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.
SCIENTIFIC NAMES AND STRUCTURE
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
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
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 18.104.22.168). 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 22.214.171.124), methylcrotonyl-CoA carboxylase (EC 126.96.36.199),
propionyl-CoA carboxylase (EC 188.8.131.52), and two isoforms
of acetyl-CoA carboxylase (EC 184.108.40.206), denoted I
and II, which are also known as ACC and ACC. Each
H2C (CH2) C N (CH2) 4
Figure 1 Protein-bound biotin with arrow showing the amide bond to the
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
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
oxaloacetate pyruvate acetyl CoA
tricarboxylic acid cycle
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.
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 220.127.116.11)] 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.
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
(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
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
protein-bound biotin plays an important role in biotin nutriture
of the brain.
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
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.
OCCURRENCE AND DIAGNOSIS OF BIOTIN DEFICIENCY
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
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
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,
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
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
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.
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
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
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
INDICATIONS AND USAGE
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).
TREATMENT OF BIOTIN DEFICIENCY
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.
1. Mock DM, Biotin. In: Ziegler EE, Filer LJ Jr, eds. Present
Knowledge in Nutrition.Washington, DC: International Life
Sciences Institutes–Nutrition Foundation, 1996:220–235.
2. Miljkovic D, Velimirovic S, Csanadi J, et al. Studies directed
towards stereospecific synthesis of oxybiotin, biotin, and
their analogs. Preparation of some new 2,5, anhydro-xylitol
derivatives. J Carbohydr Chem 1989; 8:457–467.
3. Deroose FD, DeClercq PJ. Novel enantioselective syntheses
of (+)-biotin. J Org Chem 1995; 60:321–330.
4. Mock DM. Biotin. In: Shils ME, Olson JA, Shike M, et al., eds.
Modern Nutrition in Health and Disease. Baltimore, MD:
Williams &Wilkins, 1999:459–466.
5. Lewis B, Rathman S, McMahon R. Dietary biotin intake modulates
the pool of free and protein-bound biotin in rat liver. J
Nutr 2001; 131:2310–2315.
6. Wolf B. Disorders of biotin metabolism. In: Scriver CR,
Beaudet AL, Sly WS, et al., eds. The Metabolic and Molecular
Basis of Inherited Disease. New York: McGraw-Hill, Inc.,
7. Wolf B, Heard G, McVoy JRS, et al. Biotinidase deficiency:
the possible role of biotinidase in the processing of dietary
protein-bound biotin. J Inherit Metab Dis 1984; 7(suppl
8. Said H. Cell and molecular aspects of the human intestinal
biotin absorption process. J Nutr 2008; 139(1):158–162.
9. Prasad PD, Ramamoorthy S, Leibach FH, et al. Characterization
of a sodium-dependent vitamin transporter mediating
the uptake of pantothenate, biotin and lipoate in human placental
choriocarcinoma cells. Placenta 1997; 18:527–533.
10. Prasad PD, Wang H, Kekuda R, et al. Cloning and functional
expression of acDNAencoding a mammalian sodiumdependent
vitamin transporter mediating the uptake of
pantothenate, biotin, and lipoate. J Biol Chem 1998; 273:
11. Said HM. Recent advances in carrier-mediated intestinal absorption
of water-soluble vitamins. Annu Rev Physiol 2004;
12. Mock DM. Biotin. In: Brown M, ed. Present Knowledge
in Nutrition. Blacksburg, VA: International Life Sciences
Institute–Nutrition Foundation, 1990:189–207.
13. Wolf B, Grier RE, McVoy JRS, et al. Biotinidase deficiency:
a novel vitamin recycling defect. J Inherit Metab Dis 1985;
14. Chauhan J, Dakshinamurti K. Role of human serum biotinidase
as biotin-binding protein. Biochem J 1988; 256:265–
15. Mock DM, Lankford G. Studies of the reversible binding of
biotin to human plasma. J. Nutr 1990; 120;375–381.
16. MockDM,Malik MI. Distribution of biotin in human plasma:
most of the biotin is not bound to protein. Am J Clin Nutr
17. Nagamine T, Takehara K, Fukui T, et al. Clinical evaluation
of biotin-binding immunoglobulin in patients with Graves’
disease. Clin Chim Acta 1994; 226(1):47–54.
18. Mardach R, Zempleni J, Wolf B, et al. Biotin dependency
due to a defect in biotin transport. J Clin Invest 2002;
19. Bowers-Komro DM, McCormick DB. Biotin uptake by isolated
rat liver hepatocytes. In: Dakshinamurti K, Bhagavan
HN, eds. Biotin. New York: New York Academy of Sciences,
20. Said HM, Ma TY, Kamanna VS. Uptake of biotin by human
hepatoma cell line, Hep G(2): a carrier-mediated process
similar to that of normal liver. J Cell Physiol 1994; 161(3):
21. Balamurugan K, Ortiz A, Said HM. Biotin uptake by human
intestinal and liver epithelial cells: role of the SMVT system.
Am J Physiol Gastrointest Liver Physiol 2003; 285(1):G73–
22. Zempleni J, Mock DM. Uptake and metabolism of biotin by
human peripheral blood mononuclear cells. Am J Physiol
Cell Physiol 1998; 275(2):C382–C388.
23. Daberkow RL, White BR, Cederberg RA, et al. Monocarboxylate
transporter 1 mediates biotin uptake in human peripheral
blood mononuclear cells. J Nutr 2003; 133:2703–2706.
24. Ozand PT, Gascon GG, Al Essa M, et al. Biotin-responsive
basal ganglia disease: a novel entity. Brain 1999; 121:1267–
25. Zeng W, Al-Yamani E, Acierno JS, et al. Mutations
in SLC19A3 encoding a novel transporter cause biotinresponsive
basal ganglia disease. American Society of
Human Genetics Meeting Web site. http://faseb.org/
genetics/ashg01/f101.htm. Accessed April 15, 2010.
26. Subramanian VS, Marchant JS, Said HM. Biotin-responsive
basal ganglia disease-linked mutations inhibit thiamine
transport via hTHTR2: biotin is not a substrate for hTHTR2.
Am J Physiol Cell Physiol 2006; 291(5):C851–859.
27. Bowman BB, McCormick DB, Rosenberg IH. Epithelial transport
of water-soluble vitamins. Ann Rev Nutr 1989; 9:187–
28. Baur B, Baumgartner ER. Na(+)-dependent biotin transport
into brush-border membrane vesicles from human kidney
cortex. Pflugers Arch 1993; 422:499–505.
29. Balamurugan K, Vaziri ND, Said HM. Biotin uptake by human
proximal tubular epithelial cells: cellular and molecular
aspects. Am J Physiol Renal Physiol 2005; 288(4):F823–F831.
30. Mock NI, Malik MI, Stumbo PJ, et al. Increased urinary excretion
of 3-hydroxyisovaleric acid and decreased urinary
excretion of biotin are sensitive early indicators of decreased
status in experimental biotin deficiency.AmJ Clin Nutr 1997;
31. Mock DM, Henrich CL, Carnell N, et al. Indicators of
marginal biotin deficiency and repletion in humans: validation
of 3-hydroxyisovaleric acid excretion and a leucine
challenge. Am J Clin Nutr 2002; 76:1061–1068.
32. Baumgartner ER, Suormala T, Wick H. Biotinidase deficiency:
factors responsible for the increased biotin requirement.
J Inherit Metab Dis 1985; 8(suppl 1):59–64.
33. Baumgartner ER, Suormala T,Wick H. Biotinidase deficiency
associated with renal loss of biocytin and biotin. J Inherit
Metab Dis 1985; 7(suppl 2):123–125.
34. Spector R, Mock DM. Biotin transport through the blood–
brain barrier. J Neurochem 1987; 48:400–404.
35. Spector R, Mock DM. Biotin transport and metabolism in the
central nervous system. Neurochem Res 1988; 13(3):213–219.
36. Bogusiewicz A, Stratton SL, Ellison DA, et al. Distribution of
biotin in cerebrospinal fluid of children: most of the biotin is
bound to protein. FASEB J 2008; 22:1104.4.
37. Mantagos S, Malamitsi-Puchner A, Antsaklis A, et al. Biotin
plasma levels of the human fetus. Biol Neonate 1998; 74:72–
38. Karl PI, Fisher SE. Biotin transport in microvillous membrane
vesicles, cultured trophoblasts and the isolated perfused
cotyledon of the human placenta. Am J Physiol 1992;
39. Schenker S, Hu ZQ, Johnson RF, et al. Human placental biotin
transport: normal characteristics and effect of ethanol.
Alcohol Clin Exp Res 1993; 17(3):566–575.
40. Hu ZQ, Henderson GI, Mock DM, et al. Biotin uptake by
basolateral membrane of human placenta: normal characteristics
and role of ethanol. Proc Soc Exp Biol Med 1994;
41. Mock DM, Mock NI, Langbehn SE. Biotin in human milk:
methods, location, and chemical form. J Nutr 1992; 122:535–
42. Mock DM, Mock NI, Dankle JA. Secretory patterns of biotin
in human milk. J Nutr 1992; 122:546–552.
43. Mock DM, Stratton SL, Mock NI. Concentrations of biotin
metabolites in human milk. J Pediatr 1997; 131(3):456–458.
44. Zempleni J, Mock D. Marginal biotin deficiency is teratogenic.
Proc Soc Exp Biol Med 2000; 223(1):14–21.
45. Mock DM, Stadler DD, Stratton SL, et al. Biotin status
assessed longitudinally in pregnant women. J Nutr 1997;
46. Mock DM, Stadler DD. Conflicting indicators of biotin status
from a cross-sectional study of normal pregnancy. J Am Coll
Nutr 1997; 16:252–257.
47. Mock DM, Quirk JG, Mock NI. Marginal biotin deficiency
during normal pregnancy. Am J Clin Nutr 2002; 75(2):295–
48. Mock DM. Marginal biotin deficiency is common in normal
human pregnancy and is highly teratogenic in the mouse. J
Nutr 2009; 139(1):154–157.
49. Mock DM, Mock NI, Stewart CW, et al. Marginal biotin deficiency
is teratogenic in ICR mice. J Nutr 2003; 133:2519–2525.
50. Watanabe T, Endo A. Biotin deficiency per se is teratogenic
in mice. J Nutr 1991; 121:101–104.
51. Mock DM, Henrich C, Carnell N, et al. Lymphocyte
propionyl-CoA carboxylase and accumulation of odd-chain
fatty acid in plasma and erythrocytes are useful indicators of
marginal biotin deficiency. J Nutr Biochem 2002; 13(8):462–
52. Mock DM, Henrich-Shell CL, Carnell N, et al. 3-
hydroxypropionic acid and methylcitric acid are not reliable
indicators of marginal biotin deficiency. J Nutr 2004; 134:317–
53. Velazquez A, Martin-del-Campo C, Baez A, et al. Biotin deficiency
in protein-energy malnutrition. Eur J Clin Nutr 1988;
54. Krause K-H, Berlit P, Bonjour J-P. Impaired biotin status in
anticonvulsant therapy. Ann Neurol 1982; 12:485–486.
55. Krause K-H, Berlit P, Bonjour J-P. Vitamin status in patients
on chronic anticonvulsant therapy. Int J Vitam Nutr Res 1982;
56. Mock DM, Dyken ME. Biotin catabolism is accelerated in
adults receiving long-term therapy with anticonvulsants.
Neurology 1997; 49(5):1444–1447.
57. Wang K-S, Mock NI, Mock DM. Biotin biotransformation to
bisnorbiotin is accelerated by several peroxisome proliferators
and steroid hormones in rats. J Nutr 1997; 127(11):2212–
58. Mock DM, Mock NI, Lombard KA, et al. Disturbances in
biotin metabolism in children undergoing long-term anticonvulsant
therapy. J Pediatr Gastroenterol Nutr 1998;
59. Said HM, Redha R, Nylander W. Biotin transport in the human
intestine: inhibition by anticonvulsant drugs. Am J Clin
Nutr 1989; 49:127–131.
60. Nisenson A. Seborrheic dermatitis of infants and Leiner’s
disease: a biotin deficiency. J Pediatr 1957; 51:537–548.
61. Nisenson A. Seborrheic dermatitis of infants: treatment with
biotin injections for the nursing mother. Pediatrics 1969;
62. Erlichman M, Goldstein R, Levi E, et al. Infantile flexural
seborrhoeic dermatitis. Neither biotin nor essential fatty acid
deficiency. Arch Dis Child 1981; 567:560–562.
63. Johnson AR, Hood RL, Emery JL. Biotin and the sudden
infant death syndrome. Nature 1980; 285:159–160.
64. Heard GS, Hood RL, Johnson AR. Hepatic biotin and the
sudden infant death syndrome. Med J Aust 1983; 2(7):305–
65. Yatzidis H, Koutsicos D, Alaveras AG, et al. Biotin for neurologic
disorders of uremia. N Engl J Med 1981; 305(13):
66. Livaniou E, Evangelatos GP, Ithakissios DS, et al. Serum
biotin levels in patients undergoing chronic hemodialysis.
Nephron 1987; 46:331–332.
67. DeBari V, Frank O, Baker H, et al. Water soluble vitamins
in granulocytes, erythrocytes, and plasma obtained from
chronic hemodialysis patients. Am J Clin Nutr 1984; 39:410–
68. Yatzidis H, Koutisicos D, Agroyannis B, et al. Biotin in the
management of uremic neurologic disorders. Nephron 1984;
69. Braguer D, Gallice P, Yatzidis H, et al. Restoration by biotin
in the in vitro microtubule formation inhibited by uremic
toxins. Nephron 1991; 57:192–196.
70. Colombo VE, Gerber F, Bronhofer M, et al. Treatment of brittle
fingernails and onychoschizia with biotin: scanning electron
microscopy. J Am Acad Dermatol 1990; 23:1127–1132.
71. Sander JE, Packman S, Townsend JJ. Brain pyruvate carboxylase
and the pathophysiology of biotin-dependent diseases.
Neurology 1982; 32:878–880.
72. Suchy SF, Rizzo WB, Wolf B. Effect of biotin deficiency and
supplementation on lipid metabolism in rats: saturated fatty
acids. Am J Clin Nutr 1986; 44:475–480.
73. Suchy SF,Wolf B. Effect of biotin deficiency and supplementation
on lipid metabolism in rats: cholesterol and lipoproteins.
Am J Clin Nutr 1986; 43:831–838.
74. Mock DM. Evidence for a pathogenic role of 6 polyunsaturated
fatty acid in the cutaneous manifestations of biotin
deficiency. J Pediatr Gastroenterol Nutr 1990; 10:222–
75. McMahon RJ. Biotin in metabolism and molecular biology.
Annu Rev Nutr 2002; 22:221–239.
76. Zempleni J. Biotin. In: Bowman BB, Russell RM, eds. Present
Knowledge in Nutrition.Washington, DC: International Life
Sciences Institutes–Nutrition Foundation, 2001.
77. Zempleni J, Mock DM. Chemical synthesis of biotinylated
histones and analysis by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis/streptavidinperoxidase.
Arch Biochem Biophys 1999; 371(1):83–88.
78. Zempleni J, Mock DM. Chemical synthesis of biotinylated histones
and analysis by SDS-PAGE/streptavidin peroxidase.
Biomol Eng 2000; 16(5):181.
79. Stanley JS, Griffin JB, Zempleni J. Biotinylation of histones in
human cells: effects of cell proliferation. Eur J Biochem 2001;
80. Hassan YI, Zempleni J. Epigenetic regulation of chromatin
structure and gene function by biotin. J Nutr 2006;
81. Gravel R, Narang M. Molecular genetics of biotin
metabolism: old vitamin, new science. J Nutr Biochem 2005;
82. Narang MA, Dumas R, Ayer LM, et al. Reduced histone
biotinylation in multiple carboxylase deficiency patients: a
nuclear role for holocarboxylase synthetase.HumMol Genet
83. Zempleni J. Chromatin remodeling events at theSMVTlocus.
J Nutr 2008; 139(1):163–166.
84. Bailey LM, Ivanov RA,Wallace JC, et al. Artifactual detection
of biotin on histones by streptavidin. Anal Biochem 2007;
85. Healy S, Heightman TD, Hohmann L, et al. Nonenzymatic
biotinylation of histone H2A. Protein Sci 2008; 18:314–
86. Healy S, Perez-Cadahia B, Jia D, et al. Biotin is not a natural
histone modification. Biochem Biophys Acta 2009; 1789:719–
87. National Research Council. Dietary reference intakes for thiamin,
riboflavin, niacin, vitamin B-6, folate, vitamin B-12,
pantothenic acid, biotin, and choline. In: Recommended Dietary
Allowances, Food and Nutrition Board, Institute of
Medicine, ed. Washington, DC: National Academy Press