Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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internet by ProMED-mail, the Program for Moni-

toring Emerging Diseases. ProMED-mail is a pro-

gram of the International Society for Infectious

Diseases (ISID) and acts as a global electronic

reporting system for outbreaks of emerging infec-

tious diseases. These outbreaks are all unconfirmed

and the only source for these outbreaks is media

reports.

0016 Two large unconfirmed botulism outbreaks have

been reported in the north African countries Algeria

and Morocco. Algerian state-run radio was cited as

reporting that an outbreak of botulism killed 17

people in July 1988 in eastern Algeria. One of the

foods suspected in this outbreak was a processed

meat known as kashir. Another unconfirmed out-

break was reported by Associated Press, stating that

mortadella sausage was responsible for a suspected

botulism outbreak in Morocco during September

1999 with at least 10 deaths.

0017 Reuters news agency had reported an unconfirmed

botulism outbreak involving 90 cases with four

deaths from consumption of fish at a wedding recep-

tion in the former Soviet republic of Azerbaijan. The

Russian news agency Itar-Tass has reported an out-

break of botulism in southern Siberian Buryat, with

48 cases and four deaths after consumption of fish

from Lake Baikal. More than 70 cases of botulism,

including eight deaths, had been reported in Buryatia

in 1999.

Conclusions Regarding Epidemiology

0018 Most botulism outbreaks have occurred in the north-

ern hemisphere, particularly in countries north of the

Tropic of Cancer. Argentina is the only country in the

southern hemisphere which has reported several out-

breaks. Seasonal trends in outbreak frequencies are

apparent in some areas. In Canada, Alaska, and

Poland most outbreaks occur from May until Octo-

ber, whereas in China most outbreaks occur in the

late winter and spring. There is a strong association

between the prevalent outbreak type and the

prevalent environmental type, as well as the impli-

cated food. In colder regions, Canada, Alaska,

Greenland, Scandinavia, parts of Russia, Iran, and

northern Japan, type E causes most botulism out-

breaks and is the prevalent environmental type.

The implicated foods are usually fish or marine

mammals. In central Europe, type B causes most out-

breaks and is the prevalent environmental type.

Meats, particularly home-cured smoked ham, are

the major cause of botulism. In the western USA,

Argentina, and China, type A causes the majority of

outbreaks and is the prevalent environmental isolate.

In these areas, the most frequently implicated foods

are vegetables.

Clinical Aspects

Symptoms

0019The disease may vary from a mild illness which may

be overlooked or misdiagnosed to a serious disease

which may be fatal within 24 h. The onset of symp-

toms typically occurs 12–36 h after ingestion of toxin,

with a range from a few hours to 14 days. Usually,

the earlier symptoms appear, the more serious the

disease. The first symptoms are generally nausea

and vomiting. Mainly neurological signs and symp-

toms appear next, including visual impairments

(blurred or double vision, ptosis, fixed and dilated

pupils), loss of normal mouth and throat functions

(difficulty in speaking and swallowing, dry mouth,

throat and tongue, a sore throat), general fatigue

and lack of muscle coordination, and respiratory im-

pairment. Other gastrointestinal symptoms include

abdominal pain, diarrhea, and constipation. Diarrhea

occurs relatively early in the course of the disease,

whereas constipation persists in the advanced stages.

Respiratory failure and airway obstruction are the

main causes of death. Fatality rates in the first half

of the century were about 60%, but with the avail-

ability today of antisera and modern respiratory sup-

port systems, they have decreased to about 10%.

Treatment

0020Initially, treatment of botulism is directed toward

removing or inactivating the toxin by (1) neutralizing

circulating toxin with antiserum; (2) enema or treat-

ment with cathartics to remove residual toxin from

the bowel; and (3) in the absence of vomiting, gastric

lavage or treatment with emetics. Induced emesis

or gastric lavage is recommended if exposure has

occurred within several hours. Treatment with anti-

serum is most effective in the early stages of the

illness. The impact of antiserum is obvious from the

Chinese data; before the availability of antisera in

1960, the death rate in China was approximately

50%, but it was only 8% in the nearly 4000 patients

who received antitoxin. Subsequent treatment is

mainly to counteract the paralysis of the respiratory

muscles by artificial ventilation.

Diagnosis

0021The initial diagnosis of foodborne botulism is based

on the patient’s signs and symptoms, and perhaps

food history. It must be confirmed by detecting

toxin or viable C. botulinum in a suspect food or

clinical sample, or by epidemiological association

with a laboratory-confirmed case. Serum, feces,

enema fluid, stomach contents, and autopsy sections

of the small and large intestines, and of the liver, are

1416

CLOSTRIDIUM

/Botulism

suitable specimens for neurotoxin detection. Except

for serum, these specimens are also suitable for

detecting viable C. botulinum. The methods for

detecting the neurotoxin and C. botulinum are given

in the previous article Clostridium: Occurrence of

Clostridium botulinum. Other than neutralizing any

acidic samples, little special treatment is required.

Occasionally, extracts prepared from feces cannot be

filter-sterilized. In that case, tetracycline should be

added to 200 p.p.m. to control infection of the mouse.

Case History

0022 One of the most severe, nonfatal cases of botulism

was documented in a patient in the UK. Within ap-

proximately 10 h of eating the suspect food, he had

blurred and double vision followed by nausea and

vomiting, difficulty in swallowing and talking, dry-

ness of the mouth, and arm weakness. He suffered a

respiratory arrest on arrival at the hospital and was

intubated. A progressive flaccid paralysis ensued. He

was given polyvalent antitoxin, a tracheostomy was

done and total parenteral nutrition (TPN) was

started. There was little change in his condition for

100 days. His ptosis did not resolve until 46 days after

onset of symptoms. TPN was stopped on day 158,

and respiratory support was withdrawn after 173

days. He was not discharged until after 237 days.

Prevention

0023 Usually, the preservation of high-moisture foods is

geared toward control of C. botulinum, which usually

involves inhibition rather than destruction. Such con-

trol generally also insures control of other foodborne

pathogens and of many spoilage microorganisms. The

effects of different factors on the growth of C. botuli-

num in foods are described in the previous entry Clos-

tridium: Occurrence of Clostridium botulinum.

Control of C. botulinum in commercial foods is gener-

ally achieved by one of the following methods:

0024 Low-acid shelf-stable canned foods are preserved

by a full thermal process.

0025 Shelf-stable canned cured meats are preserved by a

combination of thermal processing and addition

of salt and nitrite.

0026 Canned acid foods are preserved by a pasteurizing

thermal process and acidity.

0027 Products such as dry fermented sausages are pre-

served by reduced water activity (a

) and pH, and

added nitrite.

0028 Packaged raw or cooked meats or fish and shell-

fish are preserved by refrigeration alone < 3



0029 Many meat and fish products are preserved by a

combination of added salt and refrigeration.

0030Many cured meat products are preserved by a

combination of added salt and nitrite, and refriger-

ation.

0031Vacuum-packaged smoked fish are preserved by a

combination of thermal processing, added salt,

smoking, and refrigeration.

0032Foodborne botulism, caused by home-prepared

foods, can be prevented by strict adherence to proper

home-canning instructions. The type of container,

levels of acidity, and salt and heat treatments are

especially important for home-canning. All home-

canned foods should be brought to a boil before

they are eaten. An effective botulinal toxoid is avail-

able from the US Centers for Disease Control for

immunization. Immunization of high-risk popula-

tions, such as Alaskan and Canadian Inuits, has

been repeatedly proposed, but never implemented.

At present, only laboratory workers at risk are

immunized.

Neurotoxin

0033As previously stated, seven serologically different

neurotoxins are produced by various strains of C.

botulinum. Some strains of C. butryricum produce

type E neurotoxin and some strains of C. barati can

produce type F neurotoxin. In general, the designa-

tion of the strain is that of the toxin it produces.

Strains of subtypes. AB, AF, BA, and BF are rare and

produce, in addition to the first toxin type, lesser

amounts of toxin of the second type. Type C and D

strains often produce small amounts of D and C

toxin, respectively. They may also produce a toxin,

C2, which is distinctly different from the neurotoxin

and will not be discussed here.

Structure

0034The neurotoxins are all very similar proteins with a

molecular weight of approximately 150 kDa. They

are synthesized as a single-chain protein with rela-

tively low toxicity, which is activated when it is

‘nicked’ by a number of proteases, including prote-

ases of group I C. botulinum, into a double-chain

molecule which is held together by a disulfide bond.

The two components of the nicked toxin, a light

(L) and heavy (H) chain, have molecular weights

of approximately 50 and 100 kDa, respectively.

Individually, the two components are not toxic, but

toxicity can be restored by reestablishing the disulfide

bond.

0035The neurotoxins exist as complexes of four

molecular sizes: 7S, 12S, 16S, and 19S, designated S

(small), M (medium), L (large), and LL (extra large)

toxins, respectively. The molecular weights range

CLOSTRIDIUM

/Botulism 1417

from 150 to 900 kDa. The M form is the most

common natural form, found in foods and cultures,

along with the L form. It is a complex of the S form

with an atoxic component. In the L form, a hemag-

glutinin is also part of the complex. The LL form is

only known for type A, and was the first form of the

botulinum toxin to be purified and crystallized. It has

not been found in cultures and is probably an artifi-

cial aggregate. The M and L forms are referred to as

progenitor toxins. They dissociate under mild alka-

line conditions into the S, or derivative toxin, which is

the single-chain protein.

Mode of Action

0036 Botulinum neurotoxins cause paralysis by blocking

acetylcholine release at neuromuscular junctions.

Cholinergic systems are affected the most, but adre-

nergic systems may also be affected by high concen-

trations of toxin. Paralysis appears to be caused by a

four-step process. First, the toxin binds to a type-

specific receptor on the presynaptic membrane. This

step is mediated by the carboxy-terminus of the H

chain. Second, the toxin is internalized into the nerve

cell by an initial receptor-mediated endocytosis. Acid-

ity in the endosome is believed to induce the amino-

terminus of the H chain to form channels in the

endosome membrane which permit the L chain to

enter the cytoplasm. Finally, the light chain acts as a

zinc endopeptidase and hydrolyzes the SNARE pro-

teins responsible for synaptic vesicle fusion: synapto-

brevin, syntaxin, or SNAP-25. Cleavage of the

SNARE proteins results in inhibition of acetylcholine

release, ultimately resulting in flaccid paralysis.

Inactivation

0037 The most effective means of inactivating botulinum

toxins in foods is by heat. Heat inactivation curves

are biphasic, with an initial steep decline that levels

off with time. Foods, especially those high in protein,

colloidal components, or ionic strength, have a pro-

tective effect. The toxin is most stable between pH 4

and 5. For the safe thermal inactivation of toxin at

concentrations up to 10

per gram, time/tem-

perature combinations of 20 min at 79



C or 5 min at



C have been recommended. Other means of toxin

inactivation include treatment with chlorine or

ozone.

See also: Canning: Principles; Chilled Storage:

Microbiological Considerations; Packaging Under

Vacuum; Clostridium: Occurrence of Clostridium

botulinum; Fish: Spoilage of Seafood; Heat Treatment:

Chemical and Microbiological Changes; Lactic Acid

Bacteria; Marine Foods: Marine Mammals as Meat

Sources; Meat: Preservation; Microbiology: Detection of

Foodborne Pathogens and their Toxins; Nisin; Nitrates

and Nitrites; Nitrosamines; Smoked Foods: Principles;

Water Activity: Principles and Measurement

Further Reading

Centers for Disease Control and Prevention (1998) Botu-

lism in the United States, 1899–1966. Handbook for

Epidemiologists, Clinicians, and Laboratory Workers.

Atlanta, GA: Centers for Disease Control and Preven-

tion.

Dodds KL and Austin JW (1997) Clostridium botulinum.

In: Doyle MP, Beuchat LR, and Montville TJ (eds)

Food Microbiology. Fundamentals and Frontiers,

pp. 288–304. Washington: ASM Press.

Galazka A and Przybylska A (1999) Surveillance of food-

borne botulism in Poland: 1960–1998. Eurosurveillance

4: 69–72.

Gao QY, Huang YF, Wu JG, Liu HD and Xia HQ (1990) A

review of botulism in China. Biomedical and Environ-

mental Sciences 3: 326–336.

Hauschild AHW and Dodds KL (1993) Clostridium botuli-

num: Ecology and Control in Foods. New York: Marcel

Dekker.

Pellizzari R, Rossetto O, Schiavo G and Montecucco C

(1999) Tetanus and botulinum neurotoxins: mechanism

of action and therapeutic uses. Philosophical Transac-

tions of the Royal Society of London Series B – Bio-

logical Sciences 354(1381): 259–268.

Proulx JF, Milot-Roy V and Austin J (1997) Four outbreaks

of botulism in Ungava Bay, Nunavik, Quebec. Canada

Communicable Disease Report 23: 30–32.

Shapiro RL, Hatheway CL and Swerdlow DL (1998) Botu-

lism in the United States: a clinical and epidemiologic

review. Annals of Internal Medicine 129: 221–228.

Smith LDS and Sugiyama H (1988) Botulism: The Organ-

ism, Toxins, the Disease, 2nd edn. Toronto: Academic

Press.

St Louis ME, Peck SH, Bowering D et al. (1988) Botulism

from chopped garlic: delayed recognition of a major

outbreak. Annals of Internal Medicine 108: 363–368.

Therre H (1999) Botulism in the European Union. Euro-

surveillance 4: 2–7.

Villar RG, Shapiro RL, Busto S et al. (1999) Outbreak of

type A botulism and development of a botulism surveil-

lance and antitoxin release system in Argentina. Journal

of the American Medical Association 281: 1334–1338,

1340.

Weber JT, Hibbs RJ Jr, Darwish A et al. (1993) A massive

outbreak of type E botulism associated with traditional

salted fish in Cairo. Journal of Infectious Diseases 167:

451–454.

1418

CLOSTRIDIUM

/Botulism

COBALAMINS

Contents

Properties and Determination

Physiology

Properties and Determination

MJa

gerstad and K Arkba

ge, Swedish University of

Agricultural Sciences, Uppsala, Sweden

Discovery

0001 The history of cobalamin (vitamin B

) started almost

two centuries ago, when a fatal anemia, resulting

from some disorder of the digestive and assimilative

organs, was described. The symptoms of this anemia,

named pernicious anemia, was further outlined during

this century, but a successful treatment of this fatal

disease was reported first in 1926, when raw liver

was introduced as therapy. In 1929, it was shown

that the intestinal absorption of the antipernicious

anemia principle in liver (extrinsic factor) required

prior binding to an ‘intrinsic factor’ secreted by the

stomach. The search for the active principle in liver

culminated with the isolation of vitamin B

in 1948

by a team of Merck scientists in the USA, which

was confirmed by a British team 3 weeks later. Injec-

tions of the isolated vitamin B

into muscular tissues

induced a dramatic beneficial response in patients

with pernicious anemia. Hodgkin and coworkers elu-

cidated the chemical structure of cyanocobalamin

(vitamin B

) by X-ray crystallography in 1964,

for which they were rewarded the Nobel Prize in

chemistry (the second Nobel Prize designated for

this vitamin).

Structure, Nomenclature, and Physical

Properties

0002 Cobalamins have a complex chemical structure based

on a corrin ring system. Four reduced pyrrole rings

are linked together to form a macrocyclic ring desig-

nated corrin, which is chelated by the four pyrrole

nitrogens. Corrin resembles the heme of hemoglobin,

but has one less a-methene bridge and has cobalt (Co)

instead of iron at the center. All the compounds con-

taining this ring are designated corrinoids. The fifth

co-ordinate covalent bound to cobalt is a nitrogen

of a dimethylbenzimidazole moiety. Thus, as with

nucleic acids, cobalamins contain a nucleotide, in

this case 5,6-dimethylbenzimidazole, rather than

various purine or pyrimidine bases. And the sugar,

ribose, has an a-glycosidic linkage, unlike the b-link-

age in the nucleic acids. The d-1-amino-2-propanol

moiety of the molecule is esterified to the nucleotide

and joined in amide linkage to the porphyrin-like

nucleus. The sixth position of cobalt may be

occupied by different anionic ligands, (R) according

to Figure 1 and Table 1. This ligand can be one of the

following types, e.g., – CN(cyanocobalamin), – OH

(hydroxocobalamin), – H

O (aquacobalamin), –NO

(nitritocobalamin), – SO

(sulfitocobalamin), – CH

(methylcobalamin), or – 5

-deoxy-5

-adenosyl (adeno-

sylcobalamin). Approximately 20 naturally occurring

analogs of cobalamins have been identified. Some of

these have no biological activity in mammals,

whereas others exhibit at least partial vitamin activity

but are often poorly absorbed. Human serum con-

tains not only the active cobalamins (mainly the

hydroxo-, methyl-, and adenosyl-cobalamins) but

also a variable number of analogs that are biologic-

ally inactive noncobalamin corronoids. These vita-

min B

analogs have their base of nucleotide,

5,6-dimethylbenzimidazole replaced by other nucle-

otides, e.g., adenine, guanine, 5-methyl-adenine, or

5-hydroxybenzimidazole.

0003According to the nomenclature of the

corrinoids, a cobalamin is a cobamide in which 5,6-

dimethylbenzimidazole is the aglycon attached by a

glycosyl link. Consequently, the systemic name of

cobalamin given by IUNS (International Union of

Nutritional Sciences) and IUPAC (International

Union of Pure and Applied Chemistry) is: Coa-

[a-(5,6-dimethylbenzimidazolyl)]-Cob-‘R’ cobamide,

where ‘R’ denotes ‘cyano,’‘aqua,’‘hydroxo,’

‘nitrito,’‘adenosyl,’‘methyl,’ etc) IUNS and IUPAC

state that the term ‘vitamin B

’ should be used as the

generic description for all corrinoids exhibiting quali-

tatively the biological activity of cyanocobalamin,

whereas the term ‘corrinoid(s)’ should be used as the

generic descriptor for all compounds containing the

corrin nucleus and thus chemically related to cyano-

cobalamin. The term ‘vitamin B

’ has two meanings.

COBALAMINS/Properties and Determination 1419

To the chemist, it means only cyanocobalamin,

whereas in the nutrition and pharmacology literature,

vitamin B

is a generic term for all the cobamides

exerting biological activity in humans. So far, all the

cobamides found to play a role in human metabolism

have been cobalamins. Table 1 summarizes the

generic or trivial names for different cobalamins,

together with their abbreviations, CAS numbers,

and physical properties.

Chemical Properties, and Stability

Cyanocobalamin

0004 Cyanocobalamin, a synthetic form of vitamin B

,is

commercially available for medical use, food fortifi-

cation, and nutrient supplements. The original iden-

tification of vitamin B

as cyanocobalamin involved

its formation as an artefact of the isolation procedure.

Little or no naturally occurring cyanocobalamin

exists in foods. Thus, cyanocobalamin is normally

not present in humans except in very small amounts

in smokers. It is a tasteless, odorless, red crystalline

substance with good water solubility. The color of the

compound may pose a limitation in the possible add-

ition of cyanocobalamin to certain foods, especially

light-colored products (e.g., white bread).

0005 The cyanide group of cyanocobalamin can be

replaced by other ions to form hydroxycobalamin,

chlorocobalamin, nitritocobalamin, thiocyanatoco-

balamin, and others; for example, bisulfite ions cause

conversion of aquacobalamin to sulfitcobalamin, and

similar reactions can occur to form cobalamins sub-

stituted with ammonia and nitrite. These reactions

have little influence on the net vitamin B

activity

of foods, since all the cobalamines are readily recon-

verted to cyanocobalamin after treatment with

cyanide, except methylcobalamin.

0006Cyanocobalamin appears to be the most stable of

the various vitamin B

analogs, being stable in air

and in dry form, and is even relatively stable at 100



for a few hours. Aqueous solutions at pH 4–7 can be

autoclaved at 120



C. Cyanocobalamin is slowly de-

composed by ultraviolet or strong visible light. The

cyano group is split off, yielding hydroxocobalamin,

which is also a biological active form of vitamin B

(and is available for medical use and used in fortifica-

tion by the food industry). Prolonged exposure to

light, however, causes irreversible decomposition

and inactivation.

0007Mild acid hydrolysis of cyanocobalamin induces

the removal of the nucleotide, and additional frag-

mentation occurs as the severity of the acidic condi-

tions increases. Exposure to alkaline conditions

causes hydrolysis of amides, yielding biologically in-

active carboxylic acid derivatives of vitamin B

.In

solution, thiamin and nicotinamide, or nicotinic acid,

destroy cyanocobalamin slowly, whereas the addition

of small amounts of iron or thiocyanate appear to

protect it. However, the literature is not definitive.

−

NOC

CONH

NOC

−

NOC

CONH

NOC

fig0001 Figure 1 Left: Chemical structure of cyanocobalamin; right: Coenzyme B

/adenosylcobalamin (R ¼5

-deoxy-5

-adenosyl); methyl-

cobalamin (R ¼methyl); aquacobalamin (R ¼H

); hydroxocobalamin (R ¼OH), with kind permission of Kra

utler.

1420 COBALAMINS/Properties and Determination

tbl0001 Table 1 Nomenclature and physical properties of cobalamins (vitamin B

)

Generic name Ligand (R) Abbreviation Molar mass Formula Solubility Crystal form l

max

(nm) Absorption

1cm

)

e10

3

Solvent

Cyanocobalamin

(vitamin B

)

– CN CNCbl 1355.38 C

CoN

P 1 g in 80 ml of water Dark red

hygroscopic

278 115 [15.6] Water

361 204 [27.6] Water

CAS No. 68-19-9

551 64 [8.7] Water

Aquacobalamin

(vitamin B

12a

)

–H

OCbl

1347.0 C

CoN

P 274 [153] 20.6 Water

351 [197] 26.5 Water

CAS No. 13422-52-1 499 [60] 8.1 Water

Hydroxocobalamin

(vitamin B

12b

)

– OH HOCbl 1346.37 C

CoN

POH Moderately soluble

in water; insoluble

in acetone,

ether, petroleum

ether, benzene

Dark red

orthorombic

279 [141] 19.0 Water

325 [85] 11.4 Water

CAS No. 13422-51-0 359 [153] 20.6 Water

516 [66] 8.9 Water

537 [71] 9.5 Water

Nitritocobalamin

(vitamin B

12c

)

–NO

1374.6 C

CoN

P Red crystalline

solids

352 153 [21.0] Water

528 60 [8.4] Water

CAS No. 20623-13-6

Sulfitocobalamin – SO

1409.5 C

CoN

PS 275 328 [46.2] Water, pH 7

CAS No. 15671-27-9 365 130 [18.3] Water, pH 7

516 61 [8.6] Water, pH 7

Adenosylcobalamin (cobamide

or coenzyme B

)

-deoxy-

-adenosyl

AdoCbl 1579.6 C

100

CoN

P Soluble in

ethanol, phenol;

insoluble in

acetone, ether,

dioxane

Yellow–orange

six-faced crystal

288 [115] 18.1 Water, pH 7

340 [78] 12.3 Water, pH 7

375 [60] 10.9 Water, pH 7

CAS No. 13870-90-1 522 [51] 8.0 Water, pH 7

Methylcobalamin

(methyl B

)

–CH

MeCbl 1344.4 C

CoN

P Bright red 266 [148] 19.9 Water

342 [107] 14.4 Water

CAS No. 13422-55-4 522 [70] 9.4 Water

CAS No., Chemical Abstract Service Number.

Values in brackets are calculated from the corresponding e value or E

1cm

value.

Modified from Eitenmiller.

Coenzyme B

0008 The coenzyme forms of vitamin B

are methyl-

cobalamin and adenosylcobalamin. Methylcobala-

min functions coenzymatically in the transfer of a

methyl group (from 5-methyltetrahydrofolate to

homocysteine) in methionine biosynthesis, whereas

-deoxyadenosylcobalamin serves as the coenzyme

in an enzymatic rearrangement reaction catalyzed by

methylmalonyl-CoA mutase. Both forms of vitamin

coenzymes are converted to the biological active

aquacobalamin upon light exposure. In fact, vitamin

coenzymes exhibit extreme photosensitivity,

which necessitates the use of subdued lighting and

low actinic glassware during vitamin B

analyses.

The photochemical degradation of vitamin B

coen-

zymes interferes with experimental studies of vitamin

metabolism and function, but this conversion has

no influence on the total vitamin B

activity of foods,

because aquacobalamin retains vitamin B

activity.

Surprisingly, methylcobalamin and adenosylcobala-

min are relatively stable in neutral aqueous solution

in the dark and can be heated for 20 min at 100



0009 Adenosylcobalamin is unstable under acidic and

alkaline conditions, whereas methylcobalamin is

stable in the presence of dilute acid or alkali. In

general, the natural forms of cobalamins (hydroxo-,

aqua-, methyl-, or adenosylcobalamin) are inacti-

vated by severe alkaline conditions, heavy metals,

strong oxidizing agents, or strong reducing agents

(like ascorbate). A possible interaction with ascorbic

acid has been reported to be detrimental to the vita-

min, especially under heating conditions. The signifi-

cance of these observations for the processing of food

remains unclear (see further discussion below). The

mechanism of vitamin B

degradation has not been

fully determined, in part because of the complexity of

the molecule and the very low concentration in foods.

Cobalamins in Food

0010 Only microorganisms are able to synthesize cobala-

min, and therefore, higher plants are devoid of

vitamin B

unless processed microbiologically or

contaminated by fecal material, e.g., from fertilizer.

Certain legumes have been reported to absorb small

amounts of vitamin B

produced by bacteria associ-

ated with root nodules, but little enters the seeds.

Food from animal origin, especially organs like liver

and kidney, contain > 30 mg vitamin B

per 100 g.

Other rich sources are meat muscles, fish, dairy prod-

ucts, and eggs. Bovine milk contains about 0.4 mgof

vitamin B

per 100 g (range 0.24–0.74 mg per 100 g).

Human milk contains only about 15% as much vita-

min B

as bovine milk. The predominant source of

vitamin B

in bovine milk is biosynthesized by

microorganisms in the rumen. Hydroxocobalamin is

the predominant form of vitamin B

in bovine milk.

There may also be some of the light-sensitive forms of

vitamin B

, adenosylcobalamin and methylcobala-

min, present. In milk, most of the vitamin B

protein-bound. The significance of this is still un-

known, although a protective effect against microbial

breakdown has been suggested. The vitamin B

most animal tissues consists mainly of the coenzyme

forms, methylcobalamin and adenosylcobalamin, in

addition to aquacobalamin. Some foods or microor-

ganisms contain other corrinoids, with varying bio-

logical activity, some of which may have antivitamin

activity. Foods are classified according to their

vitamin B

content, as shown in Table 2.

Process and Storage Losses

0011Under most conditions of food processing and preser-

vation, there is little nutritionally significant loss of

vitamin B

. Cyanocobalamin added to breakfast

cereal products has been reported to undergo an aver-

age loss of 17% during processing, with an additional

17% loss during storage for 12 months at ambient

temperature. When milk is pasteurized for 2–3s, it

loses 7% of its available vitamin B

, and boiling

for 2–5 min destroys 30%. Sterilization in a bottle

for 13 min at 119–120



C causes a loss of 77%, and

rapid sterilization (3–4 s) with superheated steam at

143



C, i.e., ultrahigh temperature (UHT) destroys

about 10% of the vitamin.

0012There are also processes like fermentation used for

manufacture of certain dairy products, e.g., yogurt

tbl0002 Table 2 Grouping of food according to vitamin B

content

Food Vitamin B

(mg per 100 g edible weight)

Very rich sources: organ meats (liver, kidney, heart), bivalves (clams and oysters) > 10

Moderately rich sources: some fish, crabs, egg yolk 3–10

Moderate sources: muscle meats (beef), some fish, cheese, egg, milk powder 1–3

Good sources: milk, yogurt, muscle meats (pig) <1

Poor sources: vegetables, cereals, fruits and berries n.d.

n.d., not detectable.

1422 COBALAMINS/Properties and Determination

and cheeses, which might result in either increased or

decreased vitamin B

concentrations depending on

the vitamin B

-producing or -consuming micro-

organisms.

0013 Studies indicate that the storage temperature and

time have a significant influence on the rate of break-

down. Thus, if UHT milk is stored at a low tempera-

ture (þ7



C), as for pasteurized milk, the losses of

vitamin B

are low for up to at least half a year.

However, if storage is at ambient temperature,

which is a normal condition for an aseptic product

such as UHT milk, breakdown of the vitamin may be

considerable even within a few weeks. Fermented

dairy products might lose or gain vitamin B

during

storage for the reason given above.

0014 Like all water-soluble vitamins, vitamin B

is sub-

ject to losses through leaching into the cooking water.

This indicates the potential for substantial losses of

vitamin B

during prolonged heating of foods at or

near neutral pH. Typical oven heating of commer-

cially prepared convenience dinners has been shown

to yield 79–100% retention of vitamin B

0015 Ascorbic acid has long been known to accelerate

the degradation of vitamin B

, especially during

high-temperature heating, although this may be of

little practical significance, because foods containing

vitamin B

usually do not contain significant

amounts of ascorbic acid. In curing solutions for

ham, neither ascorbate nor erythorbate has been

found to have any influence on vitamin B

retention,

probably since this process does not include high-

temperature heating.

Determination of Cobalamins

General

0016 For detection purposes, cobalamins are all UV ab-

sorbers. Unfortunately, their absorption spectra vary

between different congeners, and detection is compli-

cated by the low concentration at which they occur

normally in foods. Available methods for assay of

cobalamins developed over the years include both

general methods, e.g., polarographic, spectrophoto-

metric, various chromatographic procedures (paper,

thin-layer, and open-column chromatography), and

more specific and/or sensitive methods, e.g., micro-

biological, protein-binding, and high-performance

liquid chromatography (HPLC) procedures.

0017 Almost all available data on vitamin B

in food

have been obtained by microbiological assay (MA)

using Lactobacillus delbrueckii as the test organism.

Still, the microbiological assay is the reference

method for assaying cobalamins in foods. For clinical

tissues and serum samples, rapid radioprotein-

binding assays with

Co-labeled cyanocobalamin

are routine. However, radioassay kits for clinical

assay are not reliable for assay of food unless care-

fully validated for matrix effects. A more recent de-

velopment of this protein-binding assay is the use of a

biosensor technique to quantify cobalamins in food.

Although the various forms of vitamin B

can be

separated chromatographically, HPLC methods are

not readily suitable for food analysis, even if precon-

centration steps are used, because of the very low

concentrations typically found. Below, only methods

currently in use for the assay of naturally occurring

cobalamins in foods and/or fortified foods are pre-

sented and discussed.

Extraction

0018All methods for cobalamin analyses of food samples

need a preparative step prior to analysis. Since animal

products are the sole food sources of vitamin B

, the

vitamin must be extracted from protein-rich matrices

under conditions that will not lead to its destruction.

As a result, extraction procedures often involve diges-

tion with protease followed by extraction in hot

(80



C) ethanol. For quantitation as total vitamin

, heating in a cyanide- or sulfate-containing buffer

has been used to extract the endogenous vitamin B

forms, thereby converting them to the more stable

cyano- or sulfitocobalamin forms. The AOAC official

method (952.20) recommends mixing approximately

1 g or 1 ml of the sample in 25 ml of a freshly

prepared solution containing sodium phosphate,

citric acid, and metabisulfite. The extraction is com-

pleted by homogenizing the sample in the extraction

solution, autoclaving the mixture at 121–123



C for

10 min, adjusting the cooled mixture to pH 6.0,

filtering and/or centrifugation, and diluting with

water to a vitamin B

concentration of approxi-

mately 0.2 ng ml

1

. However, most extraction pro-

cedures described in the literature add either sodium

cyanide (NaCN) or potassium cyanide (KCN) to

0.1 M acetate buffer, pH 4.5–4.7, during homogen-

ization and extraction. The heating time varies be-

tween 3 and 30 min, and a heating temperature of

either 100



C (boiling water) or 121



C (autoclava-

tion) is used. The heat-extraction procedure including

centrifugation and/or filtration are enough of sample

preparation for microbiological assays and protein-

binding assays (immunoassays). In the use of

HPLC, further clean-up work might be necessary,

e.g., ultracentrifugation, ultrafiltering, affinity chro-

matography, or adsorption on to charcoal or chroma-

tography on anion exchange resin and/or solid-phase

extraction on C

cartridges.

0019The extraction conditions have been shown to affect

significantly the efficiency of the overall extraction

COBALAMINS/Properties and Determination 1423

process. It has therefore been suggested that the ex-

traction conditions should be optimized for each type

of food sample in order to achieve reproducible and

good recoveries of the complete analysis.

Microbiological Assay

0020 Over the years, the growth response of various micro-

organisms has been used to analyze cobalamins in

foods and other biological material. The micro-

organisms that have been used are Lactobacillus

delbrueckii (previously, leichmanni), Escherichia

coli, Euglena gracilis,andOchromonas malhamen-

sis. Below, only the AOAC (Association of Official

Analytical Chemists) Official method (reference

method) based on Lactobacillus delbrueckii is pre-

sented. The other microorganisms have different dis-

advantages compared with Lactobacillus delbrueckii,

i.e., less sensitive and/or specific also towards less

active biological analogs. In addition, Euglena graci-

lis and Ochromonas malhamensis grow more slowly

and are thus more painstaking.

0021 The AOAC Official Method 952.20: Cobalamin

(Vitamin B

-activity) in Vitamin preparations use a

microbiological assay with Lactobacillus delbrueckii

(ATCC 7830) (45.2.02). This method was originally

collaborated for use on vitamin preparations, but the

AOAC Task Force on Methods for Nutrition Label-

ing recommended the procedure for use on all food

matrices. The Lactobacillus delbrueckii growth re-

sponse is sufficiently sensitive to quantify cyanoco-

balamin at concentrations approaching 1.0 pg per

milliliter of assay growth medium (corresponding to

less than 0.5 mg per 100 g). It has a variable response

to various cobalamins. A similar growth response has

been reported for cyanocobalamin, hydroxocobala-

min, sulfitocobalamin, nitritocobalamin, and dicya-

nocobalamin. However, adenosylcobalamin produces

a greater response, and methylcobalamin a lower

growth response. This will generally not cause a prob-

lem, since excess of cyanide or sulfite is used during

the extraction procedure prior to analysis, thereby

converting all different native forms of cobalamins

except methylcobalamin into one stable form.

0022 An important aspect in all methods of analysis is

their specificity. Lactobacillus delbrueckii can utilize

vitamin B

analogs, e.g., deoxyribonucleotides and

deoxyribonucleosides, in addition to biologically

active cobalamins. Older literature suggests that dilu-

tion of deoxyriboside levels (e.g., thymidine) to less

than 1 mg per milliliter of the assay medium will elim-

inate the effect.

Radioprotein-Binding Assays

0023 Radioprotein-binding assays have been used rou-

tinely for blood and tissue analysis since early work

by Rothenberg and several others during the 1960s.

The principle of this assay is based on the competition

of added isotope-labeled cyanocobalamin (

Co)

versus cyanocobalamin from the unknown sample

for a limited number of binding sites on cobalamin-

binding proteins (often intrinsic factor from hog). The

amount of labeled cobalamin bound to the binding

protein is quantified in a gamma counter, and the

concentration of cobalamin in the unknown sample

is inversely proportional to the concentration of

labeled cobalamin, and calculated from a calibration

curve.

0024Early types of radioprotein-binding assays for de-

termination of vitamin B

in clinical specimens and

foods were often inaccurate because the binding pro-

tein could bind to active forms of vitamin B

as well

as biologically inactive analogs. The specificity of

such assays has been greatly improved through

the use of a vitamin B

-binding protein (generally

porcine intrinsic factor, IF) that is specific for the

biologically active forms of the vitamin. Radioligand

binding methods based on monospecific antisera that

eliminate any cross-reactions with other cobalamins

are now available and should be used routinely in all

clinical laboratories.

0025The relative binding affinities of porcine IF for

various cobalamins have also been studied. An

equivalent affinity has been found for cyano-,

dicyano, nitrito-, and methylcobalamins, whereas

significantly different binding affinities have been

found for hydroxo-, sulfito, and adenosylcobala-

mins. Therefore, with cyanocobalamin as the calibra-

tion standard, it is necessary to add cyanide to the

samples prior to quantification during the extraction

procedure to convert all native forms to cyanocobal-

amins.

0026Studies by several research groups indicate that

radioprotein-binding assays with IF can be used for

food analysis. When optimized extraction procedures

have been used, radioprotein binding assays and

microbiological procedures show agreement for

most foods; however, differences have been noted

often enough to conclude that the two methods

are not universally interchangeable for the assay of

all foods. Thus, recent work has shown that the

radioprotein binding assay could be reliable for food

analysis, providing careful method validation includ-

ing control of selectivity. One should bear in mind

that not only the food matrix itself but also sub-

stances used during the extraction procedure, e.g.,

buffer strength, pH, cyanide concentration, and

added enzymes needed to release vitamin B

bound

to the food matrix, might cause nonspecific binding

and therefore must be accounted for in order to

achieve reliable results.

1424 COBALAMINS/Properties and Determination

0027 An alternative to the protein-binding assay is an

enzyme-linked protein-binding assay that appears to

be useful for fortified foods in which cyanocobalamin

is the predominant cobalamin. The procedure uses

an R-protein–enzyme conjugate and microtiter plate

techniques for quantitation of cyanocobalamin. Since

R-proteins bind cobalamins and their analogs, the

assay has not been applied to nonfortified foods.

Another drawback is that the R-protein enzyme con-

jugate is not commercially available. Applications to

(a)

(

)

(b)

Vitamin-specific molecules are added to sample extract

Vitamin-specific

molecule

Vitamin Vitamin sensor

chip

Measurement of unbound

vitamin-specific molecules

Vitamin concentration

Response (RU)

Light-

source

Sensor chip with

gold film

Prism

Optical

detection

unit

Intensity

Angle

Time

Resonance

signal

Sensorgram

Flow channel

III

Polarized

light

Reflected

light

10 000

12 000

14 000

16 000

18 000

20 000

200 400 600

Time (s)

Sensorgram

Cobalamin, 0.6 ng/ml

Response (RU)

800 1000 1200

fig0002 Figure 2 Assay principle for determination of vitamin B

in food using optical biosensor technology (BIACORE).

COBALAMINS/Properties and Determination 1425