 23, 2018
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
11

12. Intrinsic factor
Formation of theIF-vitamin B12
complex protects the vitamin
from catabolism by intestinal
bacteria andprotects IF from
hydrolytic attack by pepsin
and chymotrypsin.
April 23, 2018
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
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13. April 23, 2018
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
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Mechanisms of absorption

14. Active transport
The carrier-mediatedabsorption of vitamin B12 is highly
efficientand quantitatively important at low doses(1-2 µg).
Such doses appear in the bloodwithin 3-4
hours of consumption.
The activetransport of vitamin B12 depends on the
interactions of the IF-vitamin B12 complexwith a specific
receptor in the microvilli of theileum.
April 23, 2018
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
14

15. Active transport
The ilealIF receptor, a
glycoprotein,binds the IF-
vitamin B12 complex, but
little,if any, free IF or free
vitamin B12.
April 23, 2018
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
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16. Active transport
Receptor binding occurs atneutral
pH and depends on Ca
2+
, in the
presence of which it forms a stable
IF-vitaminB12-IF receptor complex.
The receptor isanchored to the brush
border membraneand effects the enteric
absorption of vitaminB12 through the
endocytoticinternalizationof the
receptor-bound complex.
April 23, 2018
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
16

17. Active transport
•Theabsorption 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 theenterocyte, the vitamin is
bound to anintracellular protein that is
immunologicallysimilar to IF, and eventually
transferred to theportal circulation bound to a
specific carrierprotein, transcobalaminII (TCII).
April 23, 2018
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
17

18. Active transport
Human patients who
lack IF have very low
abilities to absorb
vitamin B12, excreting
inthe feces 80-100%
of oral doses (versus
the30-60% fecal
excretion rates of
individualswith
adequate IF).
April 23, 2018
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
18

19. Simple diffusion
Diffusion of the vitaminoccurs with low efficiency
(1%) throughoutthe small intestine and becomes
significant onlyat higher doses.
Such doses appear in the bloodwithin
minutes of consumption.
This passivemechanism is utilized in therapy for
perniciousanemia, in which patients are given high
doses(>500µg/day) of vitamin B12 per os.
The vitamin must be given an hourbefore
or after a meal.
April 23, 2018
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
19

20. Transport
II.
April 23, 2018
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
20

21. Transport proteins
On absorption from the intestine, vitamin
B12 is initially transported in the plasma,
most of which isbound as the
adenosylcobalaminand methylcobalaminto
an R protein called transcobalaminI (TCI).
Most of the remainder is bound to another
binding protein transcobalaminII (TCII)
synthesizedin several tissues, including the
intestinal mucosa,liver, seminal vesicles,
fibroblasts, bone marrowand macrophages.
April 23, 2018
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
21

22. TranscobalaminI (TCI)
•It is a 60-kDa α-glycoprotein.
•This is an R protein, also referredto as
haptocorrin.
•Vitamin B12 bound to thisprotein appears to
turn over very slowly (half-lifeis9-10 days),
becoming available for cellularuptake only over
fairly long time frames.
•TCI occurs at very high concentrationsin saliva,
breast milk, tears and othersecretions.
April 23, 2018
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
22

23. TranscobalaminII (TCII)
•It is smaller (38 kDa).
•TCII serves as the chief transport protein of the
vitamin, binding it stoichiometricallyin a 1:1 molar
ratio.
•Only10-25% of plasma vitamin B12is bound to this
transporter.
•The rapid turnover(half-lifeis90 min) of the
protein-ligand complexrenders TCII the only
functional source of thevitamin for cellular uptake.
April 23, 2018
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
23

24. TranscobalaminIII (TCIII)
It is electrophoreticallysimilar to
TCI,but antigenically similar to TCII.
Its metabolic role is less clear.
April 23, 2018
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
24

25. 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.
•TCIIis necessary for normal cellular maturation
of the hematopoietic system.
•Because cobalaminis lost within days from TCII, the
amount bound tothat protein can be a useful
parameter of early-stagevitamin B12 deficiency.
April 23, 2018
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
25

26. Transcobalamin receptor
•Membrane-bound receptor proteins for TCII occur
in all cells.
•The TCII receptor is structurally similar toTC.
•It is a 50-kDa glycoprotein with a single binding
site for the TCII-vitamin B12 complex.
•The binding isof high affinity and requires Ca
2+
.
•The cellular uptake of vitamin B12 might involve such
TCIIreceptors mediating the pinocytotic entrance of
thevitamin-TCII complex into the cell.
April 23, 2018
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
26

27. Role of R proteins
Mostrecently absorbed vitamin B12 istransferred to
the plasma R protein TCI, which binds approximately
three-quarters of the circulating vitamin B12.
Owing to the specificity of theTCI for
methylcobalamin,that vitamer
predominatesin the circulation of humans.
Congenital deficiency of this R protein resultsin low
concentrations of vitamin B12 in the plasma, but not in
detectable losses infunction, as cobalamins bound to TCI
do not appearto be available for cellular uptake.
April 23, 2018
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
27

28. 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-FH
4
methyltransferase) in the cytosol
●Methylmalonyl-CoA mutase in mitochondria
April 23, 2018
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
28

29. Distribution in tissues
•Vitamin B12 is the best stored of the
vitamins.
•Underconditions of non-limiting intake,
the vitamin accumulates to very
appreciable amounts in the body,
mainlyin the liver (about 60% of the
total body store) andmuscles (about
30% of the total).
April 23, 2018
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
29

30. Distribution in tissues
Totalhepatic reserve isabout 1,5 mg.
Meantotal body stores of vitamin
B12 are in therange of 2-5 mg.
Pituitarygland,kidneys, heart,spleen
and brain also contain substantial
amounts: 20-30µg of vitamin B12.
April 23, 2018
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
30

31. Distribution in tissues
The great storage and long biological half-life
(350-400 days) of the vitamin providesubstantial
protection against periods of deprivation.
The low reserve of the human infant (25µg)
is sufficient to meet physiological needs for
about a year.
April 23, 2018
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
31

32. Distribution in tissues
The predominant form in
plasma ismethylcobalamin
(60-80% of the total), owing
to thepresence of TCI that
selectively binds that vitamer.
April 23, 2018
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
32

33. Distribution in tissues
The vitamin B12 concentration ofhumanmilk
varies widely (130-320 pg/ml) and is particularly
great (10-fold that of mature milk) in colostrum.
Althoughthose products contain TCII, most of
the vitamin(mainly methylcobalamin) is bound
to R proteins.
April 23, 2018
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
33

34. Metabolism
III.
April 23, 2018
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
34

35. Activation to coenzyme forms
•Vitamin B12 is delivered to cells in the
oxidizedform, hydroxycob(III)alamin,
where it is reducedby thiol-and
reduced flavin-dependent reductionof
the cobalt center of the vitamin (to
Co
+
) to formcob(I)amin, also called
vitamin B
12s.
April 23, 2018
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
35

36. Activation to coenzyme forms
•Thevitamin is active in metabolism only as
derivativesthat 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 differentenzymatic steps.
April 23, 2018
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
36

37. Generation of methylcobalamin
This step is catalyzed by the cytosolic
enzyme 5-methyl-FH
4: 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
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
37

38. 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 entryof hydroxycobalamininto
the mitochondria andits subsequent reduction in
sequential, oneelectron steps involving NADH and NADPH
linked aquacobalaminreductases to yieldcob(II)alamin.
April 23, 2018
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
38

39. Catabolism
•VitaminB12 is excretedas the
intact cobalamin.
•Only the free cobalamins (not the
methylated or adenosylated
forms) in the plasma are available
for excretion.
April 23, 2018
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
39

40. Excretion
•Vitamin B12 is excreted via both renal and
biliaryroutes at the daily rate of about 0,1-
0,2% of totalbody reserves.
•Glomerularfiltration 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
B12status:this is called the Schilling test.
April 23, 2018
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
40

41. Excretion
•The biliaryexcretion of the vitamin is substantial,
accounting for the secretion into the intestineof
0,5-5 µg/day.
•Most (65-75%) of this amountis reabsorbed in
the ileum by IF-mediated activetransport.
•This enterohepatic circulation constitutesa highly
efficient means of conservation, with biliary
vitamin B12 contributing only a small amount
to the feces.
April 23, 2018
Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
41

42. Literature
•Combs GF. The Vitamins. Fundamental Aspects in
Nutrition and Health. Elsevier Inc. 2008.
April 23, 2018 42