Chapter 14 vitamin b12 (cobalamins)

There are two vitamin B12coenzymes with known metabolic activity in humans, namely methylcobalamin and adenosylcobalamin.. Vitamin B12deficiency is rarely, if ever, caused by a lack of di

There are two vitamin B12coenzymes 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 B12means 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 indistin-guishable 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 demyeliniza-tion 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 B12is stored in the liver Vitamin

B12deficiency is rarely, if ever, caused by a lack of dietary B12; rather it

is attributable to various disorders of absorption and transport Absorp-tion of vitamin B12is 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

275

© 2006 by Taylor & Francis Group, LLC

atrophies to such an extent that virtually no intrinsic factor (or hydro-chloric acid) is secreted In patients with diverticula, strictures, and fistu-las 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 auto-immune reaction with formation of antibodies against intrinsic factor Vitamin B12is nontoxic when taken orally

Physicochemical Properties

14.2.1 Structure and Potency

In accordance with the literature on nutrition and pharmacology, the term vitamin B12is 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 contain-ing a corrin rcontain-ing system substituted with numerous methyl, acetamide,

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-dimethyl-benzimidazole (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 pseudonucleo-tide (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 cyanocobala-min (C63H88O14N14PCo, MW¼ 1355.4) or, depending on the chemical environment, to some other group (e.g., 22OH in hydroxocobalamin and22HSO3in sulfitocobalamin)

There are two vitamin B12coenzymes with known metabolic activity in humans, namely, methylcobalamin and 50-deoxyadenosylcobalamin (frequently abbreviated to adenosylcobalamin and also known as coen-zyme B12) The methyl or adenosyl ligands of the coenzymes occupy the X position in the corrin structure The coenzymes are bound intra-cellularly to their protein apoenzymes through a covalent peptide link,

or in milk and plasma to specific transport proteins

(Figure 14.1)

14.2.2 Physicochemical Properties

14.2.2.1 Appearance and Solubility

Cyanocobalamin is an artificial product used in pharmaceutical prep-arations 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

N N

N N

R′ H 3 C

R′

R

CH2

R

R ′

CH 3

Co+

H 3 C X

R

CH 3

CH3

H 3 C

H 3 C

CH 3

CH2 CO NH

CH 2

CHCH 3

O P O

O –

O O

N N

CH3

CH 3

H

OH H H HOH 2 C H

A

C D

OH O

OH

–CH 2

N

N N N

5 ′-Deoxyadenosylcobalamin

NH2

R = CH2CONH2

R ′ = CH 2 CH2CONH2

Cyanocobalamin Hydroxocobalamin Aquocobalamin Methylcobalamin

–CN

–OH

–H 2 O

–CH 3

X group

(Coenzyme B 12 )

B

FIGURE 14.1

Structures of vitamin B 12 compounds.

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 cobal-amins 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 B12activity

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 Adenosyl-cobalamin and methylAdenosyl-cobalamin are reduced cob(I)alamin derivatives, which are easily oxidized by light to the hydroxo compound [3]

14.3.1 Occurrence

Naturally occurring vitamin B12originates solely from synthesis by bac-teria 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 B12is ubiquitous in foods of animal origin and is derived from the animal’s ingestion of cobalamin-containing animal tissue or micro-biologically contaminated plant material, in addition to vitamin absorbed from the animal’s own digestive tract The vitamin B12 contents of some the outstanding dietary source of the vitamin, followed by kidney and heart Muscle meats, fish, eggs, cheese, and milk are other important food foods in which the vitamin is found are listed inTable 14.1 [4] Liver is

sources Vitamin B12activity 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 B12metabolism

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 proces-sing 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

TABLE 14.1

Vitamin B12Content of Various Foods

Food

mg Vitamin B 12 per 100 g Edible Portion Cow milk, whole, pasteurized 0.9

Cheese, cheddar, average 2.4

Egg, chicken, whole, raw 2.5

Beef, trimmed lean, raw, average 2

Lamb, trimmed lean, raw, average 2

Pork, trimmed lean, raw, average 1

Chicken meat, raw Tr

Liver, lamb, fried 83

Kidney, lamb, fried 54

Cod, raw, fillets 1

Herring, grilled 15

Pilchards, canned in tomato sauce 13

Salmon, grilled 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.

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 B12have 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 B12are 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 B12at 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 manufac-ture of cottage cheese, about 82% of the original vitamin B12was 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 dres-sing (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

B12was 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 B12with the whey The added mold did not contain any detectable vitamin B12 After a ripening period of 5– 6 weeks, 45%

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 B12content

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 Cyano-cobalamin is a synthetic stable form of the vitamin and is used in fortifica-tion The potential vitamin B12activity of a food sample is represented by the total cobalamin content, regardless of the ligand attached The deter-mination of total vitamin B12may 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 B12activity 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

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 vitamin-containing 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 B12present

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

absorbed and transported in the same way; structurally similar but bio-logically 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 B12vitamers 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 cobal-amins leave the stomach and enter the duodenum bound to haptocorrin and accompanied by free intrinsic factor In the mildly alkaline environ-ment of the jejunum, pancreatic proteases, particularly trypsin, partially degrade both free haptocorrin and haptocorrin complexed with cobal-amins Intrinsic factor, which is resistant to proteolysis by pancreatic enzymes, then binds avidly to the released B12vitamers

The intrinsic factor– B12complex is carried down to the ileum where it binds avidly to specific receptors on the brush border of the ileal entero-cyte 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 – B12complex 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 B12is 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

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 B12is 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 B12excreted 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 rela-tively free of analogs

The binding of vitamin B12with specific plasma proteins (transcobala-mins I and II) prevents the vitamin molecule from being excreted in the urine as it passes through the kidney Only if the circulating B12exceeds 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

14.5.1 Efficiency of Absorption

The percentage of ingested vitamin B12that 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 B12is 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

absorption is 16% (range, 0 –34%) [22] When 100 mg or more of crystalline vitamin B12is 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

B12and whole body counting or stool counting techniques The mean per-centage absorption of the extrinsic vitamin B12label 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 B12was absorbed as efficiently as a comparable amount of crystalline cyanocobalamin administered orally in aqueous solution The relatively poor absorption of vitamin B12in eggs was attrib-uted 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 utiliz-ation by the rat The results showed that cellulose or pectin added to purified vitamin B12-deficient diets increased the fecal excretion of radio-active 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]