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

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fortified breakfast cereals have given a limit of detec-

tion of 9 pg per well and a quantification limit of

0.09 mg per 100 g.

Optical Biosensor-based Immunoassay

0028 This assay is the latest methodological development

for vitamin B

in foods and is based on biospecific

recognition of the analyte, cyanocobalamin, by a

vitamin B

binding protein. The event of recognition

is transduced to a signal by means of an optical

sensing mechanism. Real-time biomolecular inter-

action analysis recently developed by BIACORE is a

label-free technology for monitoring biomolecular

interaction as they occur. A special kit, Qflex

Kit Vitamin B

, is commercially available and uses

an inhibition assay/indirect assay for the analyses.

In an inhibition assay, the analyte or an analog is

immobilized onto the surface of a sensor chip. A

high-molecular-weight detecting molecule such as a

binding protein in a defined concentration is added to

the sample. The binding proteins bind to the analyte,

but at equilibrium, some binding proteins will remain

in solution free to bind to the sensor surface. A stand-

ard curve is prepared from solutions with known

concentrations. The concentration of analyte in an

unknown sample is then derived from a standard

curve.

0029 The detection principle relies on surface plasmon

resonance (SPR), an electronic charge-density wave

phenomenon that arises at the surface of a metallic

film, usually based on gold, when light is reflected at

the film under specific conditions. The resonance is a

result of energy and momentum being transformed

from incident photons into surface plasmons and is

sensitive to the refractive index of the medium on the

opposite side of the film from the reflected light. A

scheme of the principle of the optical biosensor

technology is shown in Figure 2.

0030 Method validation data using milk-based infant

formulas fortified with cyanocobalamin have given

the following results; precision (RSD

): 4.4; recov-

ery: 93–106%; detection limit (LOD 3 SD):

0.07 ng ml

1

and quantification limit (LOQ

10 SD): 0.17 ng ml

1

. Samples can be quantified

in the range of 0.1–4ng ml

1

, corresponding to an

LOQ of * 0.5 m g per 100 g, depending on sample

preparation.

0031 Samples analyzed by MA and Biacore Q show a

good agreement, but the BIACORE method is less

sensitive so far. This restricts its use at the moment

to fortified foods, e.g., infant milk formulas and

gruels, and pharmaceutical vitamin mixtures. The

biosensor-based BIA:SPR technique provides a con-

venient, label-free, and automated quantitative

analysis amenable to routine compliance monitoring

in the food laboratory. Advantages of this technique

include the speed and simplicity of operation for rou-

tine compliance analyses and an enhanced precision.

HPLC Methods

0032Liquid chromatography can be conveniently applied

to the assay of vitamin B

in high-concentration

supplements and pharmaceutical preparations (usu-

ally cyano- or hydroxocobalamin forms). A few such

HPLC methods have been published since 1980. It

can also effectively resolve natural cobalamins.

However, owing to the low levels of analytes,

radioligand-binding assays are necessary to quantify

eluted cobalamins. For milk, which is a good source

of vitamin B

(containing around 4 mg of vitamin

per liter), an HPLC method based on UV detec-

tion (550 nm) and with a detection limit of 0.2 mgof

vitamin B

per liter of milk is available. Recently, a

novel HPLC method for the determination of vitamin

(cyanocobalamin) with fluorescence detection

has been reported. The method has been used success-

fully to determine vitamin B

in pharmaceutical

preparations, e.g., vitamin B

-containing multivita-

min tablets and fermentation medium. The recovery

varies from 94 to 102%, and the relative standard

deviation is in the range of 1.8–4.1%.

Certified Reference Material

0033Two Bureau Commission Reference materials, milk

powder (CRM 421) and lyophilized pig’s liver (CRM

487) are available by the European Commission in

Brussel with a certified vitamin B

content analyzed

by microbiological assay (Lactobacillus leichmanni,

ATCC 7830) and radioprotein-binding assay. The

two different methods show similar vitamin B

con-

centrations in these reference materials.

See also: Biosensors; Chromatography: High-

performance Liquid Chromatography; Cobalamins:

Physiology; Coenzymes

Further Reading

AOAC International (1999) Official Methods of Analysis,

17th edn. Arlington, VA: AOAC.

Eitenmiller RR and Landen WO Jr. (1999) Vitamin Analysis

for the Health and Food Sciences, pp. 467–478. New

York: CRC Press.

Ellenbogen L and Cooper BA (1991) Vitamin B

. In:

Machlin LJ (ed.) Handbook of Vitamins, 2nd edn,

pp. 491–505. New York: Marcel Dekker.

Finglas PM, Scott KJ, Wittho

ft CM, van den Berg H and

de Froidmont-Go

rtz I (1999) The Certification of the

Mass Fractions of Vitamins in Four Reference Materials:

1426 COBALAMINS/Properties and Determination

Wholemeal Flour (CRM 121), Milk Powder (CRM 421),

Lyophilized Mixed Vegetables (CRM 485) and Lyophil-

ized Pig’s Liver (CRM 487). European Commision. BCR

Information/Reference materials: EUR 18320 EN.

Gregory JF (1996) Vitamins. In: Fennema OR (ed.) Food

Chemistry, 3rd edn. pp. 603–606. New York: Marcel

Dekker.

Herbert V (1996) Vitamin B

. In: Ziegler EE and Filer LJ

(eds) Present Knowledge in Nutrition, 7th edn,

pp. 191–205. Washington, DC: ILSI Press.

Kra

utler B, Arigoni D and Golding BT (1998) Vitamin B

and B

-Proteins. Weinheim, Germany: Wiley-VCH.

Lindemans J and Abels J (1992) Cobalamins. In: De Leen-

heer AP, Lambert WE and Nelis HJ (eds) Modern

Chromatographic Analysis of Vitamins, 2nd edn. New

York: Marcel Dekker.

Machlin LJ and Hu

ni JES (1994) Vitamin B-12. In: Vita-

mins Basics, pp. 40, 56. Basel, Switzerland: La Roche.

Muhammad K, Briggs D and Jones G (1993a) The appro-

priateness of using cyanocobalamin as calibration stand-

ards in competitive binding assay of vitamin B

. Food

Chemistry 48: 423–430.

Muhammad K, Briggs D and Jones G (1993b) Comparison

of a competitive binding assay with Lactobacillus leich-

manni ATCC 7830 assay for the determination of vita-

min B

in foods. Food Chemistry 48: 431–439.

Physiology

C J Bates, MRC Human Nutrition Research,

Cambridge, UK

Introduction

0001 The importance of vitamin B

in human nutrition

has been dominated by the potentially fatal disease,

pernicious anemia, which results from a failure of

secretion of the specific cobalamin-binding protein,

intrinsic factor, by the parietal cells of the stomach.

This protein is essential for the subsequent recogni-

tion, binding, and transport of vitamin B

at the ileal

wall. Transcobalamin II then carries it to the portal

circulation. The treatment of pernicious anemia with

massive amounts of dietary liver, followed by the

isolation, characterization, and X-ray crystallo-

graphic structure determination of vitamin B

, were

major achievements of the first half of the 20th cen-

tury. During the past few years, new evidence has

been accumulating to suggest that more subtle, insidi-

ous and easily overlooked forms of vitamin B

deficiency may occur in modern-day western society,

and that the challenges of their recognition and treat-

ment are by no means entirely solved. The fact that

prolonged vitamin B

depletion can lead to irrevers-

ible neural damage, coupled with the prediction that

defects of absorption can arise slowly and remain

entirely unnoticed as people grow older may be of

special relevance at a time when the quality of life in

old age is becoming a major priority issue. (See

Anemia (Anaemia): Megaloblastic Anemias.)

0002Dietary intake by omnivores is much greater than

by vegetarians or vegans (who depend to a large

extent on contamination sources, unless they deliber-

ately take B

supplements). Some B

-like corrinoids

in certain seaweeds have limited B

activity. In vitro,

vitamin B

may be destroyed by contact with vita-

min C (ascorbic acid), but in vivo this probably does

not occur, which suggests that the catalysis of destruc-

tion may depend on free transition metal ions which

are not usually present in living tissues. (See Vegetar-

ian Diets.)

0003Although B

intakes are much lower in Third

World countries than in western countries, severe

deficiency is surprisingly rare in the Third World.

However, a moderate degree of deficiency has re-

cently been reported from parts of South America,

currently attributed to a combination of low intakes

and impaired absorption due to gastrointestinal para-

sites. Fully breast-fed infants of mothers with very

low intakes, or of mothers with vitamin-B

malab-

sorption syndromes (usually pernicious anemia), may

develop overt B

deficiency. Older omnivores usually

accumulate considerable amounts in their tissues, and

in these subjects the effect of a switch to diets with

low contents only becomes apparent several decades

after the B

supply to the internal milieu has virtually

ceased.

0004There are still some problems with the assay of

vitamin B

levels in food, and 5–30% of the reported

content may be biologically inactive corrinoids.

Small amounts of B

arising from bacteria in the

ileum can be absorbed, but B

produced by bacteria

in the colon is not. An efficient enterohepatic circula-

tion of B

occurs in normal people, and if this cycle is

interrupted, as it is in cases of impaired intestinal

absorption, this results in an additional source of

losses for subjects with impaired B

absorption.

(See Microflora of the Intestine: Role and Effects.)

0005Large-scale industrial production of vitamin B

makes use of the capacity of organisms such as Pro-

pionibacterium spp. to synthesize as much as 40 mg

per liter of growth medium. World consumption

was around 5000 kg in the early 1980s.

Absorption and Tests of Status

0006The key stages in vitamin B

utilization from food

sources are depicted in Table 1.

COBALAMINS/Physiology 1427

0007 In addition to classical pernicious anemia (lack of

secretion of intrinsic factor due to atrophy of gastric

mucosa or the production of autoantibodies to intrin-

sic factor or its intestinal binders), there are several

other medical conditions that can result in impaired

absorption, as well as a variety of congenital abnor-

malities of transport of the vitamin, which require

both specialized detection procedures and specialized

therapeutic measures. These include:

0008 surgical procedures such as total gastrectomy,

removal of ileum, or presence of blind loops of

the small gut with an abnormal bacterial flora

0009 the effects of certain parasites, drugs, or sub-

stances like nitrous oxide

0010 the Imerslund–Gra

sbeck syndrome which appears

to result from lack of ileal receptors

0011 pancreatic insufficiency leading to failure of diges-

tion of R proteins

0012 lack of circulating transcobalamin II

0013 The classical procedure for the recognition of

impaired absorption, specially designed to detect per-

nicious anemia, is the urinary excretion (Schilling)

test (Table 2). Around 57% of people with pernicious

anemia also have circulating antibodies to intrinsic

factor in their blood, and their detection can provide

further corroborative evidence of the presence and

type of pernicious anemia. In conjunction with a

macrocytic anemia and a standard assay of circulat-

ing plasma vitamin B

or of holo-transcobalamin II,

the measurement of these antibodies can be a useful

screening procedure to be performed before the more

complex Schilling test is undertaken.

0014Serum vitamin B

levels are usually measured

nowadays by radioisotope dilution assay, generally

using pure intrinsic factor as the specific binder.

Normal levels are above 200 pg ml

1

, and levels

below 160 pg ml

1

(118 pmol l

1

) are indicative of

deficiency. Many commercial kits are now available

for the radioisotope dilution assay. (See Immuno-

assays: Radioimmunoassay and Enzyme Immuno-

assay.)

0015An older functional test of vitamin B

or folate

status is the deoxyuridine suppression test, usually

performed in vitro on bone marrow aspirates. This

looks directly at the cells’ ability to synthesize thymi-

dine from deoxyuridine. For B

-deficient cells, B

cofactor is the only addition which will partially

overcome the block in conversion. This test has the

potential advantage of being a functional test and of

probing a specific tissue site, but it is time-consuming,

and it is now mainly of research, rather than diagnos-

tic, importance. (See Coenzymes.)

0016Another functional test involves the metabolism of

an oral load of valine: if B

deficiency is present, then

methylmalonyl CoA mutase activity falls (see below)

so that methylmalonic acid accumulates, and in-

creased amounts of this byproduct are found in the

plasma and excreted in the urine. Moderately raised

plasma levels of methylmalonic acid can also arise

in subjects with marginal vitamin B

status, even

without the loading dose, and this is becoming the

basis for a useful diagnostic procedure for marginal

status. The most commonly used assay procedure

for serum or urinary methyl malonic acid is based

on mass spectrometry, although other methods, such

tbl0001 Table 1 Stages of normal vitamin B

absorption in humans

. Release from dietary protein by gastric acid and pepsin (this can often become impaired in older people)

. Binding to ‘R-binders’: glycoproteins secreted by saliva

. Transfer to intrinsic factor (IF) when R-binders are degraded by pancreatic enzymes in the intestine

. Attachment of the B

–IF complex to ileal receptors, followed by endocytotic transfer across the intestinal wall. This has very precise

specificity requirements and is the rate-limiting step in absorption. It is most efficient in the range of 1–2 mgB

, whereas passive

absorption occurs with only c. 1% the efficiency, but over a much wider range of intakes, so that useful amounts are absorbed when

intakes are high

. Circulation in the plasma in several forms: transcobalamins (TC) I, II and III, and R-proteins (haptocorrins), of which TC II is the main

tissue uptake precursor, with a half-life of only c. 6 min

. Storage, mainly in the liver; recycling by enterohepatic circulation; turnover at a rate of c. 0.1% of the body pool per day

tbl0002 Table 2 The Schilling test for vitamin B

absorption efficiency

. Measures percentage recovery of an oral dose of labeled vitamin B

, using an intravenous dose to flush the newly absorbed

vitamin largely into the urine

. Cobalt-labeled vitamin B

is used, and there are three usable, g-emitting isotopes:

Co (half-life 5 years);

Co (half-life 270 days),

and

Co (half-life 71 days)

. After a parenteral injection of unlabeled B

, normal absorbers excrete c. 15% of the labeled dose within 24 h, whereas those with

impaired absorption excrete less than 5%

. By using two different isotopes of cobalt simultaneously, it is possible to distinguish the different types of malabsorption, e.g., by

comparing free B

against B

bound to intrinsic factor, R-binders, or binding proteins in food

1428 COBALAMINS/Physiology

as capillary electrophoresis, are now also becoming

available. The assay is still too demanding of skill and

expensive equipment for routine clinical chemistry

laboratories, but it is becoming the index of choice

for the demonstration of mild functional vitamin B

deficiency. Circulating levels of homocysteine can

provide further evidence, but since folate and vitamin

status can also affect homocysteine economy, the

evidence that it provides is less specific, and requires

the investigation of the other vitamins.

Biological Functions of Vitamin B

0017 Three vitamin B

-dependent enzymes occur in

animals. The first, methionine synthase or synthetase,

has the Enzyme Commission (EC) official name 5-

methyltetrahydrofolate-homocysteine S-methyltrans-

ferase, EC 2.1.1.13, and requires methylcobalamin as

the cofactor or cosubstrate. The other two vitamin

-dependent enzymes both require adenosyl coba-

lamin: they are methylmalonyl-coenzyme A (CoA)

mutase EC 5.4.99.2, responsible for conversion of

methylmalonyl-CoA to succinyl CoA during the oxi-

dation of propionate (see above), and leucine 2,3-

aminomutase, EC 5.4.3.7, which converts l-a-leucine

to aminoisocaproate as the first step in leucine deg-

radation or synthesis. There is a rare congenital dis-

order in which the enzyme methylmalonyl-CoA

mutase is almost absent, and this is characterized by

methylmalonic aciduria, homocysteinuria, muscle

cramps, mental retardation, and lethargy. Large par-

enteral doses of vitamin B

may be helpful.

0018 The most important biochemical reaction cata-

lyzed by vitamin B

in humans and animals is the

conversion of homocysteine to methionine. However,

the most obvious result of deficiency in vivo is a block

in DNA synthesis, leading characteristically to mega-

loblastosis, and macrocytic anemia, which is indistin-

guishable, at least at the cytological level, from the

megaloblastosis and anemia of folate deficiency. A

good test for response of a probable case of pernicious

anemia, or other B

-deficiency syndrome, is an

increase in circulating reticulocyte numbers follow-

ing B

treatment. (See Amino Acids: Metabolism;

Anemia (Anaemia): Megaloblastic Anemias.)

0019 The most generally accepted explanation for the

connection between B

deficiency and the failure of

cell division leading to anemia is the ‘methylfolate

trap hypothesis’, in which methylene tetrahydrofolate

polyglutamate, the intracellular cofactor for thymi-

dine synthesis, becomes depleted by excessive con-

version (i.e., reduction) to methyltetrahydrofolate

polyglutamate. The latter is unable to transfer its

carbon unit (methyl group) to homocysteine and

thus complete the folate cycle back to the methylene

form, because of the absence of B

cofactor (methyl-

cobalamin). At the same time, short-chain methylfo-

late glutamates from the diet are poorly utilized and

therefore excreted, so that intracellular folate levels

decline. (See Folic Acid: Physiology.)

0020An alternative interpretation, the ‘formate starva-

tion hypothesis’, maintains that because B

defi-

ciency causes a reduction in methionine levels, an

important consequence of its deficiency is a reduced

conversion of the methyl group of methionine to for-

myltetrahydrofolate, which is a good precursor of

folate polyglutamates and hence of active methylene

groups for thymidine synthesis. In the absence of

vitamin B

, formate accumulates in blood, liver,

and brain, and there is increased formate excretion

in the urine. In support of this hypothesis, formylfo-

late, but not tetrahydrofolate, can correct the func-

tional evidence of B

deficiency, and the organism is

still able to oxidize the methyl group of methylfolate

to methylene- and formyl-folates. One very useful

experimental tool has been the use of nitrous oxide,

which can chemically inactivate vitamin B

in vivo,

e.g., in experimental animals. This procedure has

permitted the rapid induction of tissue B

deficiency,

without the tedious and protracted use of B

deficient diets.

0021An important functional result of vitamin B

defi-

ciency is the failure of maintenance of nerve tissue

myelin, which accounts for the irreversible neuro-

logical damage seen after prolonged B

deficiency.

This lesion may also be connected biochemically with

the block in methionine formation (and hence in

phospholipid metabolism), but its etiology has not

yet been fully elucidated. There may also be other

disturbances in lipid metabolism in B

-deficient

subjects.

Human Vitamin B

Requirements

0022In the absence of the metabolic abnormalities that can

affect B

absorption and utilization, human dietary

requirements for the vitamin are small. A purely nu-

tritional deficiency may, however, occur in strict

vegans and lifelong vegetarians such as strict Hindus

who have very low intakes because they do not eat

any animal products. Studies of the amount of B

needed to cure this, together with studies of popula-

tions with low intakes who, nevertheless, do not

show deficiency signs, have indicated that the require-

ment is generally less than 1 mg day

1

for adults,

and is probably in the range 0.1–1 mgday

1

. There is

little firm evidence for a major increase in require-

ment during pregnancy and lactation, but modest

increments in the recommended dietary allowances

(RDAs) are usually applied to these physiological

COBALAMINS/Physiology 1429

states. The amount secreted in breast milk ranges

from 0.1 to 3 mg day

1

. Infants who were breast-fed

by mothers who were themselves deficient and were

secreting milk with a very low content of the vitamin

have in several reported instances been diagnosed

with B

-responsive megaloblastic anemia. (See Lac-

tation: Human Milk: Composition and Nutritional

Value; Physiology.)

0023 The early recognition of B

deficiency in certain

immigrant vegetarian groups, especially Asians,

merits attention, especially in western countries

where bacterial contamination sources of the vitamin

are eliminated more completely than in developing

countries.

0024 Typical B

intakes by omnivorous people in west-

ern countries are 3–30 mgday

1

0025 Food table values of B

contents of foods have

been obtained mainly by microbiological assays and

some further verification is needed. Vitamin B

stored mainly in the liver (60%) and muscles (30%).

Methylcobalamin is the most abundant form in

human plasma, whereas in most human tissues,

deoxyadenosyl cobalamin is the most abundant,

with aquacobalamin coming second.

0026 At high intakes, there is little evidence of toxicity,

and injections of as much as 3 mg day

1

have been

used in an attempt to treat fatigue and neurological

disorders. However, there is no evidence that such

high doses can have any benefit for normal subjects

and they may occasionally result in allergic reactions.

Degradation of B

to B

antagonists in multinutri-

ent preparations has been claimed.

0027 The RDAs from the World Health Organization

(WHO) currently stand at 1 mg day

1

for adults. In

the UK the reference nutrient intake for vitamin B

1.5 mg day

1

for adults, and in the USA, the dietary

reference intake is 2.4 mgday

1

. Thus there is a rather

large variation between the different sources of refer-

ence values. Values for pregnancy and lactation are

generally slightly higher than for nonpregnant, non-

lactating adults. A typical treatment schedule for per-

nicious anemia would be 500 mg by injections every

2–3 months.

Vitamin B

: Increased Vulnerability in

Older People

0028 The aging process is characterized by progressively

increasing vulnerability to serious impairment of

vitamin B

absorption, and hence the development

of functional deficiency. The most widely known

example of this is pernicious anemia, often result-

ing from autoantibodies which interfere with the

functions of intrinsic factor, and which can be

detected by blood antibody analysis. Another

common cause is atrophic gastritis, which reduces

gastric acid production and thus interferes with the

liberation of the vitamin from the protein binders in

food. Several recent studies have highlighted the evi-

dence for a relatively common, mild vitamin B

defi-

ciency in older people living in western countries.

There is recent evidence of racial differences in vul-

nerability to vitamin B

deficiency in older Ameri-

cans: white and Latin subjects are more vulnerable

than black or Asian ones. Raised methylmalonic acid

levels and lowered levels of holotranscobalamin II are

found. As absorption becomes more severely

impaired, the normal B

enterohepatic cycle is also

affected, which increases the rate of loss of B

(nor-

mally c. 0.1% day

1

). The longer a deficiency state

goes undetected, the greater the danger of permanent

neurological damage. In the absence of the highly

efficient intrinsic factor-catalyzed absorption path-

way, the alternative passive absorption route is only

about 1% as efficient; therefore an intake at least

100-fold greater than the normal requirement is

needed. At around 100–200 mg day

1

this is not avail-

able (in the UK) in nonprescribed vitamin supple-

ments; therefore diagnosis and treatment are of

critical importance. There is some concern that recent

public health interventions designed to increase the

level of food fortification with folate may mask the

anemia of incipient vitamin B

deficiency, and thus

delay diagnosis.

See also: Amino Acids: Metabolism; Anemia (Anaemia):

Megaloblastic Anemias; Coenzymes; Folic Acid:

Physiology; Immunoassays: Radioimmunoassay and

Enzyme Immunoassay; Lactation: Human Milk:

Composition and Nutritional Value; Physiology;

Macrobiotic Diets; Microflora of the Intestine: Role and

Effects; Vegetarian Diets

Further Reading

Chanarin I (1990) The Megaloblastic Anaemias, 3rd edn.

Oxford: Blackwell.

Mann NJ, Li D, Sinclair AJ et al. (1999) The effect of diet on

plasma homocysteine concentrations in healthy male

subjects. European Journal of Clinical Nutrition 53:

895–899.

Nilsson-Ehle H (1998) Age-related changes in cobalamin

(vitamin B

) handling. Implications for therapy. Drugs

and Aging 12: 277–292.

Savage DG and Lindenbaum J (1995) Neurological compli-

cations of acquired cobalamin deficiency: clinical

aspects. Bailliere’s Clinical Haematology 8: 657–678.

Scott JM (1997) Bioavailability of vitamin B

. Euro-

pean Journal of Clinical Nutrition 51: (suppl. 1): S49–

S53.

Scott JM (1999) Folate and vitamin B

. Proceedings of

the Nutrition Society 58: 441–448.

1430 COBALAMINS/Physiology

Seetharam B (1999) Receptor-mediated endocytosis of

cobalamin (vitamin B

). Annual Review of Nutrition

19: 173–195.

Stabler SP, Lindenbaum J and Allen RH (1997) Vitamin B

deficiency in the elderly. American Journal of Clinical

Nutrition 66: 741–749.

Woodson RD (ed.) (1990) New frontiers in vitamin B

metabolism. American Journal of Hematology 34:

81–139.

Zittoun J and Cooper BA (eds) (1989) Folates and Cobala-

mins. Berlin: Springer-Verlag.

COBALT

A MacPherson and J Dixon, The Scottish Agricultural

College, Auchincruive, Ayr, UK

Background

0001 Cobalt was first isolated by a Swedish chemist,

Brandt, in 1735 and characterized more fully by Berg-

man in 1780. However, it had been used as an ore for

millennia to give a bright blue color to Egyptian pot-

tery and Iranian glass from 2600 to 2250 bc, respect-

ively. The name is derived from the German word

kobold, meaning an evil spirit or gnome, as copper

miners working in the Harz mountains believed that

these spirits delighted in exposing ores that looked like

copper but yielded none when smelted.

0002 Cobalt is widely distributed in the earth’s crust but

it is only 30th in order of abundance and thus less

common (at 25 mg kg

1

) than all other elements in

the first transition series with the exception of scan-

dium (22 mg kg

1

). There appears to be some overall

loss of cobalt during soil formation as the world mean

value is quoted as 12 mg kg

1

and Scottish soils at

8mgkg

1

. World consumption of cobalt was 31 400

tonnes in 1997 and is projected to increase by 50–

100% by 2012. Asia, including China, is the largest

consuming region, and the chemical sector is the

largest end user. The use of cobalt in chemicals for

rechargeable batteries is having a significant impact

on total consumption.

0003 The metal itself is silvery in appearance with a

bluish tinge and considerably harder than iron. It

has only one naturally occurring isotope,

Co, but

this can be converted to radioactive

Co by bom-

bardment with thermal neutrons. This has a half-life

of 5.271 years and decays to nonradioactive

Ni.

The radioactive isotope

Co is used medically in

cancer therapy, where it can selectively destroy

abnormal areas in the brain, such as tumors or

arteriovenous malformations, while leaving normal

tissue unaffected. This procedure has been termed

‘radiosurgery.’ Nitrosylcobalamin shows potential as

a biologically compatible and selective chemothera-

peutic agent as tumor cells have surface receptors for

vitamin B

Co also affords a concentrated source

of radiation for research purposes.

Co is used in the

assay of vitamin B

by radioisotope dilution.

0004Like iron, cobalt is a ferromagnetic element, but is

considerably less chemically reactive. It is stable to

oxygen, and the range of oxidation states is consider-

ably fewer than for earlier members of the first tran-

sition series. The commonest are þ2andþ3, but, as

the latter is a strong oxidizing agent, it decomposes

rapidly in aqueous solution with the production of

oxygen. As a result, only a few Co

3þ

salts exist, and

these tend to be unstable. However, Co

3þ

forms a

large number of coordination complexes, particularly

with nitrogen donor ligands, and this is important in

its biological role as a structural component of vita-

min B

. This structure is characterized by a ‘corrin’

ring, where the cobalt is coordinated to four coplanar

nitrogen atoms with another (imidazole) nitrogen in

the fifth position. The sixth position, however, makes

vitamin B

unique as, here, the cobalt is bonded to

carbon, making it to date the only naturally occurring

organometallic vitamin.

0005The incorporation of cobalt into the corrin ring

affects its reduction potential and gives it the three

consecutive oxidation states shown in eqn (1). The

reduced Co

species is a highly reactive compound

and can liberate hydrogen from water. Both vitamin

12r

and vitamin B

12s

are very labile in the presence of

air and are instantly oxidized to the Co

3þ

Vitamin B

(Co

) Vitamin B

12r

(Co

)

NaBH

,OH

−

Vitamin B

12s

(Co

)

Red Brown

Blue−gray green

(1)

COBALT 1431

compound cobalamin. The sixth coordination site is

crucial to the biological role of vitamin B

, where it

is involved as a hydrogen carrier in substitution

reactions of the type shown in eqn (2):

(2)

Occurrence in Soil

0006 This is primarily dependent on the geological nature

of the parent material from which the soil is derived.

Thus, soils formed from old red sandstone, granitic,

and other acid igneous, limestone, and predominantly

sandy rocks are liable to be cobalt-deficient.

0007 The availability of soil cobalt and hence its uptake

by pasture and crops are affected by the natural

drainage conditions of the soil, and so cobalt defi-

ciency is most likely to be found on freely drained

soils. Improvement of upland pasture by the removal

of heather and the introduction of more productive

grass species, raising the soil pH by liming, and

improving the drainage tend to lower the availability

of soil cobalt and thus induce cobalt deficiency in

the grazing animal where none existed previously.

Soil maps have been produced based on the cobalt

content of the B horizon and these categorize the

concentrations (in mg kg

1

) as follows: low < 5,

medium 5–15 and high > 15. Areas falling into

the ‘low’ category have the highest risk of cobalt

deficiency. Soils that have a high content of secondary

manganese or iron oxides adversely affect cobalt

availability, as they strongly absorb native cobalt.

Concentrations in Food and Dietary

Intakes

0008Cobalt, like other trace elements, tends to be concen-

trated in young actively growing parts of plants, with

green leafy material often having the highest concen-

trations. Concentrations tend to be lower in stems,

roots, tubers, and cereal grains and also tend to di-

minish as maturity is reached. Typical values for some

foods are given in Table 1 but are scarce. A dietary

and nutritional survey of British adults conducted in

1995 concluded that average dietary intakes of cobalt

were lower than those reported previously. The Com-

mittee on Toxicology stated that levels of cobalt in the

UK diet are not a cause for toxicological concern.

0009There is, however, a wide margin of safety between

the cobalt concentration of ordinary diets and toxic

levels, which are around 250–300 mg kg

1

and thus

more than 1000 times normal dietary concentrations.

High cobalt levels have, however, been implicated as

a triggering factor in cases of severe cardiac failure in

heavy beer drinkers (up to 12 l daily). Cobalt had

been added as a foaming agent in concentrations of

1.2–1.5 mg l

1

, but high alcohol and cobalt intakes,

together with poor-quality diets, combined to pro-

duce a distinctive cardiomyopathy, and so this prac-

tice has been discontinued. Cardiomyopathy has also

been reported in humans following industrial expos-

ure to cobalt. Studies in Germany have suggested that

tbl0001 Table 1 Range of cobalt concentrations in foods

Food Co (mgkg

1

)

Co (mgkg

1

)

Co (mgkg

1

)

Green leafy vegetables 0.20–0.60 0.009 0.01–0.12

Dairy products 0.01–0.03 0.004 0.0005–0.001

Liver and kidney 0.15–0.25 0.06 0.004–0.47

Muscle meat 0.06–0.12 0.004 0.001–0.007

Cereal grains 0.01–0.04 0.01 0.02–0.085

Sugar 0.01–0.03 0.03

Fish <0.10–0.03 0.01 0.004–0.06

Milk 0.50 mgl

1

0.002 0.0008

White bread 0.02

Wholemeal bread

Fruit 0.004 0.002–0.15

Eggs 0.002 0.06–0.35

Peas/beans 0.009 0.03–0.08

Root vegetables 0.006 0.006–0.02

Poultry 0.003 0.019–0.02

Nuts 0.09 0.09–0.34

Values (dry matter) from Mertz W (1987) Trace Elements in Human and Animal Nutrition, 5th edn. London: Academic Press.

Data (fresh weight) from Ministry of Agriculture, Fisheries and Food Total Diet Study (1994) and published by Ysart G, Miller P, Crews H et al. (1999) Food

Additives and Contaminants 16: 391–403.

Data (fresh weight) from Souci SW, Fachmann W and Kraut H (1994) Food Composition and NutritionTables, 5th revised edn. Stuttgart: Scientific Publishers.

1432 COBALT

solutions used for total parenteral nutrition (TPN) are

virtually all contaminated with cobalt, but, even so,

several preparations include supplemental cobalt in

addition. Serum cobalt concentrations in preterm

babies on TPN at 1.44 + 0.48 ng l

1

have been

found to be significantly higher than those

(0.67 + 0.47 ng l

1

) of age-matched controls. No

toxic consequences have been reported, but any clin-

ical consequences of long-term cobalt surplus have

yet to be studied systematically. Most species studied

have a high tolerance to cobalt, particularly sheep,

but recent evidence suggests that this does not extend

to lambs < 6 weeks old. Cattle are more susceptible to

toxic effects from excess cobalt intakes.

0010 Cobalt is present in very low concentration in body

fluids and tissues, with the total content of an adult

human body being < 1.5 mg, with liver, heart, and

bone containing the highest concentrations. Refer-

ence levels are said to be 0.22, < 0.16, 0.06, 0.11,

and < 0.08 mg of cobalt per kilogram (dry matter) in

liver, spleen, kidney, heart, and pancreas, respectively.

Cobalt deficiency in ruminants has been diagnosed on

the basis of liver concentrations of < 0.06 mg kg

1

(dry matter) with values > 0.1 mg kg

1

being regarded

as adequate. Deficiency during pregnancy will result,

with the offspring being born with inadequate con-

centrations, and this has an adverse effect on their

viability. Supplementation of the dams diet or by

direct treatment overcomes this problem.

0011 Cobalt differs from all other essential trace elem-

ents in that it is required by the body in a preformed

compound, vitamin B

, whereas the other elements

are required in ionic form and then converted into

their metabolically active species. Animals and

humans are unable to synthesize vitamin B

. This

ability is found only in some bacteria and algae.

Some of these occur in ruminants, so that, provided

there is an adequate supply of dietary cobalt salts

(> 0.1 mg kg

1

, dry matter), vitamin B

is synthe-

sized in the rumen and then absorbed lower down

the intestinal tract. Humans derive their essential

vitamin B

through the consumption of animal-

related foods. It has been thought that animals do

not produce or require these cobalt-related com-

pounds, and strict vegetarians and vegans are at risk

from pernicious anemia, unless they take synthetic

supplements. Recent evidence suggests that this may

not be strictly true, but it remains practically so, as

the amounts involved would be of little nutritional

significance.

0012 The cobalt atom in vitamin B

catalyzes the reac-

tions of two B

coenzymes – adenosyl cobalamin and

methyl cobalamin – which are essential cofactors in

the activity of methylmalonyl-coenzyme A mutase

and methionine synthetase. The unique nature of the

cobalt–carbon bond in adenosylcobalamin means

that it can undergo reversible homolytic splitting

with the production of free radicals, which are stabil-

ized by the cobalt atom. This allows the intramolecu-

lar rearrangements of the type referred to earlier to

take place. Methylcobalamin has a similar cobalt–

carbon bond that is involved in the synthesis of

methionine. The mechanism has not yet been fully

established, but it is thought that vitamin B

12s

(in

the Co

form) is involved, as it can undergo rapid

addition and substitution reactions.

Analytical Methods

Sample Preparation

0013Since many of the available analytical methods re-

quire cobalt to be in solution, solid samples such as

foods and clinical and biological materials need to be

subjected to a primary dissolution process. Most

commonly, this involves either acid digestion or dry-

ashing procedures, which mineralize cobalt through

destruction of the organic matter present. The most

frequently used ashing technique is by muffle furnace

at temperatures within the range 450–550



C. Alter-

natively, there are low-temperature ashers that oper-

ate between 100 and 200



C under low pressure using

a gaseous oxidant, usually oxygen or an oxygen/tetra-

fluoromethane mixture; the oxidant is activated by a

high-frequency electromagnetic field. Dissolution of

cobalt from the ashed material is usually achieved

using dilute acid.

0014There are a number of different combinations of

acids and oxidizing chemicals that have proved to

be effective digestion mixtures, namely nitric acid/

perchloric acid, nitric acid/perchloric acid/sulfuric

acid, and sulfuric acid/hydrogen peroxide. These

operations have been traditionally performed in an

open Kjeldahl-type flask; however, much more rapid

digestion procedures have recently been developed

using Teflon bomb vessels and microwave heating.

0015Dissolution of cobalt from soils and related mater-

ials can be performed using strong acid digestion,

normally hydrofluoric acid or a hydrochloric acid/

nitric acid mixture (aqua regia), whereas the more

readily available fractions of cobalt are extracted

with dilute acetic acid. Water samples and biological

fluids such as urine and blood plasma can be analyzed

directly using one of the more modern sensitive

techniques such as electrothermal atomization atomic

absorption spectrometry (ETAAAS).

0016Where limited sample preparation of solid mater-

ial, other than drying and milling, is required, a solid

sample presentation can be made using direct current

arc emission spectrometry (DCarcES), direct current

COBALT 1433

plasma atomic emission spectrometry (DCPAES) and

ETAAAS; presentation in slurry form can also be

made with the last two techniques. Nondestructive

analysis can be performed using X-ray fluorescence

(XRF).

Analytical Techniques

0017 Traditional gravimetric and titrimetric methods have

much poorer levels of detection for cobalt than

modern instrumental techniques and are subject to

interferences. Two of the more commonly used pre-

cipitating reagents are 1-nitroso-2-naphthol (interfer-

ence from copper and iron) and anthranilic acid

(interference from iron, nickel and zinc). Typical

titrants are potassium cyanide (interference from

copper, mercury, and zinc), potassium hexacyanofer-

rate (interference from manganese), and ethylene-

diaminetetra acetic acid (EDTA) (interferences from

nickel, zinc, and copper).

0018 Prior to the rapid expansion of flame atomic

absorption spectrometry (FAAS) in the 1960s, the

analysis of cobalt at the microgram level was per-

formed routinely by colorimetric techniques using

spectrophotometry in the ultraviolet and visible

wavelength range (300–700 nm). However, this tech-

nique is also subject to chemical interference from a

wide range of other elements. These have to be elim-

inated either by the addition of masking agents or

buffers, or by solvent extraction of the cobalt com-

plex. The more widely used complexing agents are

ammonium thiocyanate (interferences from iron and

copper), nitroso-R-salt (interferences from iron, chro-

mium, nickel, vanadium, and copper), 1-nitroso-2-

naphthol (interference from copper and iron), and

sodium diethyldithiocarbamate (interferences from

iron, aluminum, chromium, titanium, manganese,

copper, and nickel). By applying modern chemo-

metric procedures to spectrophotometric measure-

ments, cobalt can be determined simultaneously

with other trace elements. Flow injection analysis

has automated many cobalt colorimetric methods.

Recently, sensitive spectrophotometric methods have

been developed that depend on the catalytic effect of

the cobalt iron on the oxidation reaction between

hydrogen peroxide and o-dihydroxybenzene deriva-

tives such as tiron (disodium 1,2-dihydroxybenzene-

3,6-disulfonate), catechol, quinazarin, and gallocya-

nine; detection limits at the picogram level are obtain-

able. A more recent novel utilization of catalysis

incorporates chemiluminescence sensors that meas-

ure the catalytic activity of cobalt on the reaction

between luminol and certain oxidizing agents such

as hydrogen peroxide or periodate salts.

0019 The following spectrographic techniques have been

used for the analysis of cobalt (approximate detection

limits have been quoted to an order of magnitude):

FAAS (detection limit, 0.01 mgg

1

), ETAAAS (detec-

tion limit, 0.0002 mgg

1

), inductively coupled plasma

atomic emission spectrometry ICPAES, detection

limit, 0.01 mgg

1

), inductively coupled plasma

mass spectrometry (ICPMS, detection limit, 0.0002

mgg

1

), DCarcES (detection limit, 0.3 mgg

1

spark source mass spectrometry (SSMS, detection

limit, 0.005 mgg

1

) and DCPAES (detection limit,

0.01 mgg

1

0020FAAS has been the most extensively used technique

for the routine determination of cobalt at the

microgram level in food, agricultural, and biological

materials. The technique is relatively free from

chemical and spectral interferences, and a large

number of preconcentration chemical procedures

have been developed to improve detection levels.

Ammonium pyrrolidinedithiocarbamate and sodium

diethyldithiocarbamate are the two principal che-

lating agents that have been used for multitrace elem-

ent complexation prior to solvent extraction,

although a more cobalt-specific chelate is 1-nitroso-

2-naphthol. A range of different organic solvents have

been used for extraction of the chelated cobalt com-

pounds, the two most common being methyl isobutyl

ketone and chloroform. Ion-exchange resins such as

chelex-100 or C-18 sorbant materials can be used as a

method of on-line preconcentration in FIA systems

for FAAS.

0021ETAAAS has proved to be sufficiently sensitive for

the determination of cobalt at the nanogram level in

clinical and biochemical samples. The technique is

more susceptible to sample matrix interferences than

FAAS, but these have been significantly reduced by

using FAAS-type preconcentration techniques as

separation procedures for the removal of major elem-

ents. Alternatively, matrix modifiers such as palla-

dium, magnesium, or certain inorganic salts such as

ammonium phosphate or thiocyanate have proved

effective. Graphite tubes are normally used for atom-

ization, but more recently, tungsten tubes have been

found to be equally effective. Automatic on-line

preconcentration systems have been developed that

utilize a microcolumn packed with Muromac A-1 che-

lating resin. Analysis of solid samples is now possible

through the incorporation of minature cups that

can hold powdered samples. The resonant absorption

wavelength for cobalt lies in the ultraviolet region at

240.7 nm. Measurements are therefore subject to

background interferences that occur to a greater

degree in electrothermal rather than flame atomiza-

tion. All modern AAS instruments have automatic

background correction; there are four different

types: Zeeman, deuterium continuum, Smith–

Hieftje, and xenon continuum in the simultaneous

1434 COBALT

multielement atomic absorption continuum instru-

ments. The normal detection limit for cobalt as deter-

mined by ETA-AAS has been further improved

through the coupling of laser technology with electro-

thermal atomization. This is either in the form of

laser-excited atomicfluorescence spectrometry or

with the laser-enhanced ionization technique.

0022 The 1980s saw a rapid growth in the number of

commercial ICPAE spectrometers being used for mul-

tielement analysis. Since the detection limit for cobalt

by ICPAES is similar to that for FAAS, similar pre-

concentration techniques have been applied. The two

most prominent emission lines are almost equal in

sensitivity; however, since the line at 238.8 nm is

subject to strong spectral interference from iron, the

cobalt line at 228.6 nm has been preferentially used,

but this suffers from a significant spectral interference

from titanium and slight spectral interference from

nickel and iron. The exact magnititude of the inter-

fering spectral overlap depends on the spectral

resolution of each particular type of spectrometer.

Interfering elements that produce strong spectral

overlap used to be removed by chemical separation

prior to instrumental analysis, whereas those that

exhibit a small degree of spectral overlap can be

corrected for by appropriate interelement spectral

subtraction procedures.

0023 Since the mid 1980s, there has been a steady

growth in the application of ICPMS to multielement

ultratrace analysis of clinical and biochemical

samples. Detection limits are about the same as for

ETAAAS, and therefore, cobalt can be determined at

the nanogram level. Interferences occur from the pro-

duction of polyatomic ions within the plasma that

have the same atomic mass as

Co, e.g., calcium–

oxygen and argon–sodium. These are corrected for

by measuring the background levels of such ions in

simulated blank solutions. Automatic on-line pre-

concentration systems have also been developed for

ICPMS.

0024 DCarcES, SSMS, and XRF have mainly been

applied to the direct analysis of cobalt in solid

samples such as soils and related materials; with

these techniques, matrix matching of samples with

calibration standards and choice of internal standards

are the most critical aspects of the determination. The

application of the DCarcES to plant and agricultural

materials has been largely superceded by AAS and

ICPAES.

0025 Electrochemical techniques have proved to be very

effective in the determination of cobalt at the micro-

gram and nanogram levels in food, biological, and

environmental samples, being among the most sensi-

tive techniques for the determination of cobalt in

natural waters. During the 1980s, the technique of

adsorptive stripping voltametry was developed as the

most sensitive of the polarographic methods for trace

metal analysis. For the determination of cobalt, the

square wave stripping is more sensitive than the dif-

ferential stripping mode. Electrolytic preconcentra-

tion of cobalt at the mercury electrode is carried out

with chelated cobalt, since the free cobalt ion would

be irreversibly adsorbed. The most widely used che-

lating agent has been dimethyl glyoxime; adsorption

of the cobalt dimethyl glyoximate occurs at 0.7 V,

and the voltammetric peak at 1.12 V in a cell system

using a silver/silver chloride/potassium chloride elec-

trode as the nonworking electrode. Interference from

zinc can be masked by the addition of nitrilotriacetic

acid or sodium iminodiacetate; nickel interferes if

present in great excess, but this is reduced by using

alternative chelating agents such as 1,2-cyclohexane-

dione dioxime or a-furil dioxime for complexing the

cobalt. More recently, cathodic stripping voltamme-

try coupled with FIA has become a successful add-

ition to the range of electrochemical techniques used

for cobalt analysis.

0026High-performance liquid chromatography (HPLC)

has been successfully applied to the determination

of cobalt and other trace metals in plant materials,

natural waters, and environmental samples, and

detection levels of nanogram quantities are obtain-

able. The use of disubstituted dithiocarbamates

such as sodium diethyldithiocarbamate, ammonium

pyrrolidinedithiocarbamate, and ammonium bis(2-

hydroxyethyl)dithiocarbamate has been favored,

since they rapidly form stable complexes with a

wide range of trace metals. These metal complexes

can be separated chromatographically using alkyl-

bonded silica columns and quantitatively determined

with ultraviolet/visible diode array detectors or

electrochemical detectors. Precolumn derivitization

can be done off-line using solvent extraction for

water-insoluble complexes or in-line using a presen-

tation column loaded with an appropriate ion-pair

reagent such as cetyltrimethylammonium bromide-

dithiocarbamate. On-column derivatization can be

performed by including the dithiocarbamate reagent

in the mobile phase, usually a water/methanol

mixture; however, this process is less sensitive than

precolumn derivatization. Other complexing chelat-

ing agents that can be used include EDTA or 4-(2-

pyridylazo)resorcinol. Metal ion chromatography

can be applied to cobalt analysis using an appropriate

complexing chelating agent such as pyridine-2-

carboxyaldehyde phenylhydrazone as a stationary

phase immobilized on a silica matrix.

0027HPLC has been successfully interfaced with a wide

range of spectroscopic instruments (FAAS, ETAAAS,

ICPAES, and ICPMS) to provide powerful techniques

COBALT 1435