In by Raphikammer


Biotin is usually classified as a B-complex vitamin. “Biotin

is by far the most widely used term for this vitamin.

However, discovery of biotin by different approaches has

also led to names such as Bios IIB, protective factor X, vitamin

H, coenzyme R, factor S, factorS, and vitamin BW.

This entry reviews the biochemistry of biotin and summarizes

the clinical findings of deficiency. Readers are encouraged

to use the references for further information.


The molecular weight of biotin is 244.31 Da. The structure

of biotin was elucidated independently by Kogl and du

Vigneaud in the early 1940s and is shown in Figure 1 (1).

Biotin is a bicyclic compound. The imidazolidone contains

an ureido group (–N–CO–N–). The tetrahydrothiophene

ring contains sulfur and has a valeric acid side chain attached

to the C2 carbon of the sulfur-containing ring. This

chain has a cis configuration with respect to the ring that

contains the nitrogen atoms. The two rings are fused in

the cis configuration, producing a boat-like structure.With

three asymmetric carbons, eight stereoisomers exist; only

one [designated D-(+)-biotin or, simply, biotin] is found in

nature and is active when covalently joined via an amide

bond between the carboxyl group of the valeric acid side

chain of biotin and the ε-amino group of a lysine residue of

an app carboxylase. Biocytin (ε-N-biotinyl-L-lysine) is the

product of digestion of protein-bound dietary biotin and

cellular turnover of biotin-containing carboxylases and histones;

biocytin is as active as biotin on a molar basis in

mammalian growth studies.

Goldberg/Sternbach synthesis or a modification

thereof is the method by which biotin is synthesized commercially

(1). Additional stereospecific methods have been

published (2,3).


Biotin was discovered in nutritional experiments that
demonstrated a factor present in many foodstuffs that was
capable of curing the scaly dermatitis, hair loss, and neurologic
signs induced in rats fed dried egg white.

Avidin, a glycoprotein found in egg white, binds biotin very specifically

and tightly. From An evolutionary standpoint, avidin

probably serves as a bacteriostat in egg white. Consistent

with this hypothesis is the observation that avidin is resistant

to a broad range of bacterial proteases in both free

and biotin-bound form. Because avidin is also resistant to

pancreatic proteases, dietary avidin binds to dietary biotin

(and probably any biotin from intestinal microbes)

and prevents absorption, carrying the biotin on through

the gastrointestinal tract.

Biotin is definitely synthesized by intestinal microbes;

however, the contribution of microbial biotin to

absorbed biotin, if any, remains unknown. Cooking denatures

avidin, rendering this protein susceptible to pancreatic

proteases and unable to interfere with the absorption

of biotin.


Biotin acts as an essential cofactor for five mammalian


Each has the vitamin covalently bound to a

polypeptide. For monomeric carboxylases, this polypeptide

is the apo carboxylase. For the dimeric carboxylases,

this monomer with a biotinylation site is designated the

chain. The covalent attachment of biotin to the app carboxylase

protein is a condensation reaction catalyzed by

holocarboxylase synthetase (EC These apo carboxylase

regions contain the biotin motif (methionine–

lysine–methionine), a specific sequence of amino acids

present in each of the individual carboxylases; this sequence

tends to be highly conserved within and between

species. One interpretation concerning conservation

of this amino acid sequence is that these residues

allow the biotinylated peptide to swing the carboxyl (or

acetyl) group from the site of activation to the receiving


All five of the mammalian carboxylases catalyze the

incorporation of bicarbonate as a carboxyl group into a

substrate and employ a similar catalytic mechanism. In

the carboxylase reaction, the carboxyl moiety is first attached

to biotin at the ureido nitrogen opposite the side

chain. Then the carboxyl group is transferred to the substrate.

The reaction is driven by the hydrolysis of ATP

to ADP and inorganic phosphate. Subsequent reactions

in the pathways of the five mammalian carboxylases release

CO2 from the product of the enzymatic reaction.

Thus, these reaction sequences rearrange the substrates

into more useful intermediates but do not violate the classic

observation that mammalian metabolism does not result

in the net fixation of carbon dioxide (4).

The five carboxylases are pyruvate carboxylase

(EC, methylcrotonyl-CoA carboxylase (EC,

propionyl-CoA carboxylase (EC, and two isoforms

of acetyl-CoA carboxylase (EC, denoted I

and II, which are also known as ACC and ACC. Each

  • 43
  • 44 Mock
  • HN
  • C
  • O
  • NH
  • HC CH
  • CH
  • O
  • S
  • H2C (CH2) C N (CH2) 4
  • O
  • H
  • 4
  • N-H
  • C-H
  • C=O
  • amino group

Figure 1 Protein-bound biotin with arrow showing the amide bond to the

-amino acid.

carboxylase catalyzes an essential step in intermediary


Pyruvate carboxylase mediates in the incorporation

of bicarbonate into pyruvate to form oxaloacetate, an intermediate

in the Krebs tricarboxylic acid cycle. Thus, pyruvate

carboxylase catalyzes an anaplerotic reaction. In gluconeogenic

tissues (i.e., liver and kidney), the oxaloacetate

can be converted to glucose. Deficiency of this enzyme (denoted

by a block in the metabolic pathway) is likely the

cause of the lactic acidosis and hypoglycemia observed in

biotin-deficient animals and humans.

Methylcrotonyl-CoA carboxylase catalyzes an essential

step in the degradation of the branch-chained

amino acid leucine. Deficient activity of this enzyme

leads to metabolism of 3-methylcrotonyl CoA to 3-

hydroxyisovaleric acid and 3-methylcrotonyl glycine by

an alternate pathway. Thus, increased urinary excretion

of these abnormal metabolites reflects deficient activity of

this carboxylase.

Propionyl-CoA carboxylase catalyzes the incorporation

of bicarbonate into propionyl CoA to form methylmalonyl

CoA, which undergoes isomerization to succinyl

CoA and enters the tricarboxylic acid cycle. In a fashion

analogous to methylcrotonyl-CoA carboxylase deficiency,

inadequacy of this enzyme leads to increased urinary excretion

of 3-hydroxypropionic acid and 3-methylcitric acid

and enhanced accumulation of odd-chain fatty acids C15:0

and C17:0. The mechanism is likely the substitution of propionyl

CoA for acetyl CoA during fatty acid elongation.

Although the proportional increase is large (e.g., 2- to 10-

fold), the absolute composition relative to other fatty acids

is quite small (<1%) and likely produces little or no functional


Acetyl-CoA carboxylases, I and II both, catalyze the

incorporation of bicarbonate into acetyl CoA to form malonyl

CoA. Acetyl-CoA carboxylase I is located in the

cytosol and produces cytosolic malonyl CoA, which is

rate limiting in fatty acid synthesis (elongation). Acetyl-

CoA carboxylase II is present on the outer mitochondrial

membrane. As demonstrated by the pioneering work of

Wakil and colleagues, acetyl-CoA carboxylase II controls a

separate mitochondrial pool of malonyl CoA that, in turn,

controls fatty acid oxidation in mitochondria through the

inhibitory effect of malonyl CoA on fatty acid transport

into mitochondria.

  • isoleucine
  • methionine
  • propionyl CoA
  • d-methylmalonyl CoA
  • succinyl CoA
  • glucose
  • oxaloacetate pyruvate acetyl CoA
  • lactate
  • malonyl CoA
  • tricarboxylic acid cycle
  • 3-methylglutaconyl CoA
  • 3-methylcrotonyl CoA
  • Methylcrotonyl-CoA
  • Carboxylase
  • Acetyl-CoA
  • Carboxylase
  • Pyruvate
  • Carboxylase
  • Propionyl-CoA
  • Carboxylase
  • leucine
  • 3-hydroxyisovalerate
  • 3-methylcrotonylglycine
  • 3-hydroxypropionate
  • methylcitrate
  • odd-chain fatty acid
  • fatty acid elongation

Pathways involving biotin-dependent carboxylases. Deficiencies (hatched bar) of pyruvate carboxylase, propionyl-CoA carboxylase, methylcrotonyl-CoA

carboxylase, and acetyl-CoA carboxylase lead to increased blood concentrations and urinary excretion of characteristic organic acids denoted by ovals.

Biotin 45

In the normal turnover of cellular proteins, holocarboxylase

are degraded to biocytin or biotin linked

to an oligopeptide containing at most a few amino acid

residues. Because the amide bond between biotin and

lysine (Fig. 1) is not hydrolyzed by cellular proteases,

the specific hydrolase biotinidase [biotin amide hydrolase

(EC] is required to release biotin for recycling.

Biotin exists in free and bound pools within the cell

that are responsive to changes in its status (5). The pool

size is likely determined by a balance between cellular uptake

and cellular release, incorporation into apo carboxylases

and histones, release from these biotinylated proteins

during turnover, and catabolism to inactive metabolites.

Regulation of intracellular mammalian carboxylase activity

by biotin remains to be elucidated.

Genetic deficiencies of holocarboxylase synthetase

and biotinidase cause the two distinct types of multiple

carboxylase deficiency that were previously designated

the neonatal and juvenile forms. The genes for holocarboxylase

synthetase and human biotinidase have been

cloned, sequenced, and characterized (6). The gene coding

for holocarboxylase synthetase is located on chromosome

21q22.1 and consists of 14 exons and 13 entrons in a span of

240 kilobase (kb). Studies of human mutant holocarboxylase

synthetase indicate that all forms of holocarboxylase

synthetase are likely encoded by one gene. Biotinidase

deficiency is particularly relevant to understanding biotin

inadequacy because the clinical manifestations appear to

result largely from secondary biotin depletion.


Digestion of Protein-Bound Biotin

The content of free and protein-bound forms of biotin in

foods is variable, but the majority in meats and cereals

appear to be protein bound via an amide bond between

biotin and lysine. Neither the mechanisms of intestinal hydrolysis

of protein-bound biotin nor the determinants of

bioavailability have been clearly delineated.Wolf et al. (7)

have postulated that biotinidase plays a critical role in the

release of biotin from covalent binding to protein. Doses

of free biotin that do not greatly exceed the estimated

dietary intake (e.g., 50–150 g/day) appear adequate to

prevent the symptoms of biotinidase deficiency. This suggests

that biotinidase inadequacy in patients causes biotin

deficiency, at least in part, through impaired intestinal digestion

of protein-bound biotin.

Intestinal Absorption

At physiologic pH, the carboxylate group of biotin is negatively

charged. Thus, the vitamin is at least modestly water

soluble and requires a transporter to cross the membranes

of enterocytes for intestinal absorption, of somatic cells for

utilization, and of renal tubule cells for reclamation from

the glomerular filtrate.

An excellent in-depth review of intestinal uptake of

biotin has been published recently (8). Two biotin transporters

have been described: (i) a multivitamin transporter

present in many tissues including the intestine and (ii) a

biotin transporter identified in human lymphocytes.

The transporter responsible for absorption of free biotin

in the small and large intestine is saturable and Na+

dependent. The transporter also transports pantothenic

acid and lipoate and is deemed the sodium-dependent

multivitamin transporter (SMVT). SMVT was discovered

in 1997 by Prasad et al. (9) in human placental choriocarcinoma

cells. This transporter is widely expressed in

human tissues (10). SMVT system has been cloned and

demonstrated to be exclusively expressed at the apical

membrane of enterocytes. SVMT is the main biotin uptake

system that operates in human intestinal epithelial

cells. The 5-regulatory region of the SMVT gene has also

been cloned and characterized both in vitro and in vivo

(8). Intestinal biotin uptake is adaptively upregulated in

biotin deficiency via a transcriptionally mediated mechanism

that involves KLF4 sites. The cytoplasmic C-terminal

domain of the polypeptide is essential for its targeting to

the apical membrane domain of epithelial cells (8).

In rats, biotin transport is upregulated during maturation

after weaning and by biotin deficiency (11). Carrier Mediated

transport of the vitamin is most active in the

proximal small bowel of the rat and humans (8). However,

absorption from the proximal colon is still significant, supporting

the potential nutritional significance of biotin synthesized

and released by enteric flora (11). Clinical studies

have provided some evidence that biotin is absorbed from

the human colon (12). In contrast, more rigorous studies

in swine indicate that biotin absorption from the hindgut

is much less efficient than that from the upper intestine;

furthermore, biotin synthesized by enteric flora may not

present at a location or in a form in which bacterial biotin

contributes importantly to absorbed biotin.

Exit of biotin from the enterocyte (i.e., transport

across the basolateral membrane) is also carrier mediated

(11). However, basolateral transport is independent

of Na+, is electrogenic, and does not accumulate biotin

against a concentration gradient.

Transport in Blood

Biotin dissolved in blood is carried from the site of absorption

in the intestine to the peripheral tissues and

the liver.

(1). Wolf et al. (13) originally hypothesized that

biotinidase might serve as a biotin-binding protein in

plasma or perhaps even as a carrier protein for the movement

of biotin into the cell. Based on protein precipitation

and equilibrium dialysis using 3H-biotin, Chauhan

and Dakshinamurti (14) concluded that biotinidase is the

only protein in human serum that specifically binds biotin.

However, using 3H-biotin, centrifugal ultrafiltration,

and dialysis to assess reversible binding in plasma from

the rabbit, pig, and human, Mock and Lankford (15)

found that less than 10% of the total pool of free plus reversibly

bound biotin is reversibly bound to plasma protein;

the biotin binding observed could be explained by

binding to human serum albumin. Using acid hydrolysis

and 3H-biotinyl-albumin, Mock and Malik (16) found

additional biotin covalently bound to plasma protein.

The percentages of free, reversibly bound, and covalently

bound biotin in human serum are approximately 81%,

7%, and 12%. A biotin-binding immunoglobulin has been

identified in human serum. An approximately fivefold

higher concentration of this biotin-binding immunoglobulin

was reported in patients with Graves disease than

in normal and healthy controls (17). The role of plasma

proteins in the transport of biotin remains to be definitively


Biotin concentrations in erythrocytes are equal to

those in plasma

(D.M. Mock, unpublished observation).

However, transport into erythrocytes is very slow, consistent

with passive diffusion (18).

Uptake by the Liver

Studies in a variety of hepatic cell lines indicate that

uptake of free biotin by the liver is similar to intestinal

uptake and is mediated by SMVT (19–21). Transport is

mediated by a specialized carrier system that is Na+ dependent,

electroneutral, and structurally specific for a free

carboxyl group. At large concentrations, movement is carried

out by diffusion. Metabolic trapping, for example,

biotin bound covalently to intracellular proteins, is also

important. After entering the hepatocyte, biotin diffuses

into the mitochondria via a pH-dependent process.

The biotin transporter identified in lymphocytes

is also Na+ coupled, saturable, and structurally specific

(22). Recent studies by Daberkow and coworkers provide

evidence in favor of monocarboxylate transporter 1 as the

lymphocyte biotin transporter (23).

A child with biotin dependence due to a defect in

the lymphocyte biotin transporter has been reported (18).

The child became acutely encephalopathic at the age of

18 months. Urinary organic acids indicated deficiency

of several biotin-dependent carboxylases. Symptoms improved

rapidly following biotin supplementation. Serum

biotinidase activity and biotinidase gene sequence were

normal. Activities of biotin-dependent carboxylases in

lymphocytes and cultured skin fibroblasts were normal,

excluding biotin holocarboxylase synthetase deficiency

as the cause. Despite extracellular biotin sufficiency, biotin

withdrawal caused recurrence of abnormal organic

aciduria, indicating intracellular biotin deficiency. Biotin

uptake rates into fresh lymphocytes from the child and

into his or her lymphocytes transformed with Epstein–

Barr virus were about 10% of normal fresh and transformed

control cells, respectively. For fresh and transformed

lymphocytes from his or her parents, biotin uptake

rates were consistent with heterozygosity for an autosomal

recessive genetic defect. SMVT gene sequence was

normal; regulatory regions of the SMVT gene have not

been characterized. These investigators speculated that

lymphocyte biotin transporter is expressed in additional

tissues such as the kidney and may mediate some critical

aspect of biotin homeostasis, but the complete molecular

etiology of this child’s biotin transporter deficiency

remains to be elucidated.

Ozand et al. (24) recently described several patients

in Saudi Arabia with biotin-responsive basal ganglia disease.

Symptoms include confusion, lethargy, vomiting,

seizures, dystonia, dysarthria, dysphagia, seventh nerve

paralysis, quadriparesis, ataxia, hypertension, chorea, and

coma. A mutation in SLC19A3 was identified, and defect

in the biotin transporter system across the blood–brain

barrier was postulated (25). However, in an elegant set

of experiments, Said and coworkers demonstrated that

SLC19A3 is the apical thiamine transporter and renamed

SLC19A3 appropriately as THTR2 (26), in contrast to the

basolateral thiamine transporter THTR1. The explanation

for the documented biotin responsiveness of these patients

remains unknown.

Renal Handling

Specific systems for the reabsorption of water-soluble vitamins

from the glomerular filtrate contribute importantly

to conservation of these vitamins (27). Animal studies using

brush border membrane vesicles from human kidney

cortex indicate that biotin is reclaimed from the glomerular

filtrate against a concentration gradient by a saturable,

Na+-dependent, structurally specific system (28). Using

human-derived proximal tubular epithelial HK-2 cells as

a model, Said and coworkers demonstrated that biotin uptake

by human renal epithelial cells occurs via the SMVT

system and that the process is regulated by intracellular

protein kinase C and Ca++/calmodulin-mediated pathways

(29). The uptake process is adaptively regulated by

extracellular biotin concentrations via transcriptional regulatory

mechanisms (29) consistent with previous studies

demonstrating reduced biotin excretion early in experimentally

induced biotin deficiency in human subjects


Subsequent egress of biotin from the tubular cells

occurs via a basolateral membrane transport system that

is not dependent on Na+. Biocytin does not inhibit tubular

reabsorption of biotin (28). Studies in patients with

biotinidase deficiency suggest that there may be a role for

biotinidase in the renal handling of biotin (32,33).

Transport into the Central Nervous System

A variety of animal and human studies suggest that biotin

is transported across the blood–brain barrier (1,34,35).

The transporter is saturable and structurally specific for

the free carboxylate group on the valeric acid side chain.

Transport into the neuron also appears to involve a specific

transport system as well as subsequent trapping of

biotin by covalent binding to brain proteins, presumably

the biotin-dependent carboxylases and histones.

Recently, concentrations of biotin were determined

initially as total avidin-binding substances in cerebrospinal

fluid (CSF) from 55 children, and biotin, biotin

sulfoxide, and bisnorbiotin were quantitated by highperformance

liquid chromatography (HPLC) and avidinbinding

assay in CSF samples from a subset of 11 children

(36). Concentrations of total avidin-binding substances

averaged 1.6 nmol/L with substantial variability, SD =

1.3 nmol/L. CSF concentrations of biotin and biotin analogous

varied widely, but substantial amounts of biotin

sulfoxide were detected in every sample. Of the total, biotin

accounted for 42% °æ 16%, biotin sulfoxide for 41% °æ

12%, and bisnorbiotin for 8% °æ 14%. Surprisingly, the molar

sum of biotin plus biotin sulfoxide and bisnorbiotin on

average exceeded the total avidin-binding substances concentrations

fromthe same CSF sample by>200-fold. These

investigators found no masking of detection or degradation

of biotin or biotin sulfoxide. Gel electrophoresis

and streptavidin Western blot detected several biotinylated

proteins in CSF leading to the conclusion that biotin

is bound to protein covalently, reversibly, or both; they

speculated that biotin bound to protein likely accounts

for the increase in detectable biotin after HPLC and that

Biotin 47


protein-bound biotin plays an important role in biotin nutriture

of the brain.

Placental Transport

Biotin concentrations are 3- to 17-fold greater in plasma

from human fetuses compared with their mothers in the

second trimester, consistent with active placental transport

(37). Specific systems for transport of biotin from the

mother to the fetus have been reported recently (10,38–

40). The microvillus membrane of the placenta contains a

saturable transport system for biotin that is Na+ dependent

and actively accumulates biotin within the placenta,

consistent with SMVT (10,38–40).


Transport into Human Milk

More than 95% of the biotin is free in the skim fraction of

human milk (41). The concentration of biotin varies substantially

in some women (42) and exceeds that in serum

by one to two orders of magnitude, suggesting that there

is a transport system into milk. The biotin metabolite bisnorbiotin

(see discussion of metabolism under pharmacology

section) accounts for approximately 50%. In early

and transitional human milk, the biotin metabolite and

biotin sulfoxide accounts for about 10% of the total biotin

plus metabolites (43). With postpartum maturation,

the biotin concentration increases, but the bisnorbiotin

and biotin sulfoxide concentrations still account for 25%

and 8% at 5 weeks postpartum. The concentration of biotin

in human milk exceeds the plasma concentration by

10- to 100-fold, implying that a transport system exists.

Current studies provide no evidence for a soluble biotin binding

protein or any other mechanism that traps biotin

in human milk. The location and the nature of the

biotin transport system for human milk have yet to be



Studies in which pharmacologic amounts of biotin were

administered orally and intravenously to experimental

subjects and tracer amounts of radioactive biotin were

administered intravenously to animals show that biotin

in pure form is 100% bioavailable when administered

orally. The preponderance of dietary biotin detectable

by bioassays is bound to macromolecules. Likely biotin

is bound to carboxylases and perhaps to histones. The

bioavailability of biotin from foodstuffs is not known,

whereas that from animal feeds varies but can be well

below 50%. After intravenous administration, the vitamin

disappears rapidly from plasma; the fastest phase of the

three-phase disappearance curve has a half-life of less than

10 minutes.

An alternate fate to being covalently bound to

protein (e.g., carboxylases) or excretion unchanged in

urine is catabolism to an inactive metabolite before excretion

in urine (4). About half of biotin undergoes

metabolism before excretion. 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 -oxidation. This leads to the formation

of bisnorbiotin, tetranorbiotin, and related intermediates

Table 1 Normal Range of Urinary Excretion of Biotin and Major

Metabolites (nmol/24 hr; n = 31 Males and Females)

Biotin Bisnorbiotin Biotin sulfoxide

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

The cellular site of this -oxidation of biotin is uncertain.

Nonenzymatic decarboxylation of the unstable –

keto-biotin and -keto-bisnorbiotin leads to formation of

bisnorbiotin methyl ketone and tetranor biotin methylketone,

which appear in urine. In the second pathway, the

sulfur in the thiophene ring of biotin is oxidized, leading

to the formation of biotin L-sulfoxide, biotin D-sulfoxide,

and biotin sulfone. Combined oxidation of the ring sulfur

and -oxidation of the side chain lead to metabolites such

as bisnorbiotin sulfone. In mammals, degradation of the

biotin ring to release carbon dioxide and urea is quantitatively


On a molar basis, biotin accounts for approximately

half of the total avidin-binding substances in human

serum and urine (Table 1). Biocytin, bisnorbiotin, bisnorbiotin

methyl ketone, biotin sulfoxide, and biotin sulfone

form most of the balance. Biotin metabolism is accelerated

in some individuals by anticonvulsant therapy and

during pregnancy, thereby increasing the ratio of biotin

metabolites to biotin excreted in urine.



The fact that normal humans have a requirement for biotin

has been clearly documented in two situations: prolonged

consumption of raw egg white and parenteral nutrition

without biotin supplementation in patients with

short-gut syndrome and other causes of malabsorption

(1). Deficiency of this member of the vitamin B

group also has been clearly demonstrated in biotinidase

deficiency (6).

The clinical findings and biochemical abnormalities

in cases of biotin deficiency include dermatitis around

body orifices, conjunctivitis, alopecia, ataxia, and developmental

delay (1). The progression of clinical findings in

adults, older children, and infants is similar. Typically, the

symptoms appear gradually after weeks to several years

of egg white feeding or parenteral nutrition. Thinning of

hair progresses to loss of all hair, including eyebrows and

lashes. A scaly (seborrheic), red (eczematous) skin rash

was present in the majority of reports. In several reports,

the rash was distributed around the eyes, nose, mouth,

and perineal orifices. The appearance of the rash was similar

to that of cutaneous candidiasis; Candida albicans could

often be cultured from the lesions. These manifestations

on skin, in conjunction with an unusual distribution of

facial fat, have been dubbed “biotin deficiency facies.”

Depression, lethargy, hallucinations, and paresthesias of

the extremities were prominent neurologic symptoms in

the majority of adults, while infants showed hypotonia,

lethargy, and developmental delay.

In cases severe enough to produce the classic cutaneous

and behavioral manifestations of biotin deficiency,

urinary excretion rates and plasma concentrations
of biotin are frankly decreased. Urinary excretion of the

organic acids discussed in biochemistry section and

shown in Figure 2 is frankly increased. The increase is

typically 5- to 20-fold or more. However, such a severe

degree of biotin deficiency has never been documented to

occur spontaneously in a normal individual consuming a

mixed general diet.

Of greater current interest and debate are the health

consequences, if any, of marginal biotin deficiency. Concerns

about the teratogenic effects have led to studies

of biotin status during human gestation (44–48). These

studies provide evidence that a marginal degree of deficiency

develops in at least one-third of women during

normal pregnancy. Although the degree of biotin deficiency

is not severe enough to produce overt manifestations,

the deficiency is severe enough to produce metabolic

derangements. A similar marginal degree of biotin deficiency

causes high rates of fetal malformations in some

mammals (30,49,50). Moreover, data from a multivitamin

supplementation study provide significant, albeit indirect,

evidence that the marginal degree of deficiency

that occurs spontaneously in normal human gestation is

teratogenic (44).

Valid indicators of marginal biotin deficiency have

been reported. Asymptomatic biotin shortage was induced

in normal adults housed in a general clinical

research center by egg white feeding. Decreased urinary

excretion of biotin, increased urinary excretion

of 3-hydroxyisovaleric acid, and decreased activity of

propionyl-CoA carboxylase in lymphocytes from peripheral

blood are early and sensitive indicators of biotin deficiency

(30,31,51). On the basis of a study of only five

subjects, 3-hydroxyisovaleric acid excretion in response to

a leucine challenge appears to be an even more sensitive

indicator of marginal biotin status (31). The plasma concentration

of biotin and the urinary excretion of methylglycine,

3-hydroxypropionic acid, and 3-methylcitric acid

do not appear to be good indicators of marginal biotin

deficiency (52). In a biotin repletion study, the resumption

of a mixed general diet produced a trend toward normalization

of biotin status within 7 days. This was achieved

when the supplement was started immediately at the time

of resuming a normal diet. However, supplementation

of biotin at 10 times the dietary reference intake (DRI)

(300 g/day) for 14 days reduced 3-hydroxyisovaleric

acid excretion completely to normal in only about half

of pregnant women who were marginally biotin deficient

(47) suggesting a substantial depletion of total

body biotin, a substantially increased biotin requirement,

or both.

On the basis of decreased lymphocyte carboxylase

activities and plasma biotin levels, Velazquez et al. (53)

have reported that biotin deficiency occurs in children

with severe protein-energy malnutrition. These investigators

have speculated that the effects of biotin inadequacy

may be responsible for part of the clinical syndrome of

protein-energy malnutrition.

Long-term treatment with a variety of anticonvulsants

appears to be associated with marginal biotin

deficiency severe enough to interfere with amino acid

metabolism (54–56). The mechanism may involve both

accelerated biotin breakdown (56–58) and impairment of

biotin absorption caused by the anticonvulsants (59).

Biotin deficiency has also been reported or inferred

in several other circumstances including Leiner disease

(60–62), sudden infant death syndrome (63,64), hemodialysis

(65–69), gastrointestinal diseases and alcoholism (1),

and brittle nails (70). Additional studies are needed to confirm

or refute an etiologic link of these conditions to the

vitamin’s deficiency.

The mechanisms by which biotin deficiency produces

specific signs and symptoms remain to be completely

delineated. However, several studies have given

new insights on this subject. The classic assumption for

most water-soluble vitamins is that the clinical findings of

deficiency result directly or indirectly from deficient activities

of the vitamin-dependent enzymes. On the basis

of human studies on deficiency of biotinidase and isolated

pyruvate carboxylase, as well as animal experiments regarding

biotin deficiency, it is hypothesized that the central

nervous system effects of biotin deficiency (hypotonia,

seizures, ataxia, and delayed development) are likely mediated

through deficiency of brain pyruvate carboxylase

and the attendant central nervous system lactic acidosis

rather than by disturbances in brain fatty acid composition

(71–73). Abnormalities in metabolism of fatty acids

are likely important in the pathogenesis of the skin rash

and hair loss (74).

Exciting new work has provided evidence for a potential

role for biotin in gene expression.

These findings

will likely provide new insights into the pathogenesis of

biotin deficiency (75,76). In 1995, Hymes and Wolf discovered

that biotinidase can act as a biotinyl transferase;

biocytin serves as the source of biotin, and histones are

specifically biotinylated (6). Approximately 25% of total

cellular biotinidase activity is located in the nucleus. Zempleni

and coworkers have demonstrated that the abundance

of biotinylated histones varies with the cell cycle,

that these histones are increased approximately twofold

compared with quiescent lymphocytes, and that these are

biotinylated enzymatically in a process that is at least

partially catalyzed by biotinidase (77–79). These observations

suggest that biotin plays a role in regulating DNA

transcription and regulation.

Biotinylation of histones is emerging as an important

histone modification. Recent studies from Hassan

and Zempleni provide evidence that biotinylation likely

interacts with other covalent modification of histones to

suppress gene expression and gene transposition (80). Although

the relative importance in biotinidase and holocarboxylase

synthetase in the biotinylation and biotinylation

of histones has yet to be fully elucidated, Gravel

and Narang have produced evidence that holocarboxylase

synthetase is present in the nucleus in greater quantities

than in the cytosol or the mitochondria and that holocarboxylase

synthetase likely acts in the nucleus to catalyze

the biotinylation of histones (81). Moreover, fibroblasts

from patients with HCLS deficiency are severely deficient

in histone biotinylation (82). Zempleni and coworkers

have shown that biotinylation of lysine-12 in histone H4

(K12BioH4) causes gene repression and have proposed

a novel role for HCS in sensing and regulating levels

of biotin in eukaryotic cells (83). They have hypothesized

that holocarboxylase synthetase senses biotin and

that biotin regulates its own cellular uptake by participating

in holocarboxylase synthetase–dependent chromatin

Biotin 49


remodeling events at an SMVT promoter locus. Specifically,

they hypothesize that nuclear translocation of HCS

increases in response to biotin supplementation and then

biotinylated histone H4 at SMVT promoters, silencing biotin

transporter genes. This group has shown that nuclear

translocation of HCS is a biotin-dependent process potentially

involving tyrosine kinases, histone deacetylases,

and histone methyltransferases. The nuclear translocation

of holocarboxylase synthetase correlates with biotin concentrations

in cell culture media and is inversely linked to

SMVT expression. Moreover, biotin homeostasis by holocarboxylase

synthetase–dependent chromatin remodeling

at an SMVT promoter locus is disrupted in holocarboxylase

synthetase knockdown cells.


Transposable elements such as retrotransposons

containing long-terminal repeats constitute about half of

the human genome, and the transposition events associated

with these elements impair genome stability. Epigenetic

mechanisms are important for transcriptional repression

of retrotransposons, preventing transposition events,

and abnormal regulation of genes. Zempleni and coworkers

have provided evidence that the covalent binding of

biotin to lysine-12 in histone H4 and lysine-9 in histone

H2A mediated by holocarboxylase synthetase is an epigenetic

mechanism to repress retrotransposon transcription

in human and mouse cell lines and in primary cells from a

human supplementation study. Abundance of biotinylation

at those sites depended on biotin supply and on holocarboxylase

synthetase activity and was inversely linked

with the abundance of long terminal repeat transcripts.

Knockdown of holocarboxylase synthetase in Drosophila

enhanced retrotransposition. Depletion of biotinylation

at those sites in biotin-deficient cells correlated with increased

production of transposition events and decreased

chromosomal stability.

Recently, controversy has arisen concerning the role

of biotin as an in vivo covalent modifier of histones. Bailey

and coworkers have reported that streptavidin binds

to histones independently of biotinylation (84). To further

investigate this phenomenon, 293T cells were grown in

14C-biotin; in contrast to the ready detectability of 14Cbiotin

in carboxylases, 14C-biotin was undetectable in histones

(i.e., represented no more than 0.03% of histones)

(84). In a subsequent study, Healy and coworkers demonstrated

that histone H2A is nonenzymatically biotinylated

by biotinyl-5-AMP and provided evidence that these enzymes

promotes biotinylation of histone H2A by releasing

biotinyl-5-AMP, which then biotinylated lysines in histone

H2A somewhat nonspecifically (85). Recently, this

group has proposed that biotin is not a natural histone

modifier at all. On the basis of studies that fail to find

in vivo biotin incorporation into histones using 3H-biotin

uptake, Western blot analysis of histones, or mass spectrometry

of affinity purified histone fragments, these investigators

concluded that biotin is absent in native histones

to a sensitivity of 1 part per 100,000 and that the

regulatory impact on gene expression must occur through

a mechanism other than histone modification (86). These

conclusions are likely to generate a lively debate until

definitive evidence is provided using mass spectrometric

analysis of in vivo histones harvested at various phases

of the cell cycle and at specific locations within particular



In 1998, the United States Food and Nutrition Board of the

National Academy of Sciences reviewed the recommendations

for biotin intake (87). The committee concluded that

the data were inadequate to justify setting an estimated

average requirement. However, adequate intake (AI) was

formulated (Table 2). The AI for infants was based on

an empirical determination of the biotin content of human

milk. Using the value for free biotin determined microbiologically

(6 g/L) and an average consumption of

0.78 L/day by infants of age 0–6 months, an AI of 5g/day

was calculated. The AI for lactating women has been increased

by 5 g/day to allow for the amount of biotin

secreted in human milk. Using the AI for 0–6-month-old

infants, the reference body weight ratio method was used

to extrapolate AIs for other age groups (see Table 2).



If biotin deficiency is confirmed, biotin supplementation

should be undertaken and effectiveness should be documented.

Doses between 100 g and 1 mg are likely to

be both effective and safe on the basis of studies supplementing

biotin deficiency during pregnancy, chronic

anticonvulsant therapy, and biotinidase deficiency.


Daily doses of up to 200 mg orally and up to 20 mg intravenously

have been given to treat biotin-responsive inborn

errors of metabolism and acquired biotin deficiency.

Toxicity has not been reported.


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