Chapter 14 vitamin b12 (cobalamins)

14
Vitamin B12 (Cobalamins)

14.1 Background
There are two vitamin B12 coenzymes with known metabolic activity in
humans, namely methylcobalamin and adenosylcobalamin.
Vitamin B12 deficiency has a drastic effect on folate metabolism
because methylcobalamin is a coenzyme for methionine synthetase, the
enzyme that catalyzes the methylation of homocysteine to methionine
using 5-methyl-tetrahydrofolate (5-methyl-THF) as the methyl donor.
The inability to synthesize methionine from homocysteine in the
absence of vitamin B12 means that THF cannot be regenerated from the
demethylation of 5-methyl-THF. The folate thus becomes trapped in
the form of 5-methyl-THF because the formation of this derivative by
reduction of 5,10-methylene-THF is thermodynamically irreversible. This
situation could lead to the inability to form the other THF derivatives
that are necessary for purine and pyrimidine synthesis. The consequent
lack of DNA synthesis causes many hemopoietic cells to die in the bone
marrow. In this event, a megaloblastic anemia that is clinically indistinguishable from that induced by folate deficiency results. When this type
of anemia is caused by deficiency of vitamin B12, it is called pernicious
anemia, because it is accompanied by a neuropathy which is unrelated to
folate deficiency. The neuropathy is caused by the inability to produce
the lipid component of myelin, which results in a generalized demyelinization of nerve tissue. Neuropathy begins in the peripheral nerves, affecting
first the feet and fingers, and then progressing to the spinal cord and
brain.
The body is extremely efficient at conserving vitamin B12. Unlike the
other water-soluble vitamins, vitamin B12 is stored in the liver. Vitamin
B12 deficiency is rarely, if ever, caused by a lack of dietary B12 ; rather it
is attributable to various disorders of absorption and transport. Absorption of vitamin B12 is facilitated by intrinsic factor, a protein secreted by
the parietal cells of the stomach lining. Elderly people are prone to
atrophic gastritis, a condition in which the gastric oxyntic mucosa

© 2006 by Taylor & Francis Group, LLC

275


Vitamin B12

276

atrophies to such an extent that virtually no intrinsic factor (or hydrochloric acid) is secreted. In patients with diverticula, strictures, and fistulas of the small intestine, stagnant regions of the lumen may become
contaminated with colonic bacteria. The bacteria can take up much of
the dietary vitamin B12 passing by, whether it be the free vitamin or
vitamin bound to intrinsic factor. A common inherited disorder is an autoimmune reaction with formation of antibodies against intrinsic factor.
Vitamin B12 is nontoxic when taken orally.

14.2 Chemical Structure, Biopotency, and
Physicochemical Properties
14.2.1

Structure and Potency

In accordance with the literature on nutrition and pharmacology, the term
vitamin B12 is used in this text as the generic descriptor for all cobalamins
that exhibit antipernicious anemia activity. Individual cobalamins will be
referred to by their specific names (e.g., cyanocobalamin).
The cobalamin molecule is a six-coordination cobalt complex containing a corrin ring system substituted with numerous methyl, acetamide,
and propionamide radicals (Figure 14.1). Methylene bridges link the
pyrrole rings A to B, B to C, and C to D but not A to D, which are
linked directly. The cobalt atom, which may assume an oxidation state
of (I), (II), or (III), is linked by four equatorial coordinate bonds to the

four pyrrole nitrogens, and by an axial coordinate bond to a 5,6-dimethylbenzimidazole (DMB) moiety, which extends in a-glycosidic linkage to
ribose-3-phosphate. The phosphate group is linked to the D ring of the
corrin structure via a substituted propionamide chain. The pseudonucleotide (DMB base plus sugar phosphate) is oriented perpendicularly to the
corrin structure. The remaining axial coordinate bond at the X position
links the cobalt atom to a cyano (22CN) group in the case of cyanocobalamin (C63H88O14N14PCo, MW ¼ 1355.4) or, depending on the chemical
environment, to some other group (e.g., 22OH in hydroxocobalamin
and 22HSO3 in sulfitocobalamin).
There are two vitamin B12 coenzymes with known metabolic activity in
humans, namely, methylcobalamin and 50 -deoxyadenosylcobalamin
(frequently abbreviated to adenosylcobalamin and also known as coenzyme B12). The methyl or adenosyl ligands of the coenzymes occupy
the X position in the corrin structure. The coenzymes are bound intracellularly to their protein apoenzymes through a covalent peptide link,
or in milk and plasma to specific transport proteins.
© 2006 by Taylor & Francis Group, LLC


Vitamins in Foods: Analysis, Bioavailability, and Stability
H3C H3C

R′

R

R
A

H3C
H3C

N X N

277

R = CH2CONH2
R′ = CH2CH2CONH2

R′

B

Co+
N

N

D

R

CH3

C

CH3
CH2 CH3 CH
3
CH2

R′

CO

NH
CH2

N

CH3

N

CH3

CHCH3
O–
O
P
O OH
O
H
H
HOH2C

H
O

H

Cyanocobalamin
Hydroxocobalamin
Aquocobalamin
Methylcobalamin

5′-Deoxyadenosylcobalamin
(Coenzyme B12)

X group
–CN
–OH
–H2O
–CH3

–CH2

OH OH

O
N
N

N
N
NH2

FIGURE 14.1
Structures of vitamin B12 compounds.

14.2.2
14.2.2.1

Physicochemical Properties
Appearance and Solubility

Cyanocobalamin is an artificial product used in pharmaceutical preparations because of its stability. It is a tasteless, dark red, crystalline
hygroscopic powder, which can take up appreciably more than the 12%
of moisture permitted by the British Pharmacopoeia. The anhydrous
material can be obtained by drying under reduced pressure at 1058C.
Cyanocobalamin is soluble in water (1.25 g/100 ml at 258C) and in lower
alcohols, phenol, and other hydroxylated solvents like ethylene diol; it is
© 2006 by Taylor & Francis Group, LLC


Vitamin B12

278

insoluble in acetone, chloroform, ether, and most other organic solvents.
Aqueous solutions of cyanocobalamin are of neutral pH.

14.2.2.2

Stability in Aqueous Solution

Cyanocob(III)alamin is the most stable of the vitamin B12-active cobalamins and is the one mostly used in pharmaceutical preparations and
food supplementation. Aqueous solutions of cyanocobalamin are stable
in air at room temperature if protected from light. The pH region of
optimal stability is 4.5– 5 and solutions of pH 4 –7 can be autoclaved at
1208C for 20 min with negligible loss of vitamin activity. The addition
of ammonium sulfate increases the stability of cyanocobalamin in aqueous
solution. Heating with dilute acid deactivates the vitamin owing to
hydrolysis of the amide substituents or further degradation of the
molecule. Mild alkaline hydrolysis at 1008C promotes cyclization of the

acetamide side chain at C-7 to form a biologically inactive g-lactam or, in
the presence of an oxidizing agent, a g-lactone [1]. The vitamin B12 activity
in aqueous solutions is destroyed in the presence of strong oxidizing agents
and high concentrations of reducing agents, such as ascorbic acid, sulfite,
and iron(II) salts. On exposure to light, the cyano group dissociates from
cyanocobalamin and hydroxocob(III)alamin is formed. In neutral and
acid solution, hydroxocobalamin exists in the form of aquocobalamin [2].
This photolytic reaction does not cause a loss of activity. Adenosylcobalamin and methylcobalamin are reduced cob(I)alamin derivatives,
which are easily oxidized by light to the hydroxo compound [3].

14.3 Vitamin B12 in Foods
14.3.1

Occurrence

Naturally occurring vitamin B12 originates solely from synthesis by bacteria and other microorganisms growing in soil or water, in sewage,
and in the rumen and intestinal tract of animals. Any traces of the
vitamin that may be detected in plants are due to microbial contamination
from the soil or manure or, in the case of certain legumes, to bacterial
synthesis in the root nodules.
Vitamin B12 is ubiquitous in foods of animal origin and is derived from
the animal’s ingestion of cobalamin-containing animal tissue or microbiologically contaminated plant material, in addition to vitamin absorbed
from the animal’s own digestive tract. The vitamin B12 contents of some
foods in which the vitamin is found are listed in Table 14.1 [4]. Liver is
the outstanding dietary source of the vitamin, followed by kidney and
heart. Muscle meats, fish, eggs, cheese, and milk are other important food
© 2006 by Taylor & Francis Group, LLC


Vitamins in Foods: Analysis, Bioavailability, and Stability

279

TABLE 14.1
Vitamin B12 Content of Various Foods
mg Vitamin B12 per 100 g
Edible Portion

Food
Cow milk, whole, pasteurized
Cheese, cheddar, average
Egg, chicken, whole, raw
Beef, trimmed lean, raw, average
Lamb, trimmed lean, raw, average
Pork, trimmed lean, raw, average
Chicken meat, raw
Liver, lamb, fried
Kidney, lamb, fried
Cod, raw, fillets
Herring, grilled
Pilchards, canned in tomato sauce
Salmon, grilled

0.9
2.4
2.5
2
2
1
Tr

83
54
1
15
13
5

Note: Tr, trace.
Source: From Food Standards Agency, McCance and Widdowson’s The Composition of Foods,
6th summary ed., Royal Society of Chemistry, Cambridge, 2002. With permission.

sources. Vitamin B12 activity has been reported in yeast, but this has since
been attributed to the presence of noncobalamin corrinoids or vitamin B12
originating from the enriching medium [5]. Spirulina, a type of seaweed,
is claimed to be a source of vitamin B12, but in fact is practically devoid of
the vitamin. The so-called vitamin B12 in spirulina is actually inactive
analogs, two of which have been shown to block vitamin B12 metabolism
in human cell cultures [5]. About 5–30% of the reported vitamin B12 in
foods may be microbiologically active noncobalamin corrinoids rather
than true B12 [6].
Vitamin B12 in foods exists in several forms. Meat and fish
contain mostly adenosyl- and hydroxocobalamins; these compounds,
accompanied by methylcobalamin, also occur in dairy products, with
hydroxocobalamin predominating in milk. Sulfitocobalamin is found in
canned meat and fish. Cyanocobalamin was not detected, apart from
small amounts in egg white, cheese, and boiled haddock [7]. In bovine
milk, naturally occurring vitamin B12 is bound to proteins, with a high
proportion being present in whey proteins [8].

14.3.2

Stability

The cobalamins present in food are generally resistant to thermal processing and cooking in a nonalkaline medium. There was no distinctive
destruction of vitamin B12 during sterilization and storage of canned
meals containing beef [9]. A 27– 33% loss of vitamin B12, expressed per
© 2006 by Taylor & Francis Group, LLC


280

Vitamin B12

unit of nitrogen, occurred during the cooking of beef due to the loss of
moisture and fat; the vitamin content of raw and cooked beef was,
however, similar on a moisture basis [10]. Thus there is little loss of the
vitamin in the cooking of meat provided that the meat juices are utilized.
Microwave heating of raw beef and pork and pasteurized cow’s milk
for 6 min resulted in an appreciable loss (ca. 30 – 40%) of vitamin B12
[11]. Degradation products of hydroxovitamin B12 formed in foods by
microwave heating had no vitamin activity and were nontoxic [12].
In the heat treatment of milk, the following losses of vitamin B12 have
been reported: boiling for 2– 5 min, 30%; pasteurization for 2– 3 sec, 7%;
sterilization in the bottle at 1208C for 13 min, 77%; rapid sterilization at
1438C for 3 – 4 sec with superheated steam, 10%; evaporation, 70– 90%;
and spray-drying, 20 –35% [13]. The light-sensitive coenzyme forms of
vitamin B12 are largely converted to hydroxocobalamin in light-exposed
milk, but with no loss of vitamin activity [7].
Arkba˚ge et al. [14] used a validated radio protein-binding assay to
evaluate the retention of vitamin B12 at key stages during the manufacture

of six different fermented dairy products and at subsequent ripening and
storage until “use-by” date. Two different heat treatments of milk (768C
for 16 sec and 968C for 5 min) caused no loss of vitamin B12. Milk after
heat treatment was therefore chosen as starting point for calculating the
retention of vitamin B12, and set to 100%. The addition of starter cultures
did not affect vitamin B12 concentrations in any of the fermented dairy
products tested. For the fermented milks, fermentation of heat-treated
milk resulted in vitamin B12 losses of 15% for Filmjo¨lk and 25% for
yoghurt. Storage of an unopened package of product at 48C for 14 days,
until “use-by date,” reduced the vitamin B12 concentrations further by
26 and 33% for Filmjo¨lk and yoghurt, respectively. Taken together,
Filmjo¨lk and yoghurt contained 40 –60% of the vitamin B12 originally
present in the milk, when consumed at use-by date. During the manufacture of cottage cheese, about 82% of the original vitamin B12 was removed
with the whey fraction, whereas 16% of the vitamin was retained in the
curd. A vitamin B12 addition corresponding to 6% of the starting milk
content was obtained in the final product by mixing the curd with dressing (made from skimmed milk, cream, and salt). The level of vitamin
B12 at packaging remained unaltered during storage of an unopened
package for 10 days, until use-by date. Thus, in total, the manufacture
and storage of cottage cheese retained 16% of the original vitamin B12
content. During hard cheese production, 44 –52% of the original vitamin
B12 was removed with the whey. In total, the hard cheeses that ripened
for up to 32 weeks retained about 60 –70% of the original vitamin B12
content. The manufacture of blue cheese incurred the removal of 38% of
the original vitamin B12 with the whey. The added mold did not contain
any detectable vitamin B12. After a ripening period of 5– 6 weeks, 45%
© 2006 by Taylor & Francis Group, LLC


Vitamins in Foods: Analysis, Bioavailability, and Stability

281

of the original vitamin B12 was still present in the blue cheese. The
additional 8 weeks of storage until use-by date did not seem to alter the
vitamin B12 content.
14.3.3

Applicability of Analytical Techniques

Vitamin B12 occurs intracellularly in the living tissues of animals in the
form of the two coenzymes, adenosylcobalamin and methylcobalamin,
which are covalently bound to their protein apoenzymes. In milk, these
coenzymes are bound noncovalently to specific transport proteins.
Hydroxocobalamin is present in animal-derived foods, especially in
milk, as a result of the photooxidation of the coenzyme forms. Cyanocobalamin is a synthetic stable form of the vitamin and is used in fortification. The potential vitamin B12 activity of a food sample is represented by
the total cobalamin content, regardless of the ligand attached. The determination of total vitamin B12 may be performed by microbiological assay
or by radioassay. Supplemental vitamin B12 (cyanocobalamin) may be
determined by a nonisotopic protein-binding assay. A biosensor-based
protein-binding assay has been developed for determining supplemental
and endogenous vitamin B12.
No international unit for vitamin B12 activity has been defined, and the
assay results are expressed in milligrams, micrograms, or nanograms of
pure crystalline cyanocobalamin. The measurement of biological activity
in preparations containing vitamin B12 relies on microbiological assays,
there being no animal bioassay.

14.4 Absorption and Conservation
Much of the following discussion of absorption and conservation is taken
from a book by Ball [15] published in 2004.
Humans appear to be entirely dependent on a dietary intake of vitamin

B12. Although microbial synthesis of the vitamin occurs in the human
colon, it is apparently not absorbed [16]. Strict vegetarians may obtain
limited amounts of vitamin B12 through ingestion of the vitamincontaining root nodules of certain legumes and from plant material
contaminated with microorganisms.
The absorption and transport of the vitamin B12 naturally present in
foods takes place by specialized mechanisms that accumulate the
vitamin and deliver it to cells that require it. The high efficiency of
these mechanisms enables 50 –90% of the minute amount of B12 present
in a typical omnivorous diet (ca. 10 mg) to be absorbed and delivered to
cells. The specificity is such that all natural forms of vitamin B12 are
© 2006 by Taylor & Francis Group, LLC


Vitamin B12

282

absorbed and transported in the same way; structurally similar but biologically inactive analogs, which are metabolically useless and possibly
harmful, bypass the transport mechanisms and are eliminated from the
body.

14.4.1

Digestion and Absorption of Dietary Vitamin B12

Ingested protein-bound cobalamins are released by the combined action
of hydrochloric acid and pepsin in the stomach. Gastric juice also contains
two functionally distinct cobalamin-binding proteins: (1) haptocorrin
(there are actually several haptocorrins, which are also known as R
binders or cobalophilin) and (2) intrinsic factor. Haptocorrin binds a

wide variety of cobalamin analogs in addition to vitamin B12, whereas
intrinsic factor binds B12 vitamers with high specificity and equal affinity.
The 5,6-dimethylbenzimidazole moiety is essential for recognition by
intrinsic factor [17]. The haptocorrin originates in saliva, while intrinsic
factor is synthesized and secreted directly into gastric juice by the parietal
cells of the stomach. At the acid pH of the stomach, cobalamins have a
greater affinity for haptocorrin than for intrinsic factor. Therefore cobalamins leave the stomach and enter the duodenum bound to haptocorrin
and accompanied by free intrinsic factor. In the mildly alkaline environment of the jejunum, pancreatic proteases, particularly trypsin, partially
degrade both free haptocorrin and haptocorrin complexed with cobalamins. Intrinsic factor, which is resistant to proteolysis by pancreatic
enzymes, then binds avidly to the released B12 vitamers.
The intrinsic factor – B12 complex is carried down to the ileum where it
binds avidly to specific receptors on the brush border of the ileal enterocyte. The presence of calcium ions and a pH above 5.5 are necessary to
induce the appropriate configuration of the receptor for binding [18].
The intrinsic factor – B12 complex is absorbed intact [19], but the precise
mechanism of absorption and subsequent events within the enterocyte
have yet to be elucidated. It is possible that ileal absorption of the intrinsic
factor – B12 complex is accomplished by receptor-mediated endocytosis
[20], but clathrin-coated pits or vesicles have not been found. The B12 is
subsequently released at an intracellular site, possibly in either lysosomes
or prelysosomal vesicles, both of which are acidic [21]. The intrinsic factor
appears to be degraded by proteolysis after releasing its bound B12,
there being no apparent recycling of intrinsic factor to the brush-border
membrane.
The intrinsic factor-mediated system is capable of handling between 1.5
and 3.0 mg of vitamin B12. The limited capacity of the ileum to absorb B12
can be explained by the limited number of receptor sites, there being only
about one receptor per microvillus. Saturation of the system at one meal
© 2006 by Taylor & Francis Group, LLC

Vitamins in Foods: Analysis, Bioavailability, and Stability

283

does not preclude absorption of normal amounts of the vitamin some
hours later. The entire absorptive process, from ingestion of the vitamin
to its appearance in the portal vein, takes 8 –12 h.
Absorption can also occur by simple diffusion across the entire
small intestine. This process probably accounts for the absorption of
only 1– 3% of the vitamin consumed in ordinary diets, but can provide
a physiologically significant source of the vitamin when it is administered
as free cobalamin in pharmacological doses of 30 mg or more.

14.4.2

Conservation of Vitamin B12

The body is extremely efficient at conserving vitamin B12. Unlike the other
water-soluble vitamins, vitamin B12 is stored in the liver, primarily in the
form of adenosylcobalamin. In an adult man, the total body store of
vitamin B12 is estimated to be 2000 – 5000 mg, of which about 80% is in
the liver. The remainder of the stored vitamin is located in muscle, skin,
and blood plasma. Only 2– 5 mg of vitamin B12 are lost daily through
metabolic turnover, regardless of the amount stored in the body.
Vitamin B12 excreted in the bile is reabsorbed in the ileum along with
dietary sources of the vitamin. This enteroheptic cycle allows the
excretion of unwanted nonvitamin cobalamin analogs, which constitute
about 60% of the corrinoids secreted in bile, and returns vitamin B12 relatively free of analogs.
The binding of vitamin B12 with specific plasma proteins (transcobalamins I and II) prevents the vitamin molecule from being excreted in the
urine as it passes through the kidney. Only if the circulating B12 exceeds

the vitamin-binding capacity of the blood is the excess excreted; this
typically occurs only after injection of cobalamin. The binding with
plasma proteins, negligible urinary loss, and an efficient enterohepatic
circulation, together with the slow rate of turnover, explains why strict
vegetarians normally take 20 yr or more to develop signs of deficiency.
People with absorptive malfunction develop deficiency signs within
2 –3 yr.

14.5 Bioavailability
14.5.1

Efficiency of Absorption

The percentage of ingested vitamin B12 that is absorbed decreases as the
actual amount in the diet increases. At intakes of 0.5 mg or less, ca. 70%
of the available vitamin B12 is absorbed. At an intake of 5.0 mg, a mean
of 28% is absorbed (range, 2 –50%) while at a 10-mg intake the mean
© 2006 by Taylor & Francis Group, LLC


Vitamin B12

284

absorption is 16% (range, 0 –34%) [22]. When 100 mg or more of crystalline
vitamin B12 is taken, the absorption efficiency drops to 1%, and the excess
vitamin is excreted in the urine.
The efficiency of vitamin B12 absorption from a variety of foods has
been determined in human subjects using extrinsically labeled vitamin
B12 and whole body counting or stool counting techniques. The mean percentage absorption of the extrinsic vitamin B12 label was as follows: lean

mutton, 65% [23]; chicken, 60% [24]; fish, 39% [25]; eggs, 24 –36% [26];
milk, 65% [27]; and fortified bread, 55% [27]. In all these foods, with the
exception of eggs, vitamin B12 was absorbed as efficiently as a comparable
amount of crystalline cyanocobalamin administered orally in aqueous
solution. The relatively poor absorption of vitamin B12 in eggs was attributed to the presence of distinct vitamin B12-binding proteins in egg white
and egg yolk [28].

14.5.2

Bioavailability Studies

14.5.2.1 Effects of Dietary Fiber
Vitamin B12 depletion occurs more rapidly in the presence than in the
absence of intestinal microorganisms, presumably due to competition
for available B12 between the gut flora and the host. Cullen and Oace
[29] investigated the possibility that dietary fiber, by stimulating the
growth of intestinal bacteria, might increase the rate of vitamin B12 utilization by the rat. The results showed that cellulose or pectin added to
purified vitamin B12-deficient diets increased the fecal excretion of radioactive B12 that was injected after several weeks of depletion. Thus,
both cellulose and pectin may have bound or adsorbed biliary B12 and
carried it past the ileal absorptive sites. In addition, pectin, which is
hydrolyzed to an appreciable extent by intestinal microorganisms,
might have served as a substrate for the growth of vitamin B12-requiring
bacteria.
There was no significant difference in the urinary excretion of vitamin
B12 in human subjects receiving controlled diets supplemented with or
without wheat bran [30].

14.5.2.2 Effects of Alcohol
Although alcohol ingestion has been shown to decrease vitamin B12
absorption in volunteers after several weeks of intake, alcoholics do

not commonly suffer from vitamin B12 deficiency, probably because
of the large body stores of the vitamin and the reserve capacity for
absorption [31].
© 2006 by Taylor & Francis Group, LLC


Vitamins in Foods: Analysis, Bioavailability, and Stability
14.5.2.3

285

Effects of Smoking

Several constituents of cigarette smoke react with vitamin B12 and convert
it to biologically inactive forms. For example, cyanides and hydrogen
sulfide have a high affinity for the vitamin’s cobalt atom and form cyanocobalamin and sulfocobalamin, respectively. Nitrous oxide inactivates
methylcobalamin through oxidation of the cobalt atom [32]. Cigarette
smokers, but not nonsmokers, showed a high urinary thiocyanate
excretion, which was associated with increased vitamin B12 excretion
and a relatively low serum B12 concentration [33]. Because urinary
thiocyanate excretion is an index of cyanide detoxication, it appears possible that a high plasma cyanide concentration caused through smoking
disturbs the equilibrium between plasma and urinary vitamin B12.
Piyathilake et al. [32] reported that vitamin B12 concentrations in
buccal mucosal cells of smokers were significantly lower than in cells of
nonsmokers. Salivary vitamin B12 was higher in smokers, possibly
because of leakage of cyanocobalamin from the smoke-exposed buccal
mucosal cells.

References
1. Wong, D.W.S., Mechanism and Theory in Food Chemistry, Van Nostrand

Reinhold, New York, 1989, p. 335.
2. Gra¨sbeck, R. and Salonen, E.-M., Vitamin B-12, Prog. Food Nutr. Sci., 2, 193,
1976.
3. Hoffbrand, A.V., Nutritional and biochemical aspects of vitamin B12, in The
Importance of Vitamins to Human Health, Taylor, T.G., Ed., MTP Press Ltd.,
Lancaster, U.K., 1979, p. 41.
4. Food Standards Agency, McCance and Widdowson’s The Composition of Foods,
6th summary ed., Royal Society of Chemistry, Cambridge, 2002.
5. Herbert, V., Vitamin B-12: plant sources, requirements, and assays, Am. J. Clin.
Nutr., 48, 852, 1988.
6. National Research Council, Recommended Dietary Allowances, 10th ed.,
National Academy Press, Washington, DC, 1989, p. 115.
7. Farquharson, J.N. and Adams, J.F., The forms of vitamin B-12 in foods,
Br. J. Nutr., 36, 127, 1976.
8. Hartman, A.M. and Dryden, L.P., Vitamins in milk and milk products, in
Fundamentals of Dairy Chemistry, 2nd ed., Webb, B.H., Johnson, A.H., and
Alford, J.A., Eds., AVI Publishing Co., Inc., Westport, CT, 1974, p. 325.
9. Helendoorn, E.W., de Groot, A.P., van der Mijlldekker, L.P., Slump, P., and
Willems, J.J.L., Nutritive value of canned meals, J. Am. Dietetic Assoc., 58,
434, 1971.
10. Bennink, M.R. and Ono, K., Vitamin B12, E and D content of raw and cooked
beef, J. Food Sci., 47, 1786, 1982.
© 2006 by Taylor & Francis Group, LLC


286

Vitamin B12

11. Watanabe, F., Abe, K., Fujita, T., Goto, M., Hiemori, M., and Nakano, Y., Effects

of microwave heating on the loss of vitamin B12 in foods, J. Agric. Food Chem.,
46, 206, 1998.
12. Watanabe, F., Abe, K., Katsura, H., Takenaka, S., Zakir Hussain Mazumder,
S.A.M., Yamaji, R., Ebara, S., Fujita, T., Tanimori, S., Kirihata, M., and
Nakano, Y., Biological activity of hydroxo-vitamin B12 degradation product
formed during microwave heating, J. Agric. Food Chem., 46, 5177, 1998.
13. Archer, M.C. and Tannenbaum, S.R., Vitamins, in Nutritional and Safety Aspects
of Food Processing, Tannenbaum, S.R., Ed., Marcel Dekker, New York, 1979,
p. 47.
14. Arkba˚ge, K., Wittho¨ft, C., Fonde´n, R., and Ja¨gerstad, M., Retention of vitamin
B12 during manufacture of six fermented dairy products using a validated
radio protein-binding assay, Int. Dairy J., 13, 101, 2003.
15. Ball, G.F.M., Vitamins: Their Role in the Human Body, Blackwell Publishing Ltd.,
Oxford, 2004, p. 383.
16. Shinton, N.K., Vitamin B-12 and folate metabolism, Br. Med. J., 1, 556, 1972.
17. Seetharam, B. and Alpers, D.H., Gastric intrinsic factor and cobalamin absorption, Handbook of Physiology, Section 6: The Gastrointestinal System, Vol. IV, Intestinal Absorption and Secretion, Field, M. and Frizzell, R.A., Eds., American
Physiology Society, Bethesda, 1991, p. 437.
18. Donaldson, R.M., Intrinsic factor and the transport of cobalamin, in Physiology
of the Gastrointestinal Tract, 2nd ed., Johnson, L.R., Ed., Raven Press, New York,
1987, p. 959.
19. Seetharam, B., Presti, M., Frank, B., Tiruppathi, C., and Alpers, D.H., Intestinal
uptake and release of cobalamin complexed with rat intrinsic factor, Am.
J. Physiol., 248, G326, 1985.
20. Donaldson, R.M., How does cobalamin (vitamin B12) enter and traverse the
ileal cell? Gastroenterology, 88, 1069, 1985.
21. Seetharam, B., Gastrointestinal absorption and transport of cobalamin
(vitamin B12), in Physiology of the Gastrointestinal Tract, 3rd ed., Johnson, L.R.,
Ed., Raven Press, New York, 1994, p. 1997.
22. Herbert, V., Recommended dietary intakes (RDI) of vitamin B-12 in humans,
Am. J. Clin. Nutr., 45, 671, 1987.

23. Heyssel, R.M., Bozian, R.C., Darby, W.J., and Bell, M.C., Vitamin B12 turnover
in man: the assimilation of vitamin B12 from natural foodstuffs by man and
estimates of minimal daily dietary requirements, Am. J. Clin. Nutr., 18, 176,
1966.
24. Doscherholmen, A., McMahon, J., and Ripley, D., Vitamin B12 assimilation
from chicken meat, Am. J. Clin. Nutr., 31, 825, 1978.
25. Doscherholmen, A., McMahon, J., and Economon, P., Vitamin B12 absorption
from fish, Proc. Soc. Exp. Biol. Med., 167, 480, 1981.
26. Doscherholmen, A., McMahon, J., and Ripley, D., Vitamin B12 absorption from
eggs, Proc. Soc. Exp. Biol. Med., 149, 987, 1975.
27. Russell, R.M., Baik, H., and Kehayias, J.J., Older men and women efficiently
absorb vitamin B-12 from milk and fortified bread, J. Nutr., 131, 291, 2001.
28. Levine, A.S. and Doscherholmen, A., Vitamin B12 bioavailability from egg yolk
and egg white: relationship to binding proteins, Am. J. Clin. Nutr., 38, 436, 1983.

© 2006 by Taylor & Francis Group, LLC


Vitamins in Foods: Analysis, Bioavailability, and Stability

287

29. Cullen, R.W. and Oace, S.M., Methylmalonic acid and vitamin B12 excretion of
rats consuming diets varying in cellulose and pectin, J. Nutr., 108, 640, 1978.
30. Lewis, N.M., Kies, C., and Fox, H.M., Vitamin B12 status of humans as affected
by wheat bran supplements, Nutr. Rep. Int., 34, 495, 1986.
31. Lieber, C.S., The influence of alcohol on nutritional status, Nutr. Rev., 46, 241,
1988.
32. Piyathilake, C.J., Macaluso, M., Hine, R.J., Richards, E.W., and Krumdieck, C.L.,
Local and systemic effects of cigarette smoking on folate and vitamin B-12,

Am. J. Clin. Nutr., 60, 559, 1994.
33. Linnell, J.C., Smith, A.D.M., Smith, C.L., Wilson, J., and Matthews, D.M.,
Effects of smoking on metabolism and excretion of vitamin B12, Br. Med. J.,
II, 215, 1968.

© 2006 by Taylor & Francis Group, LLC