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0006 Thiamin is readily split into the pyrimidine and

thiazole moieties by sulfite treatment at pH 6.0 or

above.

0007 In strong alkaline solution thiamin is oxidized by

oxidants (potassium ferricyanide, cyanogen bromide,

mercuric chloride, and others) to thiochrome (Figure

1). Thiochrome is a highly fluorescent compound

above pH 8, and thiamin phosphate esters are also

quantitatively converted to thiochrome phosphate

esters without affecting the phosphate bonds. All

these compounds have identical excitation maxima,

at 375 nm, and very similar fluorescence maxima, at

432–435 nm. This is the principle for the most

common and sensitive assay procedures for thiamin

as well as its phosphate esters.

0008 Hydroxyethylthiamin (Figure 1) exists in living

organisms in the form of hydroxyethyl-TDP. It is

converted to the corresponding thiochrome deri-

vative by alkaline ferricyanide oxidation but not

by cyanogen bromide oxidation. This difference

in the oxidation property is used for the assay of

hydroxyethylthiamin.

0009In aqueous solution below pH 5, thiamin is quite

stable to heat or even sterilization at 110



C. At a pH

of 5.5 or higher, it is rapidly destroyed by autoclaving,

and at pH 7 or higher by boiling or even storing at

room temperature.

0010In solution, TDP is unstable and partially decom-

poses to TMP and/or thiamin when stored for several

months at pH 5 and 37



C, but stable at pH 2–5 and



C. TTP in aqueous solution is stable for at least 6

months when stored at 80



C. In 0.1 mol l

1

hydro-

chloric acid, TTP is decomposed quantitatively to

TMP by boiling for 7 min at 100



C, indicating

H Thiamin

Thiamin

Alkali

oxidation

TMP

OHP

CHOH

OHP

TPP

TTP

Hydroxyethylthiamin

Thiochrome

fig0001 Figure 1 Structures of thiamin (vitamin B

) and its related compounds.

5768 THIAMIN/Properties and Determination

the nature of the high-energy bond of its g- and b-

phosphates.

0011 Thiamin-destroying or inactivating enzymes have

been discovered in a variety of foods. Two thiami-

nases are involved. Thiaminase I catalyzes a base

exchange between the thiazole moiety and another

base and is found in fresh fish, shellfish, ferns, and

some bacteria. Thiaminase II hydrolyzes thiamin to

its pyrimidine and thiazole moieties and is found in

certain bacteria.

0012 In addition to thiaminases, certain plants have been

shown to contain compounds which react with thia-

min in vitro to produce thiochrome-negative prod-

ucts. One such product is thiamin disulfide. Such

compounds in foods, which result in so-called thia-

min inactivation, include caffeic acid, tannic acid, a

variety of polyphenols, and some flavonoids (querce-

tin and rutin). The exact mechanisms of the inactiva-

tion reaction have not yet been elucidated. It appears

that oxidative processes are involved, since the pres-

ence of oxygen increases the amount of thiamin

inactivated.

0013 It has been discovered that the treatment of thiamin

with an extract of garlic containing allicin forms thia-

min allyl disulfide, which is thiochrome-negative, but

very active biologically. A variety of thiamin alkyl

disulfides has since been synthesized and studied,

and is now available commercially. These forms of

thiamin are more fat-soluble and are therefore more

rapidly absorbed than thiamin hydrochloride, and

result in higher blood thiamin levels when therapeut-

ically administered.

Occurrence in Foods

0014 In most animal tissues, over 90% of the thiamin

occurs in TMP, TDP, and TTP. The predominant

form (80–85%) is TDP, the active coenzyme form.

The exceptions are pig skeletal muscle and chicken

skeletal white muscle, in which TTP exists in 70–80%

of total thiamin (Figure 2). The most abundant form

in plant tissues is free thiamin.

0015 The content of thiamin in foods is relatively low.

Practically no thiamin is contained in high-fat prod-

ucts (e.g., vegetable oil) and refined products (e.g.,

sugar). It is also relatively low in green vegetables,

fruits, and seafoods. The thiamin content in a large

variety of natural and processed foods has been listed

in publications from the US Department of Agricul-

ture (USA), Medical Research Council (UK) and Re-

sources Council, Science and Technology Agency

(Japan).

0016 Table 2 lists the expected concentration in thiamin-

rich foods. Refer to individual foods.

0017In addition, average thiamin contents (m g per

100 g) in the following categorized groups of foods

are as follows: dried beans, 680; nuts, 560; whole-

grain cereals, 370; organ meats (liver, heart, kidney),

100; leaf and stem vegetables, 70; milk, 40; fruits, 30;

pork muscle, 600–800; common white fish (cod,

flounder, haddock, halibut), 50–90; hen’s whole egg,

170; egg yolk, 500. Interesting foods of high thiamin

content (mg per 100 g) are dried brewers’ yeast

(1800), wheat germ (2000), and tampala (a spinach-

like vegetable) leaves (1600).

Losses During Food Processing and

Storage

0018Factors affecting the survival of thiamin in the final

food products after harvesting, handling, and pro-

cessing the foods include pH, temperature, solubility,

oxidation, and radiation. Among these factors, the

effect of processing on the thiamin content has been

extensively reviewed by several authors. A brief sum-

mary of the findings is given here.

0019Thiamin losses in dehydrated products, such as

cereal grains, are very low if the materials are kept

dry, and thiamin undergoes only limited destruct-

ion in the commercial refrigeration of meats and

Retention time (min)

(a) (b)

fig0002Figure 2 A typical chromatogram of fresh pig skeletal muscle

extract by precolumn derivatization high-performance liquid

chromatography (HPLC). (a) Skeletal muscle extract; (b) as (a),

plus 1 pmol of authentic thiamin triphosphate (TTP) which was

converted into thiochrome triphosphate and then added to the

oxidized sample. The recovery rate of added TTP was 100%. 1,

Thiamin; 2, thiamin monophosphate (TMP); 3, thiamin diphos-

phate (TDP); 4, TTP; 5, unknown, but possibly thiamin tetra-

phosphate.

THIAMIN/Properties and Determination 5769

vegetables. Foods can be stored frozen for long

periods without significant loss of thiamin, but

losses are significant on thawing. Losses from frozen

muscle meats are 20–40% during storage for 2–8

months.

0020 On cooking and canning of meats, losses of thia-

min (25–85%) strongly depend on the processing

stresses encountered and type of meat product.

Cooking by microwaves leads to much lower losses.

Losses on cooking of vegetables vary from 0% to

60%. Vigorous boiling or other types of agitation

result in greater losses of thiamin than simmering at

the same temperature or pressure and steam cooking.

Losses are caused by degradation and by leaching.

(See Canning: Quality Changes During Canning;

Cooking: Domestic Techniques.)

0021Roasting of beef and pork involves thiamin losses

of 36–53%. Ionizing radiation used during steriliza-

tion destroys 53–88% of thiamin in meats, yet, in

certain poultry, fish, and vegetables, losses are only

5–37%. Baking of bread leads to losses of 5–35%. In

the processing of milk, losses of thiamin are 9–20%

by pasteurization, 30–50% by sterilization, 10–15%

by drying, and 40% by condensing and subsequent

canning. (See Irradiation of Foods: Applications.)

0022The milling of cereal grains, wheat flour extraction

of less than 85%, and the degerming of corn cause a

marked drop in thiamin. Parboiling of rice and drying

before milling conserve the majority of the thiamin.

(See Milling: Characteristics of Milled Products.)

Uses in Food Fortification

0023Thiamin hydrochloride is the most common additive.

(See Food Fortification.)

0024Thiamin (125 mg per 100 g) is added to polished

rice and (1.5 mg per 100 g) to pressed barley in Japan,

mostly in special cases, such as for use in school

lunches.

0025The recommended daily allowance of thiamin is

0.5 mg per 1000 kcal. The continuing need for thia-

min fortification of processed cereals has been pro-

posed in the UK (1986), in order to meet the adult

thiamin requirement, which has been determined bio-

chemically.

0026Human thiamin deficiency, called beriberi, has

been largely eliminated in those countries where for-

tification of staple foods (rice, cereal, dairy products)

is practiced, but it is still prevalent in those countries

where unfortified foods are used. However, even in

western countries it is becoming evident that subclin-

ical thiamin deficiency is more widespread than pre-

viously realized, especially among infants and the

elderly, and other groups who consume snacks and

highly refined, processed foods. Thiamin fortification

of rice, dairy products and cereals is also recom-

mended for these ’at-risk’ groups and for athletes

whose metabolic demands are greater.

Analytical Procedures

0027Alkaline oxidation of thiamin by either ferricyanide

or cyanogen bromide gives thiochrome, an intense

blue fluorescence compound. This principle is the

basis for the official standard method of the Associ-

ation of Official Analytical Chemists (AOAC). How-

ever, high-performance liquid chromatography

(HPLC) is most frequently used at the present time

for the assay of thiamin in foods. Microbiological

tbl0002 Table 2 Top sources of thiamin

To p s o u rc e s T hiami n

(mg100 g

1

)

Yeast, brewers’, debittered 15.61

Yeast, torula 14.01

Yeast, brewers’ 12.12

Sunflower seed, flour, partially defatted 3.60

Yeast, bakers’, dry 2.33

Rice bran 2.26

Wheat–soy blend (WSB)/straight grade,

wheat flour

2.02

Wheat germ 2.02

Sunflower seed kernels, dry, hulled 1.96

Rice polish 1.84

Wheat germ, toasted 1.65

Wheat–soy blend (WSB)/bulgur, flour 1.49

Pinenuts, pin

on 1.28

Coriander leaf, dried 1.25

Cottonseed flour 1.21

Cornflakes, with added nutrients 1.20

Peanuts, raw with skins 1.14

Pork, fresh, loin, lean, boiled 1.13

Safflower seed, flour, partially defatted 1.12

Soybean flour, defatted 1.09

Alfalfa seeds 1.08

Sesame seed 0.98

Bacon, Canadian, broiled or fried, drained 0.92

Sausage, links or bulk, cooked 0.79

Wheat bran 0.72

Kidneys, beef, braised 0.67

Wheat flour, all-purpose, enriched 0.64

Rye flour, dark 0.61

Nuts, mixed, shelled 0.59

Wheat flour, whole (from hard wheats) 0.55

Pork, cured, canned, ham 0.53

Cornmeal, degermed, enriched 0.44

Rice, white, enriched, raw 0.44

Soybean sprouts, cooked 0.42

Bread, white, enriched 0.40

Peas, green, immature, boiled, drained 0.28

Turkey, hamloaf 0.27

Beef liver 0.25

Luncheon meat, salami, cooked 0.25

From Ensminger AH (ed.) (1994) Foods and Nutrition Encyclopedia, 2nd edn,

pp. 2102–2108. Boca Raton: CRC Press.

5770 THIAMIN/Properties and Determination

assays are still also widely used for both foods

and animal feeds. (See Chromatography: High-

performance Liquid Chromatography.)

0028 An outline of the manual fluorimetric AOAC

method follows. Thiamin in foods is protein-bound

in both animal and plant tissues, although it occurs

principally as the diphosphate in the former and as

the free form in the latter. It is therefore necessary to

extract thiamin and its phosphates from food pro-

teins.

0029 This is carried out by boiling the finely ground or

homogenized sample in 0.1 mol l

1

hydrochloric acid

or 0.1 mol l

1

sulfuric acid. The extract is then neu-

tralized to pH 4–4.5 and treated with an enzyme

preparation exhibiting phosphatase activity (Taka-

diastase, Mylase P, or Clarase). After filtration or

centrifugation, free thiamin thus obtained is purified

through a column of cation exchange-type silica (Per-

mutit T, Decalso F, or Zepolite S/E). After washing the

column several times with almost boiling water,

the thiamin is eluted with almost boiling acid potas-

sium chloride solution.

0030 An aliquot of the above eluate is oxidized by alka-

line ferricyanide or alkaline cyanogen bromide solu-

tion and another aliquot is mixed with an alkali

solution (blank). Thiochrome thus obtained is ex-

tracted into isobutanol. Thiochrome fluorescence in-

tensity is measured spectrofluormetrically at an

excitation wavelength of 375 nm and an emission

wavelength of 435 nm. The amount of thiamin ex-

tracted is calculated from the standard curve obtained

with thiamin standard solution treated in the same

manner as above. Fortified foods and pharmaceut-

icals can be analysed for thiamine with reduced

sample preparation.

Chromatographic Methods

0031 Recent advances in HPLC techniques allow the deter-

mination of thiamin content in foods more rapidly,

accurately, sensitively, and reproducibly than the

standard AOAC method. The purification step of

the extracts can usually be omitted in the HPLC

procedure. In addition, thiamin and its phosphate

esters can be determined separately by the HPLC

method when the appropriate extraction procedure

is employed. Furthermore, thiamin, riboflavin, pyrid-

oxine, or niacin in foods can be simultaneously

assayed when appropriate detection systems are

used. This procedure has been successfully used for

quantifying these vitamins in a wide range of foods,

including cereal products, raw meat, processed meats,

fruits, and vegetables.

0032 Samples are treated, for example, by the AOAC

method and then subjected to enzymatic hydrolysis.

The filtrate or the supernatant after centrifugation

can be directly analyzed by a reversed-phase column

with ion-pairing chromatography. A wide range of

mobile phases has been employed, but those based

on octane sulfonate (as ion pair) in aqueous methanol

or acetonitrile have proved to be generally successful.

Detection is performed spectrophotometrically at

254 nm, which gives a detection limit on injection of

30 ng (90 pmol) of thiamin. This procedure is suitable

for food containing a relatively large amount of

thiamin, especially for fortified rice or cereals. (See

Spectroscopy: Overview.)

0033An HPLC system equipped with fluorometric de-

tection is suitable to quantify much smaller amounts

of thiamin in foods with a detection limit of 5–

50 fmol thiamin. Both precolumn derivatization and

postcolumn derivatization procedures of thiamin into

thiochrome are used.

0034When the extraction procedure is carried out

with cold trichloroacetic acid or perchloric acid,

thiamin and its phosphate esters are extracted intact

and can be individually measured. Both straight-

phase and reversed-phase columns are used in the

HPLC method. In the former system, thiamin,

TMP, TDP, and TTP are eluted and detected in

that order, and in the latter system the elution

order is reversed. Total thiamin is then calculated

as a sum of thiamin and its phosphates. This pro-

cedure can avoid the enzymatic hydrolysis step.

This HPLC method of determining thiamin and its

phosphates has been successfully used to quantify

the thiamin level not only in animal tissues, but also

in human blood or serum to assess thiamin status in

humans.

0035The chromatographic profile of thiamin and its

phosphates in fresh pig skeletal muscle, obtained by

the precolumn derivatization method, is shown in

Figure 2. The analytical system consisted of LiChro-

sorb NH

(150  4.6 mm inner diameter (ID), 5 mm

particle size) as the stationary phase, and acetonitrile–

90 mmol l

1

potassium-phosphate buffer (pH 8.4)

(60:40, v/v) as the mobile phase. Detection was

carried out fluorometrically (excitation, 375 nm;

emission, 430 nm) at room temperature.

Microbiological Methods

0036Microbiological methods are based on the nutritional

requirement of a particular microorganism for the

vitamin in question, in this case thiamin. A nutrient

medium which is complete in all respects except for

thiamin is prepared for the microorganism. The

growth response for standard vitamin solutions is

then compared with that achieved with extracts

of the food sample, and hence the concentration of

THIAMIN/Properties and Determination 5771

thiamin in the sample is calculated. In most instances

it is necessary to extract thiamin from foods under

hydrolytic conditions and it may also be necessary to

release bound forms with an enzymatic digestion, as

in the standard thiochrome method.

0037 Over the years, many microorganisms have been

used in the assay of thiamin. Assays based on the use

of Lactobacillus fermentum and L. viridescens are

now widely employed as these microorganisms re-

spond only to intact thiamin. Measurement of rate

of growth is usually followed by nephelometry or

measurement of turbidity, although production of

acidity may also be monitored.

0038 Microbiological methods are slowly being replaced

by more rapid instrumental techniques, e.g., HPLC.

However, they do provide simple, sensitive analyses,

especially where a high degree of precision is not

required.

See also: Canning: Quality Changes During Canning;

Chromatography: High-performance Liquid

Chromatography; Coenzymes; Cooking: Domestic

Techniques; Food Fortification; Irradiation of Foods:

Applications; Milling: Characteristics of Milled Products;

Spectroscopy: Overview

Further Reading

Budavari S (ed.) (1996) The Merck Index. An Encyclopedia

of Chemicals, Drugs, and Biologicals, 12th edn. Rah-

way. NJ: Merck.

Ensminger AH (ed.) (1994) Foods and Nutrition Encyclo-

pedia, 2nd edn, pp. 2102–2108. Boca Raton: CRC Press.

Fayol V (1997) High-performance liquid chromatography

determination of total thiamin in biological and food

products. In: McCormik DB, Suttie JW and Wagner C

(eds) Methods in Enzymology, vol. 279, pp. 57–66. San

Diego: Academic Press.

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

Chemistry, 3rd edn, pp. 531–616. New York: Marcel

Dekker.

Helrich K (1990) Official Methods of Analysis of the Asso-

ciation of Official Analytical Chemists, 15th edn,

pp. 1049–1052. Washington: Association of Official

Analytical Chemists.

Kamas E and Harris RS (1988) Nutritional Evaluation of

Food Processing, 3rd edn. New York: Van Nostrand

Reinhold.

Kawasaki T and Egi Y (2000) Vitamin B

: thiamin. In:

DeLeenheer A, Lambert W and Van Bocxlaer J (eds)

Modern Chromatographic Analysis of the Vitamins,

3rd edn, pp. 365–389. New York: Marcel Dekker.

Machlin LJ (ed.) (1991) Handbook of Vitamins, 2nd edn.

New York: Marcel Dekker.

Paul AA, Southgate DAT and Buss DH (1986) McCance

and Widdowson’s The Composition of Foods: Supple-

mentary Information. London: Her Majesty’s Stationery

Office.

Resources Council. Science and Technology Agency. Japan

(1998) Standard Tables of Food Composition in Japan,

5th edn. Tokyo: Ministry of Finance Printing Office.

US Department of Agriculture (1995) Composition of

Foods. US Department of Agriculture. Human Nutrition

Information Services, Agriculture Handbook 8. Wash-

ington, DC: US Government Printing Office.

Physiology

R Bitsch, Friedrich-Schiller-University, Jena, Germany

Background

0001The discovery of thiamin or vitamin B

as an essential

nutrient has opened a new area in the field of nutri-

tion research in two respects. First, the nomenclature

’vitamin,’ used for the whole class of the later on

discovered essential micronutrients of organic origin,

was derived from a functional group (NH

group) of

the molecule. Second, thiamine was the first nutri-

tional factor that could be identified as a curative

agent against a deficiency disease in animals.

0002At the end of the nineteenth century, the Dutch

medical officer Ch. Eijkman discovered the beneficial

effect of rice bran or unpolished rice, respectively, for

the treatment of polyneuritis in chickens resembling

beriberi in man, which had been previously induced

by feeding polished rice. In 1911, the German-Polish

chemist C. Funk isolated a crystalline substance from

rice bran and regarded the amino function as the

essential principle of the molecule. The exact chem-

ical structure of the thiamin molecule was elucidated

by R. Williams and A. Windaus. Because of the sulfur

content of the molecule or its thiazol moiety com-

bined with the amine function, the compound was

designated as thiamin instead of the former name

aneurin. Nevertheless, the classical form of thiamin

deficiency, beriberi, mainly affects human beings and

has been well known in the rice-eating countries of

East Asia for centuries. Even 25 years after the ingeni-

ous work of Eijkman, who clarified the cause of this

deleterious disease, the mortality rate from beriberi in

industrialized countries such as Japan was still more

than 25 000 per year.

Physiological Function

0003Like other water-soluble B-vitamins, thiamin is in its

primary function coenzymatically active. The thia-

min-dependent enzymes are involved in carbohydrate

and energy metabolism (Figure 1). Key enzymes in

5772 THIAMIN/Physiology

these metabolic pathways are mitochondrial pyruvate

dehydrogenase (EC 1.2.4.1) linking glycolysis with

the citric acid cycle, a-ketoglutarate dehydrogenase

(KDH) (EC 1.2.4.2) within the citric acid cycle, and

the cytosolic transketolase (EC 2.2.1.1) in the pentose

phosphate shunt. Hence, it follows that the energy

turnover of the organism is related to thiamin

demand. As dietary carbohydrates are the predomin-

ant fuel for the rapid metabolizable energy of the

organism, the thiamin requirement is often related

merely to the carbohydrates ingested. KDH was iden-

tified as the rate-limiting enzyme of the cerebral glu-

cose utilization. A low enzyme activity caused by a

reduced intracellular TDP concentration results in

lactacidosis, cerebral dysfunction of the energy me-

tabolism, and neuronal cell degeneration. Additional

thiamin-dependent enzymes are involved in the

metabolism of branched-chain amino acids, e.g., leu-

cin, isoleucin, and vallin. All enzyme reactions are

characterized by transfer of an ’active aldehyde’

from a-keto acids formed after previous decarboxyla-

tion to coenzyme A or an aldopentose.

0004The coenzymatically active form is thiamin diphos-

phate (TDP), also called thiamin pyrophosphate

(TPP) or cocarboxylase, which is formed in the muco-

sal cell of the intestinal barrier with the aid of thiamin

pyrophosphokinase (EC 2.7.6.2) in the presence of

ATP. The latter enzyme is also found in mammalian

liver and other tissues such as brain, nerves, and heart

of mammals and birds. Further phosphorylated forms

of thiamin, which are found in varying quantities in

tissue, are thiamin monophosphate (TMP) and thia-

min triphosphate (TTP). These derivatives are synthe-

sized and metabolized according to the scheme in

Figure 2.

0005Thiamin phosphorylation represents a partitioning

process in a deeper compartment. Cell membranes

are impermeable for the intracellularly active

Dietary

carbohydrates

Glycogen

Glucose

Pentoses Nucleic acids

Glycolysis

Gluconeogenesis

Pyruvate Lactate

Amino acids

(ketogenic)

Acetyl-CoA

Fatty acids

Amino acids

(glucogenic)

Oxalacetate

Succinyl-CoA

α-ketoglutarate

fig0001 Figure 1 Metabolic pathways involving thiamin.

THIAMIN/Physiology 5773

coenzyme TDP, which is released only after hydroly-

sis via TMP to thiamin. The total thiamin concen-

tration of the blood cells as well as of the

cerebrospinal fluid (CSF) has a nonlinear relation to

the corresponding plasma concentrations. A linear

increase in blood cells and CSF has been observed

only with thiamin plasma levels above 10 nM

(3 mgl

1

). Otherwise, with extremely low plasma

and CSF concentrations, a disproportionately high

TDP content of erythrocytes and TMP content of

the CSF is maintained for a longer period. Thus, the

intracellular phosphorylation should be seen as a pro-

tecting mechanism, inhibiting rapid thiamin deple-

tion by decreasing the membrane permeability.

0006 After TDP, TTP is the next most phosphorylated

metabolite, albeit the steady state concentrations in

tissues are rather low. No coenzymatic function has

been found for this metabolite. Several investigators

have demonstrated in a variety of animals (rats,

rabbits, cows, frogs, pigs) that TTP, together with

a distinct part of TDP, is neurophysiologically active

in the central and peripheral nervous system inde-

pendent of the coenzyme function of the latter. After

electrical stimulation of nerves or treatment with

neuroactive drugs, e.g., acetylcholine, tetrodotoxin,

or LSD, a release of the phosphorylated esters TDP

and TTP concomitant with a shift to TMP and free

thiamin has been observed. Furthermore, in patients

with the deleterious ’subacute necrotizing encephalo-

pathia,’ or Leigh’s disease, the TTP level in the brain

was low and tended toward zero. It has been known

for a long time that severe thiamin depletion of the

mammalian central nervous system leads to encepha-

lopathias, which are accompanied by regionally se-

lective changes in neurotransmitter function. Though

the exact function of TTP is not yet known, it plays an

essential role in nerve excitation and transmission,

possibly in gating the ion transport. From recent in-

vestigations, it has been argued that TDP might also

be involved in the inhibition of nonenzymatic glyco-

sylation processes. It is suggested that the emerging

advanced glycosylation end products (AGE) are the

reason for the segmental demyelinization observed

in neurodegenerative diseases. Studies on cultivated

cells have demonstrated that thiamin and TDP could

prevent the AGE formation. In this regard, TDP is

more effective than the standard inhibitor aminogua-

nidine that is normally used.

Absorption, Storage, and Excretion

0007Thiamin is absorbed from the gastrointestinal tract

by a dual system, as has been shown for humans

and rats. At low concentrations, it follows energy-

dependent active transport with a saturation kinetics

up to a maximal concentration of 2 mM. At higher

concentrations, a simple diffusion process is predom-

inant. However, the relative absorption decreases

with increasing vitamin doses, so that with gram

doses, only about 5% are incorporated.

0008A close association was assumed between thiamin

transport and phosphorylation as well as Na

de-

pendency. However, the coupling of thiamin trans-

port with phosphorylation is now doubtful, and

there is much evidence of a specific protein carrier

being involved in the absorption, but the following

intracellular phosphorylation seems to be a rate-

limiting step in the active transport of thiamin across

the epithelial cells of intestine. In contrast, in the

guinea-pig intestine, thiamin seems to cross the

brush-border membrane by simple diffusion through-

out the concentration range from 0.06 to 10 mM.

0009The jejunum and ileum have been found to be the

main sites of thiamin absorption in rats with a max-

imal rate in the proximal 22 cm of the small intestine.

This specificity seems to resemble that of other

animals as well as humans.

0010After uptake from the intestinal tract, thiamin

is distributed rapidly into the organs and tissues

according to the requirements. In humans and

animals, the heart contains the highest thiamin

concentration (3–8 mgg

1

fresh weight), followed by

the kidney (2.4–6 mgg

1

fresh weight) and liver (2–

7.5 mgg

1

fresh weight), and the brain (1.4–4.4

mgg

1

fresh weight). Lesser amounts are found in

other tissues. Whole blood contains 0.05–0.12 mg

of thiamin per milliliter, about 90% of this being

found in the corpuscular constituents. The total

storage capacity of man is estimated to be 25–30 mg

of thiamin, 40% of this being in muscles. Inter-

estingly, pig muscle has a relatively high thiamin con-

tent (8–12 mgg

1

) because of feeding conditions,

higher than that in muscles of other mammals

(1–2 mgg

1

) and pig liver (2–4 mgg

1

). The thiamin

contents of several plant and animal foods are given

in Table 1.

ATP AMP ATP ADP

TDP

TTP

TMP

Thiamin (T)

fig0002 Figure 2 Interconversion of thiamin phosphates (according to

Rindi, modified).

5774 THIAMIN/Physiology

0011 As with other water-solublevitamins, thiaminexcre-

tion is dependent on intake. Within the physiological

range, most of it is excreted by urine. Besides free

thiamin and small amounts of TMP, numerous metab-

olites can be detected as minor excretion products such

as thiazol, pyramin, and methylthiazol acetic acid.

0012 After ingestion of therapeutic dosages, the amount

of thiamin excreted via bile increases proportionally,

as does the unabsorbed part in feces.

Lipid-soluble Thiamin Derivatives

0013Under relatively mild conditions (e.g., in the gastro-

intestinal lumen) thiamin reacts with allicin, the

active principle of garlic and onions, to form a lipid-

soluble compound, called allithiamin, that is able to

penetrate through membranes more easily than the

water-soluble salts (Figure 3). Additional S-alkyl de-

rivatives have been identified in cruciferous plants,

and several lipid-soluble derivatives have been

synthesized with a better stability than that of the

spontaneously formed allithiamine (S-benzoyl-, S-

tetrahydrofurfuryl analogs being the most important).

0014Oral administration of these lipid-soluble analogs

results in higher thiamin blood levels than with equi-

molar thiamin doses, because the allithiamins from

the gut lumen are preferentially absorbed by passive

diffusion and rapidly converted to the active vitamin

by reducing SH-compounds. These allithiamins are

because of their improved bioavailability suitable

transfer substances with which to establish high

thiamin levels in target tissues comparable with drug

targeting. The elevated blood level results in an

improved vitamin status, as measured by enzymatic

criteria.

Thiamin Antagonists

Synthetic Antivitamins

0015Like most of the B vitamins, thiamin has a rather high

structural specificity, i.e., minor modifications of

the molecule lead to a decrease or a total loss of

Thiaminium ion

Ethylthiamin

N-propythiamin

Oxythiamin

Pyrithiamin

Amprolium

Allithiamin

Ethyl

N-propyl

Methyl

Amino

Hydroxy

Amino

Ethanol

Hydrogen

Ethanol

Sulfur

Ethene

S-alkyl

(thiazol oxidatively

splitted at C

)

⬘

fig0003 Figure 3 Thiamin derivatives and antagonists.

tbl0001 Table 1 Thiamin content of plant and animal foods (mg per

100 g fresh weight)

Content Foods

1.0–2.0 Whole wheat and rye Plant foods

0.9 Para nuts

0.7–0.8 Soy flour

0.6–0.7 White bran

0.3–0.5 Whole cereal products, lentils, oats,

unpolished rice, lima beans, whole

corn meal, cashew nuts, pea nuts,

hazel and walnuts, mung beans

0.1–0.2 Parboiled rice, sorghum, beans and

peas cooked, cauliflower, potatoes,

fennel, broccoli cooked, asparagus,

soy sprouts, mushroom

< 0.1 Fruits, milk products, white flour

products

0.9 Loin pork Animal foods

0.3–0.5 Liver, kidney (calves, beef, pork), duck

0.1–0.2 Lean meat (calves, lamb, beef), eel,

tuna, salmon, mackerel, plaice and

flounder, oysters

Modified from Souci SW, Fachmann W and Kraut H (2000) Food

Composition and Nutrition Tables, 6th revised edn. Boca Raton, FL: CRC

Press/Medpharm Scientific.

THIAMIN/Physiology 5775

physiological activity or even to antagonistic effects.

Of the numerous analogs synthesized so far, only

the 2-ethyl- and the 2-n-propyl homologs in the

pyrimidine moiety of the molecule show any

biological activity comparable with that of thiamin.

For example, when the 4-amino group is replaced

by a hydroxyl function, oxythiamin, one of the

most potent thiamin antagonists, is obtained. An-

other prominent antivitamin is pyrithiamin, which

contains an ethylene group instead of sulfur in the

thiazolium ring. Some thiamin antagonists have

been found to exhibit antiprotozoal activity, as in

the case of amprolium, which is effective as a

prophylactic agent for coccidiosis of fowl. Some

of the modified thiamin derivatives are shown in

Figure 3.

0016 The use of antagonists results in rapid and severe

deficiency symptoms in humans and animals. Ampro-

lium is already known to affect the absorption process

already, and this has been suggested as the mechanism

for its anticoccidial action in fowls. Other antivita-

mins appear to function at the conzyme level, i.e., they

bind to the apoenzyme, but inhibit the following en-

zymatic reaction. Nevertheless, there are several dif-

ferences in the action on neurological symptoms. Only

pyrithiamin (and not oxythiamin) can block the

action potential of a single myelinated nerve fiber. It

is also well known that only pyrithiamin produces

polyneuritis. In addition, differences in neurophysio-

logical action are caused by the different abilities of

both antagonists to cross the blood–brain barrier,

which has only been verified in the case of pyrithia-

min. However, it can be shown that the amino group

of the thiamin molecule is essential for the active

transport of the vitamin from the rat small intestine

and the formation of thiamin pyrophosphate.

Thiaminases and other Antithiamin Factors

0017 Two thiamin-cleaving enzymes have been identified,

called thiaminase I and thiaminase II.

0018 Thiaminase I is found in shellfish, clams (but not

oysters), some freshwater fish viscera, crustacea, and

certain ferns, but very few higher plants. Also, certain

species of Bacillus and Clostridium, which are com-

ponents of the human and animal intestinal flora,

have been found to produce this enzyme. The enzyme

catalyzes an exchange reaction, in which the thiazol

moiety of the molecule is displaced by another N-

containing base or a SH-compound. The effect of

this compound was first observed when silver foxes

were fed raw fish waste, resulting in a deleterious

thiamin deficiency disease called Chastek paralysis.

Thiaminase I may also be produced by the rumen

microflora of ruminants or by plants, and, in the

presence of suitable cosubstrates, e.g., niacin, or pyr-

idoxine, and certain antihelmintics, seems to be

responsible for the ruminant CNS disorder, polio-

encephalomalacia.

0019Thiaminase II is of bacterial origin (predominantly

Bacillus, Candida, and Oospora) and breaks down

the free vitamin, but not the thiamin pyrophosphate,

into pyrimidin and thiazol components. More preva-

lent are thermostable thiamin-inactivating factors of

plant origin, e.g., polyphenolic substances such as

flavonoids and catechol derivatives in fermented tea,

ferns, sweet potatoes, and betel nuts, and, in small

quantities, in other leaves, fruits, and roots. These can

decompose the thiazol component of the vitamin,

accelerate the oxidation to the disulfide form, or

form unabsorbable adducts with thiamin. Rats fed

on food high in polyphenolics have been shown to

develop deficiency symptoms as a result of a marked

decrease in levels of cerebral thiamin and thiamin-

dependent enzymes. Likewise in man, high tea con-

sumption leads to a deficient thiamin status, but

ascorbic acid, if present, completely inhibits the thia-

min-inactivating processes.

Status Assessment

0020In terms of biochemical status, urinary vitamin excre-

tion and blood enzymes are suitable criteria. Urinary

thiamin excretion is, as with most B vitamins, largely

dependent on intake and therefore provides an indi-

cation of the recent dietary intake but does not ad-

equately reflect the body stores. At intake levels

below 0.5 mg of thiamin per 1000 kcal per day

(0.1 mg MJ

1

) thiamin excretion varies only slightly,

and the proportion of metabolites in the urine exceeds

the vitamin content itself. Because urinary excretion

follows a diurnal rhythm, 24 h urine or the creatinine

excreted is taken as the basis for status assessment.

Occasionally, the status can be interpreted more ac-

curately by a thiamin load test. After administration

of a 5-mg dose, vitamin excretion is measured for the

following 4 h. A deficient status can be assumed if less

than 20 mg is excreted during this period.

0021Erythrocytic transketolase activity (ETKA), the

rate-limiting enzyme in the pentose phosphate path-

way, and its activation coefficient, a

ETK

are often used

as an indication of functional status as they give a

better idea of body stores than urinary excretion.

According to the saturation deficit of the apoenzyme,

the transketolase activity can be stimulated in vitro by

the addition of TDP. The ratio of stimulated to basal

activity, which increases with the degree of deficiency,

indicated by the activation coefficient (AC) or a

ETK

,is

commonly taken as a prognostic status index. The AC

is better as a comparison of status in groups, because

5776 THIAMIN/Physiology

of the large interindividual variation in basal enzyme

activity. Moreover, it can be assumed that apoenzyme

levels are not affected by the vitamin deficiency.

Nevertheless, in alcoholics, either the apoenzyme syn-

thesis or the linkage to the coenzyme TDP seems to be

impaired, and the transketolase assay may indicate

misleading results about the true status. Furthermore,

confounding factors in the enzymatic assay, irrespect-

ive of the thiamin status, may include certain cancers,

uremic neuropathy, diabetes, and treatment with

certain anticancer drugs (fluorouracil, acytoxin) or

diuretics, such as furosemide.

0022 Recently, in view of improved high-performance

liquid chromatography techniques, the TDP content

in erythrocytes has been suggested to be a better

indicator of the body stores compared with ETKA

and independent of confounding factors. The criteria

of the thiamin status indices are listed in Table 2.

Thiamin Requirement

0023 Regarding the essential role of thiamin-dependent

enzymes in carbohydrate and energy metabolism,

the requirement for this vitamin is usually related to

energy intake. Irrespective of the catalytic function of

enzymes emerging basically unchanged from a bio-

chemical or metabolic reaction, controlled studies in

man have demonstrated that static as well as func-

tional status criteria (transketolase activity and urin-

ary excretion) deteriorate with increasing energy

uptake even when the thiamin supply remains un-

changed, thus indicating an increased vitamin turn-

over. The dependence on energy metabolism seems to

be particularly evident in a marginal vitamin status.

0024 Previously dietary fat was postulated to have a

’sparing effect’ on thiamin requirements. This effect

is, however, comparatively small, even with major

increases in fat in the diet. Furthermore, in a starva-

tion or semistarvation status when the vitamin intake

is zero and increasing amounts of fatty acids are

metabolized to satisfy the body’s energy needs, the

tissue stores of thiamin are rapidly depleted.

0025International committees recommend safe levels

of intake amounting to 100–130 mg of thiamin per

megajoule, equivalent to 0.4–0.54 mg of B

per

1000 kcal. A critical intake point appears to be ap-

proximately 0.05 mg of B

per megajoule (0.2 mg per

1000 kcal), below which urinary excretion is low, and

the first clinical signs of deficiency may appear. A

minimum intake of 1 mg per day is recommended to

provide an adequate margin, even for those consum-

ing less than 8 MJ (2000 kcal) daily. The additional

values for pregnancy and lactation mostly exceed the

calculated additional requirement of pregnant and

lactating women. These extra values insure optimal

pre- and postnatal development. In the past, clinical

deficiency symptoms of infants were sporadically ob-

served in spite of an inconspicuous appearance of the

mother (infantile beri beri; see Table 4).

0026Owing to the low storage capacity, regular thiamin

intake is important. Although, within the margin of

physiological requirements, the organism attempts to

prevent considerable thiamin losses by partial tubular

reabsorption and intracellular fixation as TDP, the

biological half-life time of this vitamin is assumed to

be 9–18 days. After this period, when thiamin uptake

is restricted, early nonspecific deficiency symptoms

occur.

0027The recommended daily allowances for several

European countries together with the USA’s dietary

reference intakes are listed in Table 3.

Deficiency Diseases

0028In thiamin deficiency, primarily, organs and tissues,

such as the central and peripheral nerve system and

the heart are affected showing an increased energy

turnover or close carbohydrate dependency. Clinical

signs described in most animals refer to lesions in

these tissues.

0029Since Eijkman’s observations in fowls, the degener-

ation of the peripheral nerves was the first patho-

logical symptom of thiamin deficiency noted in

tbl0002 Table 2 Reference values of thiamin status parameters

Deficiency

Indicator Adequate Marginal Severe

Thiamin excretion in urine

(mg per gram of creatinine)

> 66 27–66 < 27

ETKA–AC < 1.15 1.15–1.25 > 1.25

TDP content in erythrocytes

(ng per milliliter of sediment)

> 38.5 29.5–38.5 < 29.5

From Finglas PM (1994) Thiamin. International Journal for Vitamin and

Nutrition Research 63: 270–274 and Frank T, Bitsch R, Maiwald J and Stein

G (2000) High thiamine diphosphate concentrations in erythrocytes can be

achieved in dialysis patients by oral administration of benfotiame.

European Journal of Clinical Pharmacology 56: 251–257.

tbl0003Table 3 National RDAs for vitamins

Country B

(mg per day)

Poland 1.9–2.0

Belgium, Denmark, Finland, France, Hungary,

Luxemburg, Portugal, Spain

1.4

Greece, Norway 1.1–1.4

Czech Republic, Sweden 1.2

Austria, Germany, Ireland, Switzerland 1.0–1.3

The Netherlands, USA 1.0–1.2

Italy, UK 0.8–1.2

THIAMIN/Physiology 5777