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so these methods are only applicable for analyses of

the biotin content of vitamin preparations, liquid

infant formula, etc.

0048 A maximal intensification of the chemolumimetric

biotin detection can be achieved by using the biolu-

minescent protein aequorin as a label in a competitive

binding assay. Aequorin is a photoprotein from the

jellyfish Aequorea victoria. Aequorin associates non-

covalently with its chromophore coelenterazine,

which is oxidized into a metastable excited state,

resulting in the emission of photons at 469 nm.

0049 In a homogenous assay, the aequorin–biotin conju-

gate competes in a one-step procedure with free biotin

for binding to avidin in solution. Binding of the

conjugate to avidin results in partial quenching of

the bioluminescence signal, and so the decrease in

luminescence can be related to the concentration of

biotin in a sample. The detection limit is reported to

be around 10

14

M biotin (¼ 10 fM).

0050 The competition assay can also be performed in a

heterogenous system by using immobilized avidin

(agarose, polyacrylamide). Both methods need special

equipment (luminometer operating in a photon-

counting mode). The amounts of avidin and biotinyl-

ated aequorin must be optimized beforehand, as the

relative amounts of avidin and biotinylated aequorin

influence the response characteristics of the assay

(e.g., detection limits and sensitivity).

Chromatographic Analysis

0051 Earlier methods attempted to separate and quantify

biotin from vitamin mixtures or feed supplements.

After dissolution of the lyophilized preparation and

addition of the internal standard (2-imidazolidone),

the sample was applied on a TLC plate and eluted

with chloroform/methanol/formic acid. Biotin was

visualized by spraying with p-dimethylaminocinna-

maldehyde and determined in situ by reflectance

measurements. Spraying with paraffin after the

coloring procedure increased the sensitivity to a

detection limit of 10 ng of biotin per spot.

0052 Quantitation by HPLC requires derivatization

of the molecule, because biotin does not absorb in

the visible or UV region. For the derivatization

9-anthryldiazomethane was used to produce fluores-

cent biotin-9-anthrylmethylester followed by separ-

ation on C 18 bonded columns with acetonitrile/

water as the mobile phase and fluorimetric detection

(excitation ¼365 nm, emission ¼425 nm). Alterna-

tively, p-bromophenacyl bromide esters and 4-

bromomethylmethoxycoumarin derivatives of biotin

were produced for UV and fluorescence detection,

respectively, after separation on RP 18 columns with

methanol/water or tetrahydrofuran/water as eluents.

0053Standards of biotin and 13 metabolites and analogs

can be separated by HPLC on a C 18 RP phase using

gradient elution with trifluoroacetic acid/acetonitril

and UV detection at 220 nm. An HPLC separation of

six vitamins including biotin from almonds has been

performed on an LC-8-DB column with hexanesulfo-

nic acid sodium salt/methanol as the eluent and de-

tection at 200 nm. The limit of biotin detection has

been found to be as low as 0.9 mg per 100 g.

0054When biotin is derivatized with panacyl bromide in

the presence of crown ether, a fluorescence compound

results with fluorescence maxima at 380 nm (excita-

tion) and 470 nm (emission). After extraction from

biological tissue with trichloroacetic acid and purifi-

cation by solid-phase extraction combined with ion-

exchange chromatography on DOWEX and TLC,

biotin is derivatized with panacyl bromide in the

presence of crown ether. The resulting panacylester

can be separated on normal-phase HPLC using

isocratic elution with methanol/dichloromethane as

well as on RP phase HPLC using gradient elution

(water/methanol). Dethiobiotin is taken as an internal

standard.

0055With this method, the biotin content in rat small

intestine has been assessed. The detection limits are

reported to be 10 pmol of biotin (normal phase

HPLC) or 100 pmol of biotin (RP-HPLC), which is

less sensitive than with protein-binding assays. Criti-

cism results from the facts, that the clean-up required

for biological sample extracts prior to derivatization

is very extensive, the derivatization should not only

include biotin, and the cross-reactivity to biotin

analogs has to be examined.

See also: Analysis of Food; Chromatography: High-

performance Liquid Chromatography; Enzymes: Uses in

Analysis; Food Composition Tables; Immunoassays:

Principles; Radioimmunoassay and Enzyme

Immunoassay; Lactic Acid Bacteria; Microbiology:

Classification of Microorganisms; Spectroscopy:

Fluorescence; Vitamins: Determination

Further Reading

Baker H, Frank O, Matovitch VB et al. (1962) A new assay

method for biotin in blood, serum, urine and tissue.

Analytical Biochemistry 3: 31–39.

Bitsch R, Salz I and Hoetzel D (1986) Biotin assessment in

foods and body fluids by a protein binding assay. Inter-

national Journal for Vitamin and Nutrition Research 59:

59–64.

Bonjour JP (1991) Biotin. In: Machlin LJ (ed.) Handbook of

Vitamins, pp. 393–427. New York: Marcel Dekker.

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

Analysis for the Health and Food Sciences. Boca

Raton, FL: CRC Press.

BIOTIN/Properties and Determination 515

Fidanza F (1991) Nutritional Status Assessment. London:

Chapman & Hall.

Green NM (1970) Spectrophotometric determination of

avidin and biotin. Methods in Enzymology 18: 418–424.

Guilarte T (1985) Measurement of biotin levels in human

plasma using a radiometric-microbiological assay.

Nutrition Reports International 31: 1155–1163.

Hentz NG and Bachas LG (1997) Fluorophore-linked

assays for high-performance liquid chromatography

postcolumn reaction detection of biotin and biocytin.

Methods in Enzymology 279: 275–286.

Lizano S, Ramanathan S, Feltus A, Witkowski A and

Dannert S (1997) Bioluminescence competitive binding

assays for biotin based on photoprotein aequorin.

Methods in Enzymology 279: 296–303.

Schroeder HR, Vogelhut PO, Carrilco RJ, Boguslaski RC

and Buckler RT (1976) Competitive protein binding

assay for biotin monitored by chemoluminescence.

Analytical Chemistry 48: 1933–1937.

Physiology

J Zempleni, University of Nebraska-Lincoln, Lincoln,

NE, USA

D M Mock, University of Arkansas for Medical

Sciences, Little Rock, AR, USA

Introduction

0001 Biotin is a water-soluble vitamin that serves as essen-

tial coenzyme for four mammalian carboxylases.

Biotin may also play a role in the replication and

transcription of DNA, possibly by an effect on bioti-

nylation of histones. Frank biotin deficiency causes

clinical findings such as hair loss and skin rash. Mar-

ginal biotin deficiency is frequently seen in pregnant

women and theoretically could have teratogenic

effects. Biotin status may be assessed using urinary

excretion of biotin and its metabolites, and certain

organic acids that are byproducts of deficiency of

biotin-dependent carboxylases. Direct measurement

of biotin-dependent carboxylases and biotin in

lymphocytes and fibroblasts also show promise as

indicators of biotin status. Inborn errors of biotin

metabolism (e.g., biotinidase deficiency and holocar-

boxylase synthetase deficiency to a variable degree)

can produce symptoms similar to biotin deficiency.

For adults, the safe and adequate daily dietary intake

of biotin is 30 mg. Available data suggest that pure

biotin administered orally is absorbed completely.

Uptake of biotin into cells of the intestine and periph-

eral tissues is mediated by a sodium-dependent trans-

porter that requires metabolic energy. Mammals

catabolize biotin by b-oxidation of the valeric acid

side chain and by sulfur oxidation in the thiophane

ring. Biotin, bisnorbiotin, and biotin, d,l-sulfoxide

are the most abundant biotinyl compounds in urine

and plasma.

Biochemical Function

0002In mammals, holocarboxylase synthetase (EC

6.3.4.10) catalyzes the covalent binding of biotin

to the E-amino group of lysine in four different apo-

carboxylases to form the active holocarboxylases

(Figure 1). For acetyl-CoA carboxylase (EC 6.4.1.2),

a cytosolic (acetyl-CoA carboxylase a) and a mito-

chondrial form (acetyl-CoA carboxylase b) have been

identified. Both the a and b forms catalyze the bind-

ing of bicarbonate to acetyl-CoA to form malonyl-

CoA; the latter is a substrate for fatty acid synthesis.

Although the a and b forms of acetyl-CoA carboxyl-

ase catalyze the same reaction, they appear to have

different roles in intermediary metabolism. Acetyl-

CoA carboxylase a controls fatty acid synthesis in

the cytosol by providing the substrate malonyl-CoA;

acetyl-CoA carboxylase b controls fatty acid oxida-

tion inside the mitochondria via malonyl-CoA inhib-

ition of fatty acid transport into mitochondria. (See

Fatty Acids: Metabolism.)

0003The three other mammalian biotin-dependent car-

boxylases are located exclusively in mitochondria:

pyruvate carboxylase (EC 6.4.1.1), propionyl-CoA

carboxylase (EC 6.4.1.3), and b-methylcrotonyl-

CoA carboxylase (EC 6.4.1.4). By synthesizing oxalo-

acetate, pyruvate carboxylase provides a tricarboxylic

acid cycle intermediate and catalyzes a step in gluco-

neogenesis. Propionyl-CoA carboxylase catalyzes an

essential step in the metabolism of isoleucine, valine,

the cholesterol side chain, and products of dietary

carbohydrate breakdown by intestinal microorgan-

isms. b-Methylcrotonyl-CoA carboxylase catalyzes

an essential step in leucine metabolism.

0004Biotin deficiency causes reduced carboxylase activ-

ities; substrates are shunted to alternative pathways

(Figure 1). For example, reduced activity of propio-

nyl-CoA carboxylase results in increased formation

of 3-hydroxypropionic acid and 2-methylcitric acid;

reduced activity of b-methylcrotonyl-CoA carboxyl-

ase results in increased formation of 3-hydroxyisova-

leric acid and 3-methylcrotonyl glycine. Increased

urinary excretion of these organic acids has been

used to diagnose biotin deficiency, as discussed below.

0005Biotin may also play a role in the regulation of

DNA transcription and replication on the basis of

the following observations:

0006Biotinidase (EC 3.5.1.12) specifically biotinylates

histones. Histones are DNA-binding proteins that

516 BIOTIN/Physiology

regulate transcription and replication of DNA.

Biotinylation of histones might play some role in

DNA packaging in analogy to other covalent

modifications such as acetylation, methylation,

phosphorylation, or adenosine diphosphate (ADP)-

ribosylation of histones. Biotinidase is ubiquitous

in mammalian cells, and 25% of the cellular bio-

tinidase activity is located in the nucleus. Indeed,

the following observations are also consistent with

a role for biotin in modifying histones and in turn

affecting the packaging of DNA: first, histones

dissociate from the DNA in biotin-deficient rats,

and second, biotin deficiency in rats results in

decreased phosphorylation and methylation of

histones as well as increased acetylation of

histones.

0007 Rates of biotin uptake are greater in proliferating

than in nonproliferating lymphocytes. This in-

crease is mediated by an increased number of

biotin transporters on the cell surface. Cell

proliferation requires a substantial increase of

both replication and transcription. Theoretically,

the observed increased uptake of biotin by

proliferating cells may be in response to increased

demand for biotin to biotinylate histones. This is a

potential mechanism by which biotin exerts an

effect on cell replication.

0008Biotin status affects gene expression. Biotin defi-

ciency causes a 40–45% reduction of rat liver

glucokinase activity; the activity is restored by

biotin administration. Injection of biotin into rats

causes a 4–19-fold increase of mRNA encoding

glucokinase.

0009In addition to this effect on mRNA synthesis,

biotin may affect the synthesis of some proteins at

the posttranscriptional level. When human-derived

liver (HepG2) cells were cultured in biotin-deficient

medium, expression of the asialoglycoprotein recep-

tor was reduced despite normal protein synthesis,

total cellular protein content, and mRNA coding

for asialoglycoprotein receptor; addition of biotin or

biocytin restored receptor expression. These observa-

tions suggest that a biotin-dependent posttranscrip-

tional event can affect the ultimate expression of

asialoglycoprotein receptor.

Cytosol and mitochondria

Mitochondria

Acetyl CoA Malonyl CoA

ACC

SCoA

CHOOC C COOH

β-Methylcrotonyl CoA

β-Methylglutaconyl CoA

3-H

drox

isovaleric acid and 3-meth

lcroton

cine

C SCoA HOOC CH

C CH C SCoA

MCC

HOOC

SCoACH

Propionyl CoA

3-Hydroxypropionic acid and 2-methylcitric acid

Biotin-

deficient

Biotin-

deficient

Methylmalonyl CoA

SCoA

PCC

Pyruvic acid Oxaloacetic acid

COOH

HOOC H

C C SCoA

fig0001 Figure 1 Biotin-dependent carboxylases. ACC, acetyl-CoA carboxylase; PC, pyruvate carboxylase; PCC, propionyl-CoA

carboxylase; MCC, b-methylcrotonyl-CoA carboxylase.

BIOTIN/Physiology 517

Storage, Metabolism, and Excretion

0010 Depletion and repletion experiments of biotin-

dependent carboxylases in rat liver provided evidence

that mitochondrial acetyl-CoA carboxylase may serve

as a reservoir for biotin. Neither cytosolic acetyl-

CoA carboxylase nor the mitochondrial pyruvate

carboxylase, propionyl-CoA carboxylase, or b-

methylcrotonyl-CoA carboxylase seem to serve as

biotin reservoirs.

0011 The liver accumulates a significant percentage of

ingested biotin. For example, rat liver accumulates

approximately 4% of [

C]biotin within 1 h of intra-

venous administration. Once steady-state conditions

are attained, biotin is located mainly in liver mito-

chondria (30% of total [

C]biotin) and cytosol

(59%); smaller amounts can be found in microsomes

and nuclei. Greater than 80% of the [

C]biotin

present in the cytosolic fraction is acid-precipitable,

suggesting that biotin has become covalently bound.

0012 During the normal intracellular turnover of pro-

teins, holocarboxylases are degraded to biotin linked

to lysine (biocytin) or biotin linked to an oligopeptide

containing at most a few amino acid residues. Bio-

tinidase catalyzes the hydrolysis of the amide bond

between biotin and lysine. Biotinidase likely serves in

both absorption of biotin (cleavage of protein-bound

dietary biotin) and recycling of biotin (cleavage of

biocytin). Biotinidase also catalyzes a specific bioti-

nylation of histones, as noted above. (See Protein:

Synthesis and Turnover.)

0013 A significant proportion of biotin undergoes cata-

bolism before excretion (Figure 2). Two principal

pathways of biotin catabolism have been identified

in mammals. In the first pathway, the valeric acid

side chain of biotin is degraded by b-oxidation. b-

Oxidation of biotin leads to the formation of bisnor-

biotin, tetranorbiotin, and related intermediates that

are known to result from b-oxidation of fatty acids.

The cellular site of this b-oxidation of biotin is uncer-

tain. Spontaneous (nonenzymatic) decarboxylation

of the unstable b-keto acids (b-keto-biotin and b-

keto-bisnorbiotin) leads to formation of bisnorbiotin

methyl ketone and tetranorbiotin methyl ketone;

these catabolites appear in urine.

0014 In the second pathway, the sulfur in the thiophane

ring of biotin is oxidized, leading to the formation

of biotin l-sulfoxide, biotin d-sulfoxide, and biotin

sulfone. Sulfur oxidation may be catalyzed by a

NADPH-dependent process in the smooth endoplas-

mic reticulum. Combined oxidation of the ring sulfur

and b-oxidation of the side chain lead to metabolites

such as bisnorbiotin sulfone. In mammals, degrad-

ation of the biotin ring to release carbon dioxide

and urea is quantitatively minor.

0015On a molar basis, biotin accounts for approximately

half of the total avidin-binding substances in human

serum and urine (Table 1). Bisnorbiotin, bisnorbiotin

methyl ketone, biotin d,l-sulfoxide, and biotin sul-

fone account for most of the balance. Using thin-layer

chromatography and staining with p-dimethyl-

aminocinnamaldehyde, tetranorbiotin l-sulfoxide

was also identified in human urine. However, avidin

binding of this metabolite was too weak to allow

quantitation.

0016The biliary route of biotin excretion is quantita-

tively minor. For example, in rats, approximately 2%

of intravenously administered [

C]biotin was ex-

creted in bile but approximately 61% was excreted

in urine.

0017The relationship of metabolite profile to biotin

nutritional status has not been fully elucidated. In

human and rat urine, the percent excretion of biotin

increases when the biotin intake is increased from

physiologic to pharmacologic amounts. This may

reflect saturation of renal reabsorption, metabolic

pathways, or both.

Deficiency

Clinical Symptoms of Frank Biotin Deficiency

0018The fact that humans have a requirement for biotin

has been clearly documented in two situations: first,

parenteral nutrition without biotin supplementation

in patients with short-gut syndrome and other causes

of malabsorption; and second, prolonged consump-

tion of raw egg white. The critical event in the egg

white-induced biotin deficiency is a highly specific

and very tight binding (k

¼10

mol l

1

) of biotin

by avidin, a glycoprotein found in egg white. Avidin

is resistant to intestinal proteolysis in both the free

and the biotin-bound form. Thus dietary avidin binds

and prevents the absorption of biotin. Cooking de-

natures avidin, rendering it susceptible to digestion

and hence unable to interfere with absorption of

biotin. (See Eggs: Dietary Importance.)

0019Biotin deficiency has also been reported or inferred

in several other circumstances, including pregnancy,

individuals undergoing dialysis, individuals suffering

from chronic gastrointestinal disease, Leiner’s dis-

ease, and sudden infant death syndrome.

0020The clinical findings of frank biotin deficiency in

adults and older children are similar regardless of

whether deficiency is caused by egg-white feeding or

omission of biotin from parenteral nutrition. Typi-

cally, the findings begin to appear gradually after an

interval of 6 months to 3 years of parenteral nutrition

or after 6 weeks to several years of egg-white feeding.

Thinning of hair, often with loss of hair color, was

518 BIOTIN/Physiology

reported in most patients. A skin rash described as

scaly (seborrhoeic) and red (eczematous) was present

in the majority; in several, the rash was distributed

around the eyes, nose, and mouth. Depression,

lethargy, hallucinations, and paresthesias of the

extremities were prominent neurological symptoms

in the majority of adults.

0021 In infants who developed biotin deficiency, the

signs of deficiency began to appear within 3–6

months of initiation of total parenteral nutrition.

The rash initially appeared around the eyes, nose,

and mouth; ultimately, the ears and perineal orifices

were involved. The appearance of the rash was simi-

lar to that of cutaneous candidiasis (i.e., an erythe-

matous base and crusting exudates); typically,

Candida could be cultured from the lesions. The

character and distribution of the biotin deficiency

rash are quite similar to the rash of zinc deficiency.

In some infants, hair loss can progress to total bald-

ness, including loss of eyebrows and lashes. The most

striking neurological findings in biotin-deficient

infants was hypotonia, lethargy, and developmental

delay. In these infants, a peculiar withdrawn behavior

was often noted and may have reflected the same

central nervous system dysfunction diagnosed as

depression in adult patients.

Biochemical Markers of Biotin Deficiency

0022Our studies provide evidence that marginal biotin

deficiency is much more common than frank biotin

Biotin

(CH

)

COOH

HN NH

Bisnorbiotin

(CH

)

COOH

HN NH

Biotin sulfoxide

(CH

)

COOH

HN NH

Tetranorbiotin

COOH

HN NH

Biotin sulfone

(CH

)

COOH

HN NH

fig0002 Figure 2 Pathways of biotin catabolism.

BIOTIN/Physiology 519

deficiency. Diagnosis of marginal biotin deficiency

requires sensitive metabolic indicators. The best val-

idated indicators of marginal biotin deficiency are

urinary excretion of biotin and metabolites and urin-

ary excretion of organic acids such as 3-hydroxyiso-

valeric acid (see above: Figure 1). These organic acids

are synthesized and excreted in increased quantities in

biotin-deficient individuals because the normal catab-

olism of their precursors by biotin-dependent carb-

oxylases is reduced. Activities of biotin-dependent

carboxylases in lymphocytes and fibroblasts and

plasma concentrations of biotin and metabolites

have been reported to be abnormally low in individ-

uals who developed frank biotin deficiency after

1 month of biotin-free intravenous feeding.

Teratogenic Effects of Biotin Deficiency

0023 Biotin deficiency is teratogenic in several animal

species at degrees of deficiency that produce no obvi-

ous findings in the pregnant animal. Hens with

marginal biotin deficiency produce eggs with higher

embryonic mortality, reduced hatchability, chrono-

dystrophy (‘parrot beak’ deformity), perosis (an ab-

normality of bone tendon formation that results in

a deformity analogous to ‘club foot’), micromelia,

and syndactyly. Effects on hatchability and viability

have been reported in turkey poults. In some strains

of mice, asymptomatic biotin deficiency during

pregnancy causes substantial increases in fetal

malformations and mortality. Ninety-four percent of

pups from biotin-deficient dams had cleft palate,

85% had micrognathia, and 41% had micromelia;

multiple malformations were common.

0024 Biotin status may be reduced during human preg-

nancy. In the majority of pregnant women, excretion

of 3-hydroxyisovaleric acid is significantly greater

than the upper limit of normal. The serum concen-

tration of biotin commonly decreases significantly

from early to late pregnancy and reaches values that

are below the lower limit of normal in some individ-

uals. Likewise, the urinary excretion rates of biotin

and bisnorbiotin are often significantly less in late

pregnancy than in early pregnancy.

Inborn Errors of Metabolism

0025Two important classes of inborn errors of biotin

metabolism have been identified:

0026Biotinidase deficiency is an inborn error of metab-

olism that is characterized by partial (70–90%)

or profound (> 90%) reduction of biotinidase

activity. The combined incidence of partial plus

profound biotinidase deficiency is 1: 60 000 new-

borns. Clinical findings of biotinidase deficiency

largely mimic biotin deficiency and include devel-

opmental delay, seizures, skin infection, hepato-

megaly, and splenomegaly. These symptoms are

thought to be a consequence of one or more im-

pairments leading to reduced biotin status: (i) de-

ficient salvage of covalently bound biotin during

holocarboxylase turnover may result from in-

complete release of biotin from biocytin; (ii) sub-

stantial amounts of biocytin are lost in urine;

(iii) release of covalently bound dietary biotin

may be impaired, leading to malabsorption.

Biotinidase-deficient patients respond well to

physiologic or pharmacologic doses of biotin.

0027Multiple carboxylase deficiency is an inborn error

of metabolism in humans that is caused by defi-

ciency of holocarboxylase synthetase, leading to

less efficient biotinylation of apocarboxylases.

Isolated carboxylase deficiencies are caused by

mutations of genes encoding the mammalian

apocarboxylases.

(See Inborn Errors of Metabolism: Overview.)

Intestinal Absorption

0028Studies in rats provided evidence that intestinal

uptake of biotin occurs by two distinct mechanisms.

The first is a saturable, structurally specific trans-

porter located in the brush border membrane. This

transporter is dependent on sodium and energy, and is

electroneutral. As judged by the maximal transport

rate (V

max

) of the transport process, the transporter is

appropriately up- and downregulated by biotin defi-

ciency and biotin excess, respectively. In the rat,

biotin transport is most active in the upper small

bowel; activity of biotin transport declines proceed-

ing from the jejunum to the ileum to the colon. How-

ever, activity in the colon is still 3% of jejunal values,

leaving open the possibility that biotin synthesized by

tbl0001 Table 1 Biotin and metabolites in human serum and urine

Compound Serum (pmoll

1

)Urine(nmol24h

1

)

Biotin 244 + 61 35 + 14

Bisnorbiotin 189 + 135 68 + 48

Biotin d,l-sulfoxide 15 + 33 5 + 6

Bisnorbiotin methyl ketone ND

9 + 9

Biotin sulfone ND

5 + 5

Total biotinyl compounds 464 + 178

122 + 66

Means + SD are reported (n ¼15 for serum; n ¼6 for urine).

ND, not determined. Bisnorbiotin methyl ketone and biotin sulfone had

not been identified at the time when this study of serum was conducted and

hence these unknowns were not quantitated against authentic standards.

Including three unidentified biotin metabolities.

From Zempleni J and Mock DM (1999) Biotin biochemistry and human

requirements. Journal of Nutrition and Biochemistry 10: 128–138, with

permission.

520 BIOTIN/Physiology

enteric flora might contribute to absorbed biotin. In

the rat, activity of the biotin transporter increases

with age from suckling to weanling to young adult

(aged 3 months). As with the other changes observed

in the biotin transporter, the mechanism leading to

increased transport appears to be an increase in the

number or activity of the carriers, rather than changes

in the affinity of the carrier.

0029 The second mechanism of intestinal biotin absorp-

tion is passive diffusion. This process predominates at

large luminal biotin concentrations; at that point, the

biotin transporter is likely saturated.

0030 The biotin transporter in human intestine is similar

to that identified in rats. The human transporter is

saturable, electroneutral, sodium-dependent, and

capable of accumulating biotin against a concentra-

tion gradient. The anatomical distribution along the

human gastrointestinal tract is similar to that ob-

served in the rat. Changes in transport activity appear

to be mediated primarily by an increase in the number

or activity of transporters, rather than by changes in

the affinity of the transporter. The exit of biotin from

the enterocyte (i.e., transport across the basolateral

membrane) is also carrier-mediated; however, biotin

export is independent of sodium, is electrogenic, and

cannot accumulate biotin against a concentration

gradient.

0031 Recently, a biotin transporter in human tissues has

been cloned, sequenced, and expressed in mammalian

cells. This multivitamin transporter binds and trans-

ports not only biotin but also pantothenic acid and

lipoic acid. Binding affinities are similar for these

nutrients.

0032 Clinical studies have also provided evidence that

biotin is absorbed from the human colon. When biotin

is instilled directly into the lumen of the colon, the

plasma concentration of biotin increases; however,

when the same dose of biotin is given orally, the

increase in plasma concentration is greater.

Bioavailability

0033 Recent reports provide evidence that biotin is

absorbed almost completely even if large doses of

biotin are ingested. However in these studies, biotin

was administered in aqueous solution rather than

being endogenous to the diet. A large percentage of

biotin in food may be bound to protein and therefore

is likely to require cleavage from the protein in order

to be transported into enterocytes by the biotin trans-

porter. Biotinidase is thought to be responsible for

release of biotin from protein. The release of biotin

might occur in or near the intestine mucosa via the

action of mucosal biotinidase; biotinidase in pancre-

atic juice might release biotin during the luminal

phase of proteolysis. Theoretically, biotinyl oligopep-

tides might also be absorbed directly, either by a

specific biotin transporter or by a peptide transporter.

However, the mechanism remains to be elucidated.

Biotinidase is present in pancreatic juice and in intes-

tinal mucosa, but biotinidase activity is not enriched

in intestinal brush border membranes. Currently, only

limited data are available regarding the bioavailabil-

ity of protein-bound biotin in food. (See Bioavail-

ability of Nutrients.)

Transport of Biotin from the Intestine to

Peripheral Tissues

0034Biotinidase has been proposed to serve as a biotin-

binding protein in plasma and as a carrier protein

for the transport of biotin into the cell. Two biotin-

binding sites on biotinidase have been identified. One

had an equilibrium dissociation constant (k

)of

3 nmol l

1

and a maximum binding capacity (B

max

)

of 0.065 mol of biotin per mol of biotinidase;

the other had a k

of 59 nmol l

1

and a B

max

0.79 mol of biotin per mol of biotinidase. However,

empirical studies with [

H]biotin provide evidence

that biotin binds primarily to albumin. Of the total

biotin in human plasma, 81% is free, 12% is cova-

lently bound, and 7% is reversibly bound.

Transport of Biotin in Liver and Peripheral

Tissues

Biotin Uptake into Liver

0035Rat liver cells accumulate biotin by a transporter-

mediated process that depends on metabolic energy;

uptake is sodium-dependent and Na-K-ATPase-

dependent. The ureido portion of the biotin molecule

plays a role in binding to the transporter. Organic

anions such as bilirubin and cholic acid compete

with biotin uptake by hepatocytes.

0036Biotin uptake into HepG2 cells is also transporter-

mediated, energy-dependent, sodium-dependent, and

Na-K-ATPase-dependent; a free carboxyl group on

the valeric acid side chain of biotin is essential for

recognition by this transporter. Apparent Michaelis

constant (K

) and V

max

in HepG2 cells are

19.2 mmol l

1

and 6.8 nmol mg protein

1

min

1

respectively.

Biotin Uptake into Lymphocytes

0037In analogy to liver cells, human lymphocytes accumu-

late biotin by a transporter that requires metabolic

energy and is dependent on Na-K-ATPase. Apparent

and V

max

values of this biotin transporter are

BIOTIN/Physiology 521

2.6 nmol l

1

and 2.9 fmol 10

cells

1

30 min

1

, re-

spectively. In lymphocytes, the thiophane portion of

the biotin molecule is more important than the ureido

portion for binding to the transporter. The biotin

transporter in lymphocytes is very specific for biotin;

inhibition of biotin transport by hexanoic acid, lipoic

acid, bilirubin, and pantothenic acid is minor. The

same transporter that mediates biotin uptake into

lymphocytes also accounts for biotin efflux from

these cells as judged by countertransport experiments.

Biotin Uptake into the Central Nervous System

0038 Biotin enters the central nervous system by a satur-

able transport system. A free carboxylic acid group is

important for transport across cerebral capillaries;

pantothenic acid and nonanoic acid compete for

uptake, suggesting that uptake is mediated by an

organic anion carrier. In the brain, biotin is covalently

bound to proteins, presumably biotin-dependent

carboxylases.

Placental Transport of Biotin

0039 A biotin transporter is present in the placental brush

border membrane. In mammals, a single protein

termed the multivitamin transporter can transport

biotin, pantothenic acid, and lipoic acid across the

placenta; this protein has been cloned and function-

ally expressed. A recent study of fetal and maternal

plasma concentrations of biotin at 18–24 weeks’ ges-

tation of normal human pregnancies reported a fetal-

to-maternal biotin ratio ranging from 3 to 17:1,

consistent with active transport across the placenta.

Requirements and Adequate Intakes

Adequate Intakes

0040 For biotin, scientific knowledge is currently insuffi-

cient to estimate average requirements and formulate

recommended dietary allowances. Notwithstanding,

the Food and Nutrition Board of the National Re-

search Council has published recommendations for

adequate intake of biotin (Table 2). These data are

based on empirical biotin intakes in a group of

healthy people.

Factors that Affect Biotin Requirements

0041 Pregnancy (see above) and lactation appear to pro-

duce an increased demand for biotin. Biotin secretion

into breast milk accounts for the higher recommen-

dations for adequate intake of biotin in lactating

compared to normal women (Table 2). At 8 days

postpartum, biotin in human milk is approximately

8 nmol l

1

and accounts for 44% of biotin plus

measured catabolites; bisnorbiotin and biotin d,l-

sulfoxide account for 48% and 8%, respectively. By

6 weeks postpartum, the biotin concentration in-

creases to approximately 30 nmol l

1

and accounts

for about 70% of biotin plus catabolites; bisnorbiotin

and biotin d,l-sulfoxides account for about 20% and

less than 10%, respectively.

0042Accumulating data provide evidence that long-

term anticonvulsant therapy in adults and children

can lead to biotin depletion severe enough to interfere

with amino acid metabolism. This conclusion was

originally based on decreased plasma concentrations

of biotin as determined by the Lactobacillus plan-

tarum bioassay; the incidence of biotin concentra-

tions below the normal range was approximately

75% in a cumulative study group of 274 adults under-

going long-term therapy with a variety of anticonvul-

sant drugs. The diagnosis of biotin deficiency was

strengthened by the demonstration of increased

urinary excretion of the characteristic organic acids

(e.g., 3-hydroxyisovaleric acid) in adults and children

receiving long-term anticonvulsant therapy. (See

Drug–Nutrient Interactions.)

0043The mechanism of biotin depletion during anticon-

vulsant therapy is not known. The anticonvulsant

drugs implicated include phenobarbital, phenytoin,

carbamazepine, and primidone. These drugs each

have a carbamide (-NH-CO-) moiety in their struc-

ture, as does biotin; in some cases they incorporate a

full ureido group (-NH-CO-NH-). Physiological con-

centrations of primidone and carbamazepine specif-

ically and directly inhibit biotin uptake by brush

border membrane vesicles from human intestine.

This finding suggests that impaired intestinal absorp-

tion of biotin may contribute to biotin deficiency. In

addition, phenobarbital, phenytoin, and carbamaze-

pine displace biotin from biotinidase and thus

tbl0002Table 2 Adequate intake of biotin

Life-stage group Adequateintake (mgday

1

)

Infants

0–6 months 5

7–12 months 6

Children

1–3 years 8

4–8 years 12

Males and females

9–13 years 20

14–18 years 25

19 years 30

Pregnancy 30

Lactation 35

From Yates AA, Schlicker SA and Suitor CW (1998) Dietary reference

intakes: the new basis for recommendations for calcium and related

nutrients, B vitamins, and choline. Journal of the American Dietary

Association 98: 699–706, with permission.

522 BIOTIN/Physiology

conceivably could have an effect on plasma transport

of biotin, renal handling of biotin, or cellular uptake

of biotin. Recently, increased urinary excretion rates of

biotin d,l-sulfoxides and bisnorbiotin have been dem-

onstrated in adults and children receiving long-term

anticonvulsant therapy, raising the possibility that

accelerated catabolism of biotin contributes to the

biotin deficiency associated with long-term anticon-

vulsant therapy.

Intake

0044 Biotin is widely distributed in foodstuffs, but the

content of even the richest sources is low when com-

pared to the content of most other water-soluble vita-

mins. Foods relatively rich in biotin include egg yolk,

liver, and some vegetables. The average dietary biotin

intake has been estimated to be 70 mg per day for the

Swiss population. These data are in reasonable agree-

ment with the estimated dietary intake of biotin in a

composite Canadian diet (62 mg per day). Calculated

intake of biotin for the British population was 35 mg

per day.

0045 Infants who ingest 800 ml of mature breast milk

per day ingest approximately 6 mg (24 nmol) of biotin.

It remains unclear whether biotin synthesis by gut

microorganisms contributes importantly to the total

biotin absorbed. However, an infant developed biotin

deficiency while consuming a biotin-free elemental

formula.

See also: Bioavailability of Nutrients; Drug–Nutrient

Interactions; Eggs: Dietary Importance; Fatty Acids:

Metabolism; Inborn Errors of Metabolism: Overview;

Protein: Synthesis and Turnover