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How to get taller with foods

NutritionSuccess Chemistry Staff
grow tall with food

The height of each person is influenced by several factors, the main one being the genetics inherited by parents, the other factors are sleep, diet, gender, life stage and the practice of physical activities.

There is a formula to know the probable height: mother's height + father's height + 13cm for boys or -13cm for girls / 2. It does not guarantee the accurate result, varying by about 10 centimeters more or less.

Men tend to be taller than women, and their growth goes up to around 20 years of age, as their puberty starts late. Women, who reach puberty earlier, stop growing earlier at around 15 years of age.

foods that support growth

For proper growth it is important to maintain good nutritional status, avoiding nutrient, protein and calorie deficiencies that would impair growth. Some nutrients participate in growth, and food sources must be contained in a varied and healthy diet. The main nutrients are calcium, vitamin D, vitamin A, iron, zinc and folic acid.

calcium-rich foods

The calcium is a mineral that is directly linked to the growth and health of bones, as part of training, strength and bone density. The source foods of this mineral are:

  • Milk and derivatives

  • Dark green vegetables

  • Sesame

  • Tofu

vitamin d rich foods

Vitamin D has several important functions, the main one being to increase calcium absorption. The main source of vitamin D is the sun, but some food sources can be inserted into the diet, such as:

  • Mushrooms

  • Fish, eggs, and meat

  • Whole milk, butter, and yogurts

  • Vitamin D fortified foods

vitamin-rich foods

This vitamin is related to the release of GH (growth hormone), besides acting on the differentiation of bone cells. Its deficiency inhibits growth and causes bone deformities.

  • Guava

  • Whole milk and derivatives such as butter

  • Foods rich in beta carotenes such as carrots, squash, papaya, mango, and spinach

zinc-rich foods

The zinc is an important mineral that participates in the production of proteins and genetic material for cell replication, is of great importance for growth. In addition, it is also involved with the regulation of GH and IGF-1 hormones, also involved with growth. In adolescence, especially in the spurt phase, the body significantly increases zinc retention.

  • Oysters and other seafood

  • Red meat

  • Pumpkin and Chestnut Seed

folic acid-rich foods

Like zinc, folic acid acts in a variety of cellular reactions, including the formation of DNA and RNA. It is known to be extremely important for pregnant women in the development of the fetus (period of rapid growth), but remains important for later growth. It is also essential for the formation of blood cells (red blood cells and leukocytes).

  • Dark green vegetables (such as spinach, broccoli, arugula)

  • Brewer's yeast

  • Legumes

iron-rich foods

The iron is essential for the formation of blood cells, it is also important for growth, especially for teenagers as it helps in building the muscles, requiring more oxygen and increased blood volume.

  • Meat

  • Oatmeal

  • Cashew nut

  • Watercress and Spinach

sports, stretching, and posture

In addition to diet, stretching and posture is very important for growth. Stretching, keeping your posture straight and steady, and doing sports that stimulate stretching and growth, such as basketball, volleyball, and swimming, all help during the growth phase.

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Beauty, Nutrition, GlossarySuccess Chemistry Staff


Bilberry, Vaccinium myrtillus L., is a shrub with edible

fruits that is native to Circumboreal regions from Europe

to Asia as well as the Rocky Mountains in North America.

Bilberries are related to other edible berries including

blueberry, cranberry, huckleberry, and lingonberry.

Bilberry fruits contain anthocyanins, which are natural

pigments, responsible for the dark blue color of the fruits

and for many of the health benefits. In vitro studies have

shown that bilberry extracts have antioxidant activity, inhibit

platelet aggregation, prevent degradation of collagen

in the extracellular matrix surrounding blood vessels and

joints, and have a relaxing effect on arterial smooth muscle.

Bilberry extracts have also demonstrated anticancer

and antibacterial actions, in vitro. Pharmacokinetic studies

in animals and humans show that a small percentage

of the anthocyanins is absorbed into the body and widely

distributed. Human clinical studies have been conducted

evaluating the potential benefits of bilberry preparations

in treating venous insufficiency and visual disorders ranging

from night vision to diabetic retinopathy as well as

cancer prevention. No serious toxicities have been associated

with preparations of the fruits in animal screens and

no serious side effects have been identified in humans.


Bilberries are edible fruits from V. myrtillus L. of the family

Ericaceae. Bilberry is the standardized common name for

the fruit in the United States, but the fruit is also known

as European blueberry, huckleberry, and whortleberry (1).

Related to bilberry, and in the same genus of Vaccinium, are

other edible berries including blueberry, cranberry, huckleberry,

and lingonberry.

Bilberry is a shrub, 1–6 dm high, found in heaths,

meadows, and moist coniferous forests in Circumboreal

regions from Europe to Asia, with populations in the

American and Canadian Rocky Mountains (2).

The blue-black berries are harvested when ripe,

usually during the months of July through September.

The berries are oblate-globose, 5–9 mm diameter, with

4–5 locules containing many seeds. The seeds are approximately

1 mm long with a yellow/brown-dimpled

surface (2).

Both the leaves and fruits of bilberry have been used

medicinally since the Middle ages.

The leaves were used topically for inflammation, infections,

and burns, as well as internally as a treatment for

diabetes. According to the herbalist Grieve, the fruits were

used to treat dysentery, diarrhea, gastrointestinal inflammation,

hemorrhoids, vaginal discharges, scurvy, urinary

complaints, and to dry up breast milk. More recently, it

was found that bilberry was used by World War II pilots

to improve their night vision (2,3).

Bilberry fruit preparations are still used to improve

vision as well as for their benefits to the circulatory system

treating fragility and altered permeability of blood

vessels that is either primary or secondary to arterial hypertension,

arteriosclerosis, or diabetes (3).


Bilberry fruits contain anthocyanins that are natural pigments

in the chemical class known as flavonoids. Anthocyanins

are glycosides or compounds with sugars

attached at the 3 position, while anthocyanidins are aglycones

(the same basic structure without the sugars attached)

(see chapter 74, “Polyphenol Overview”). The majority

(64%) of anthocyanins in the fruit are glycosides of

cyanidin and delphinidin (Fig. 1). The quantity of anthocyanin

in the fruit ranges from 300 to 700 mg per 100 g.

Bilberry fruits also contain flavonols, tannins, phenolic

acids, organic acids, sugars, vitamins, and volatile compounds


The primary commercial source of bilberry fruits is

“wild harvest” from regions in Europe and Scandinavia.

The fruits are sold fresh, frozen, or dried. Besides the

whole fruit, commercial products include cold macerates,

decoctions, and dry extracts. The dry extracts are commonly

prepared using alcohol, methanol or ethanol (2).

Until recently, a single-wavelength spectrophotometric

technique (UV) was commonly used to standardize

the anthocyanin content of bilberry products. However,

this technique did not detect adulteration of bilberry

preparations with substances of similar color (4). A high performance

liquid chromatographic technique that can

detect and quantitate both anthocyanins and anthocyanidins

has recently been developed enabling a better assurance

of product identity and quality (5).

Most studies on bilberry have been conducted using

extracts characterized as containing 36% anthocyanins or

25% anthocyanidins.


In vitro studies have shown that bilberry extracts have

antioxidant activity, inhibit platelet aggregation, prevent

  • Cyanidin 3-O-glycoside OH OH H arabinose, glucose, or galactose

  • Delphinidin 3-O-glycoside OH OH OH arabinose, glucose, or galactose

  • Malvidin 3-O-glycoside OCH3 OH OCH3 arabinose, glucose, or galactose

  • Peonidin 3-O-glycoside OCH3 OH H arabinose, glucose, or galactose

  • Petunidin 3-O-glycoside OH OH OCH3 arabinose, glucose, or galactose

  • OH

  • HO

  • O R3

  • R2

  • R1

  • R

  • O+

degradation of collagen in the extracellular matrix

surrounding blood vessels and joints, and have a relaxing

effect on arterial smooth muscle. These actions are vasoprotective,

increasing capillary resistance and reducing

capillary permeability (3). Bilberry extracts have also

demonstrated anticancer and antibacterial actions in

vitro. There is no evidence of toxicity in animals at the

effective doses.

Antioxidant Activity

Bilberry fruits have demonstrated antioxidant activity in

in vitro and in animal models. In the oxygen radical absorbance

capacity(ORAC)assay, an in vitro test measuring

free radical quenching, bilberry fruits had potent activity

compared with other fresh fruits and vegetables (44.6 +

2.3 mol Trolox equivalents (TE)/g) (6). In another assay,

a bilberry extract (25% anthocyanins) exhibited antioxidant

activity in protecting keratinocytes in culture from

damage due to UVA and UVB light (7,8). The bilberry

extract attenuated UVA-induced reactive oxygen species

formation, peroxidation of membrane lipids, and depletion

of intracellular glutathione in concentrations of 10–50

mg/L (7). In the same concentration range, the extract inhibited

UVB-induced generation of reactive oxygen and

nitrogen species, DNA strand breaks, as well as caspase-

3 and caspase-9 activity (mediators that execute apoptotic

cell death) (8).

In an animal study, a bilberry extract characterized

as containing 38% anthocyanins reduced oxidative stress

caused in mice by removal of the animal’s whiskers. The

extract administered orally at a dose of 100 mg/kg for

7 days ameliorated the increase in oxygen radicals (thiobarbituric

acid reactive substances), protein carbonyl formation,

and lipid peroxidation in the brain, heart, kidney,

and liver. The extract also suppressed the stress-induced

changes in dopamine levels (9).

A bilberry extract (42% anthocyanins) reduced oxidative

stress to the liver in a restraint-stress mouse model.

The extract, administered at a dose of 200 mg/kg for

5 days, ameliorated the increase in plasma levels of alanine

aminotransferase, a liver enzyme. The extract also reduced

plasma and liver ORAC levels and increased plasma glutathione

and vitamin C levels in the liver (10). A similar

study was conducted in mice with kidney damage induced

by potassium bromate. Oral administration of the

same extract ameliorated the increase in blood urea nitrogen

levels and the decreases in kidney malondialdehyde,

nitric oxide, and xanthine oxidase levels. The bilberry extract

also improved the kidney ORAC levels (11).

MyrtoSelectTM, an extract containing approximately

40% anthocyanins, was tested for its effects on gene expression

(DNAmicroarray) in a macrophage cell line stimulated

with lipopolysaccharide (LPS). The extract, at a

concentration of 75 g/mL, appeared to mitigate the effect

of LPS, targeting genes involved in inflammation and

immune defense. Pretreatment with the bilberry extract

affected 45% of the genes downregulated by LPS and 36%

of genes upregulated by LPS (12).


Bilberry extracts have demonstrated beneficial effects on

the circulation.

including inhibiting platelet aggregation,

reducing capillary permeability, facilitating vasodilation,

and inhibiting the development of atherosclerosis and angiogenesis.

Myrtocyan R (also known as MirtoSelectTM, containing

36% anthocyanins) inhibited platelet aggregation in

vitro induced by adenosine diphosphate (ADP), collagen,

and sodium arachidonate in rabbit platelet-rich plasma

with IC50 values ranging from 0.36 to 0.81 mg/mL. Myrtocyan

administered orally to rats (400 mg/kg) prolonged

the bleeding time in the animals, without affecting coagulation

pathways. The same dose administered to mice reduced

the adhesiveness of platelets to glass (3). Myrtocyan

administered to healthy human subjects, 480 mg/day

(173 mg anthocyanins/day) for 30–60 days, reduced the

aggregation response ex vivo to ADP and collagen (13).

In a rabbit skin model, oral treatment with 400 mg

anthocyanins per kilogram body weight 30 minutes before

topical application of chloroform reduced the capillary

permeability caused by the irritant by 66%. In rats, administration

of bilberry anthocyanins, 200 mg/kg orally,

decreased bradykinin-induced capillary permeability by

39%. The same dose reduced carrageenin-induced rat paw

edema by 45% (14). In a rat model of experimentally induced

hypertension, 500 mg anthocyanins per kilogram

body weight given orally for 12 days completely ameliorated

the increase in blood–brain barrier permeability and

reduced the increase in aortic vascular permeability by

40% (15). In a hamster cheek pouch model, 100 mg bilberry

extract per kilogram daily for 4 weeks reduced the

circulatory damage due to ischemic reperfusion (16).Arat

model suggested that bilberry anthocyanins (50mg/kgIP)

inhibited the enzymatic degradation of collagen, decreasing

the permeability of the blood–brain barrier caused by

proteases (17).

Bilberry preparations are reported to relax arterial

tissues in vitro.

Preliminary experiments pointed

to a mechanism involving prostaglandins. However, a

more recent study using porcine coronary arteries demonstrated

a mechanism involving nitric oxide (endothelialderived

relaxing factor) (18).

A bilberry extract was reported to inhibit the development

of atherosclerosis in apolipoprotein E–deficient

mice. The mice received diets supplemented with 0.02%

of a bilberry extract (52% anthocyanins) for 16 weeks. The

Bilberry 39

extract reduced lipid deposits and the development of

lesions. It did not affect plasma antioxidant capacity or

plasma lipid levels (19).

A bilberry extract (25% anthocyanins) was tested

for its effect on angiogenesis both in vitro and in vivo.

The extract at concentrations of 0.3–30 g/mL inhibited

tube formation and the migration of human umbilical

vein endothelial cells induced by vascular endothelial

growth factor A. The extract also inhibited the induction

of retinopathy in newborn mice, which was induced

with oxygen. Intravitreal administration of 300 ng extract

per eye significantly inhibited the area of neovascular

tufts (20).


Anthocyanins have been reported to mediate several

physiological functions that ultimately may result in cancer

suppression. Anthocyanins suppress the growth of

cancer cell lines in vitro, including HL60 human leukemia

calls and HCT116 human colon cancer cells. A bilberry extract

induced apoptotic cell bodies and nucleosomal DNA

fragmentation in HL60 cells (21). A bilberry extract (Mirtocyan)

has also been shown to suppress the activity of

receptor tyrosine kinases, which are thought to play a crucial

role in carcinogenesis and tumor progression. When

tested over a number of tyrosine kinases, the activity was

consistent but not specific (22).


Phenolic compounds in bilberry have demonstrated in

vitro antimicrobial effects against strains of Salmonella and

Staphylococcus possibly through interfering with adhesion

of the bacteria. Treatment of bilberry preparations with

pectinase released phenolics from the cell wall matrix and

increased the antibacterial activity (23). In experiments

with Neisseria meningitidis, the bacteria that causes meningitis

and septicemia, a bilberry juice fraction was reported

to inhibit the binding of the bacteria to epithelial cells

in culture. Fractions of the juice also bound to the bacterial

pili. The authors concluded that anthocyanins were

partly responsible for the activity but that there appeared

to be other compounds in bilberry that may also interact

directly with the pili or act synergistically with the anthocyanins


Safety Studies (Animal Toxicology)

Myrtocyan (25% anthocyanins) has been tested for acute

and chronic toxicity in animal studies. There were no

deaths with an acute dose in rats up to 20 g/kg orally

and in mice up to 25 g/kg. Six months treatment with

doses of 125–500 mg/kg in rats and 80–320 mg/kg in

dogs found no evidence of toxicity. The preparation was

tested in guinea pigs for 2 weeks and in rats for 6 weeks

with doses up to 43 mg/kg without incident (2,3).


Animal studies show that bilberry anthocyanins are absorbed

intact, or after methylation. This is unlike other

flavonoid glycosides which are hydrolyzed to their aglycones

and metabolized to glucuronidated or sulfated

derivatives (25). Following administration of 400 mg/kg

orally to rats, peak blood levels of anthocyanins were detected

within 15 minutes and afterwards declined rapidly.

Only 1% of the anthocyanins was eliminated in the urine

and 4% in the bile. The absolute bioavailability of bilberry

anthocyanins was estimated to be 1.2–5% (26). A study in

mice reported that malvidin 3-glucoside and malvidin 3-

galactoside were the principal anthocyanins in the plasma

60 minutes after oral administration of 100 mg/kg. When

the mice were maintained on a diet containing 0.5% bilberry

extract, plasma levels of anthocyanins reached 0.26

M. Anthocyanins were detected in the liver, kidney,

and lung. They were not detected in the spleen, thymus,

heart, muscle, brain, white fat, or eyes (25).

A pharmacokinetic study with six human subjects

detected anthocyanins in the plasma 1.5–6 hours following

intake of a bilberry–lingonberry puree. The study

examined the production of urinary phenolic acids and

found the greatest increase in methylated compounds.

The amount of urinary phenolic acids was low, and the authors

suggested that the fragmentation of anthocyanins

to phenolic acids was not a major metabolic pathway

(27). Another pharmacokinetic study with 20 subjects

that consumed 100 g/day of berries, including black currant,

lingonberries, and bilberries, for 8 weeks reported

an increase in serum quercetin (up to 51% higher) compared

with control subjects who did not consume berries

(28). A study with 25 subjects administered 1.4–5.6 g

Mirtocyan (25% anthocyanins) daily for 7 days reported

detection of anthocyanins as well as methyl and glucuronide

metabolites in the plasma and urine but not in the

liver (29).


Human clinical studies have been conducted evaluating

the potential benefits of bilberry preparations in treating

venous insufficiency and visual disorders ranging from

night vision to diabetic retinopathy as well as cancer prevention.

Vascular Health

Clinical studies have been conducted evaluating the potential

benefits of bilberry preparations in treating venous

insufficiency. A review of studies conducted between 1970

and 1985 included 568 patients with venous insufficiency

of the lower limbs who were treated with bilberry preparations

(30). The studies reported an improvement in circulation

and in lymph drainage resulting in a reduction

in edema. A more recent placebo-controlled study which

included 60 participants with varicose veins reported improvement

in edema in the legs and ankles, sensation of

pressure, cramps, and tingling or “pins and needles” sensations

with a dose of 160 mg Tegens R , three times daily

for 1 month (31). Tegens (Inverni della Beffa, Italy) contains

a bilberry extract named Myrtocyan or MirtoSelect

(25% anthocyanins), manufactured by Indena SpA, Italy.

Visual Health

Asystematic review was conducted on placebo-controlled

studies on the effects of bilberry preparations on night

40 Barrett

vision. Literature searches identified 30 clinical studies,

and 12 of those met the inclusion criteria of being placebo

controlled. Of the 12 studies, 5 were randomized. Healthy

subjects with normal or above average eyesight were

tested in 11 out of the 12 studies. Many of the studies

were acute, using a single dose, and the longest treatment

period was 28 days. Full characterization of the products

used in the studies was not available, but assuming

25% anthocyanin content, the doses of anthocyanin

ranged from 12 to 2880 mg. The techniques used to measure

the extent and rate of dark adaptation ranged from

visual acuity, contrast sensitivity, and critical flicker fusion

to electroretinographic monitoring of response to light

flashes. The four most recent randomized controlled studies

with rigorous methodology reported negative results.

One randomized controlled study and all seven of the nonrandomized

studies reported positive effects. The authors

concluded that the present studies do not support the use

of bilberry by those who are healthy with normal vision to

improve their night vision. However, uncontrolled studies

report a benefit for those with eye disorders, including

retinal degeneration, myopia, simple glaucoma, and

pathological fundus. Furthermore, studies with synthetic

anthocyanins suggest a positive benefit for those with myopia,

central retinal lesions, and night blindness (32).

Two studies on diabetic retinopathy, using a dose of

160 mg Tegens twice daily, demonstrated a trend toward

improvement in mild cases of the disease. The first study

was a 1-month, placebo-controlled study that included

36 subjects. At the end of the month, 10 of 13 patients in

the Tegens group with ophthalmoscopic ally detectable

retinal abnormalities (microaneurysms, hemorrhagic foci,

exudates) were improved, while all 15 patients with these

abnormalities in the placebo group remained unchanged.

A similar trend was observed among those patients with

fluoroangiography abnormalities (33). The second study

lasted 1 year and included 40 subjects who were given

Tegens or placebo in addition to the usual therapy for

retinopathy. As a result, in 50% of patients given bilberry,

the retinal lesions and associated edema were improved

compared with 20% in the control group (34).

A mixture of vitamin E and bilberry (FAR-1, Ditta

Farmigea SpA, Italy) showed a trend toward prevention

of senile cataracts after 4 months of 180 mg bilberry

anthocyanins (25% anthocyanidins) and 100 mg DLDL tocopheryl

acetate twice daily. When the placebo group

was changed from placebo to the bilberry preparation,

and the trial continued for an additional 4 months, there

was no statistical difference between the two groups. The

rationale for this study was previous indications that

antioxidants might prevent the development of senile

cataracts (35).

A mechanistic study using Myrtocyan examined

changes in pupillary reflexes to light following a single

high dose of 240 mg anthocyanosides or placebo in

40 healthy volunteers. The study was conducted to explore

the use of bilberry in work situations where exposure

to high light intensities dampens pupillary reflexes

and leads to vision fatigue. The authors of the study suggested

that the pigments in bilberry might increase sensitivity

to light and improve blood flow in the capillaries of

the eye. Improvement in pupillary reflexes was observed

in both groups, with the improvement in the treatment

group being only slightly better than that in the placebo

group (36).

Cancer Prevention

In an open label study, 25 colorectal cancer patients scheduled

to undergo surgery were given 1.4, 3.8, or 5.6 g

bilberry extract (Mirtocyan) containing 0.2–2.0 g anthocyanins

for 7 days before surgery. Availability of anthocyanins

was determined by detection in the plasma,

colorectal tissue, and urine but not in the liver. Anthocyanins

detected in the body were unaltered, or products

of metabolic glucuronidation and O-methylation. Proliferation

of cells in the tumor tissue was decreased by 7%

compared with before the bilberry intervention (29).

Side Effects and Adverse Effects

No side effects were reported in the clinical studies mentioned

earlier. In a 1987 postmarketing surveillance study

with 2295 subjects, only 94 (4.1%) complained of minor

side effects, most of which involved the gastrointestinal

track. Most of the participants took 160 mg Tegens twice

daily for 1–2 months (3).

Observed Drug Interactions and Contraindications

No drug interactions or contraindications have been reported

in the literature for bilberry.


Bilberry fruit extracts and anthocyanins have been the

subject of pharmacological studies and human clinical trials.

In vitro and in vivo studies demonstrate good evidence

for the antioxidant activity of bilberry extracts

along with strong indications of benefit to the cardiovascular

system. Animal and human pharmacokinetic studies

demonstrate bioavailability of anthocyanins, but absorption

appears to be limited. Human clinical studies

on the effects of bilberry extracts on eyesight and vascular

diseases suffer from poor methodology, including

small sample sizes and short-term exposures. While it

appears doubtful that bilberry preparations benefit the

night vision of healthy subjects, the benefit for those with

diabetic retinopathy and other eye disorders merits exploration.

Another area that appears promising is that of

benefits to the cardiovascular system, specifically vasculitis

or venous insufficiency. Bilberry products have been

safely consumed, without significant adverse events or

side effects.


Bilberry is a food and preparations are also used medicinally

In the United States, preparations of bilberry are

sold as foods and dietary supplements. The U.S. Pharmacopoeia

has published a standard monograph for powdered

bilberry extract (37). The German Commission

E completed a monograph for bilberry fruits in which

preparations of the ripe fruit are indicated orally for

Bilberry 41

nonspecific, acute diarrhea and topically for mild inflammation

for the oral and pharyngeal mucosa (38). The European

Scientific Cooperative on Phytotherapy (ESCOP)

monograph lists the internal use of bilberry fruit preparations

(enriched in anthocyanins) for symptomatic treatment

of problems related to varicose veins, such as painful

and heavy legs. The ESCOP monograph also lists the dried

fruit as supportive treatment of acute, nonspecific diarrhea

(39). In Canada, bilberry products are approved as natural

health products for traditional use orally as an astringent

and as a source of antioxidants as well as for use as a

gargle to relieve mild inflammation of the mouth and/or

throat (40).


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24. Toivanen M, Ryynanen A, Huttunen S, et al. Binding of Neisseria

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25. Sakakibara H, Ogawa T, Koyanagi A, et al. Distribution and

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anthocyanosides pharmacokinetics in rats. Arzneimittelforschung

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27. Nurmi T, Mursu J, Heinonen M, et al. Metabolism of berry anthocyanins

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29. Thomasset S, Berry DP, Cai H, et al. Pilot study of oral anthocyanins

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Nutrition, GlossarySuccess Chemistry Staff


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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50 Mock

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protein-bound biotin. J Inherit Metab Dis 1984; 7(suppl


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of a sodium-dependent vitamin transporter mediating

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choriocarcinoma cells. Placenta 1997; 18:527–533.

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expression of acDNAencoding a mammalian sodiumdependent

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pantothenate, biotin, and lipoate. J Biol Chem 1998; 273:


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of water-soluble vitamins. Annu Rev Physiol 2004;


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8(suppl 1):53–58.

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

1992; 56:427–432.

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.

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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.

Biotin 51

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

2004; 13(1):15–23.

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

Bitter Orange

Glossary, NutritionSuccess Chemistry Staff


Citrus aurantium (C. aurantium) is the Latin name for a

plant commonly referred to as bitter orange, sour orange,

Neroli, Chongcao, or Seville orange. It is a source

of synephrine and several other biogenic amines, as well

as other bioactive phytochemicals and has been used in dietary

supplements for weight loss. In this entry,we discuss

the available evidence pertaining to safety and efficacy of

C. aurantium for weight loss, as examined in animal studies,

clinical trials, and case reports.


Bitter orange is a member of the Rutaceae family, a hybrid

between Pummelo, Citrus grandis, and Mandarin, Citrus


Native to Asia, various parts of the plant are

used throughout the world for a variety of indications.

Bitter orange and its components are commercially available

in herbal weight loss supplements, ostensibly for their

adrenergic agonistic properties (1), often in combination

with other ingredients hypothesized to promote weight

loss. Its constituent p-octopamine and synephrine alkaloids

(SAs) are usually cited as the active ingredients in

such products (2). With the banning of ephedra in the

United States in 2004, bitter orange has been increasingly

included in weight loss supplement formulations. Because

of similarities in their constituents and possible mechanisms

(both sources of natural alkaloids with sympathomimetic

activity), concerns have been raised that bitter

orange may carry risks similar to those hypothesized to

exist for ephedra (3).


Bitter Orange origin is in China and appears in writing

as far back as 300 BC.

Its ancient use has also been documented

in Japan and Rome (4). It is native to eastern

Africa, Arabia, and Syria and is cultivated in various European,

North American, and South American regions. The

leaf was historically used as a tonic, laxative, or sedative

in Mexico and South America and for insomnia, palpitations,

or stomachaches by the European Basque people

(5,6). The fruit and peel are also used for stomach aches, as

well as high blood pressure (BP), spasm, and a variety of

gastrointestinal conditions by both the Basque and practitioners

of traditional Chinese medicine (7). While the

practice arose in Ancient Egypt, neroli oil is still currently

used for aromatherapy and bergamot, a subspecies of

C. aurantium, is used for flavoring and aroma in Earl Grey

teas (8). Modern uses for C. aurantium include digestive,

cardiovascular, neuromuscular, and antiseptic indications

in countries such as China, Curacao, Haiti, India, Mexico,

Trinidad, Turkey, and the United States (9). The most

common current western use, however, is as a dietary

supplement for weight loss.


Some authors (10) state that C. aurantium contains meta synephrine

(m-synephrine, m-s), whereas others (11) state

that it contains only para-synephrine (p-synephrine, ps).

However, research (I.A. Khan, oral communication,

2005) has shown that C. aurantium naturally contains

p-synephrine and does not contain m-synephrine. Allison

and colleagues reported that at least one over-the counter

(OTC) product purportedly containing SAs from

C. aurantium contains both p-synephrine andm-synephrine

(12), raising concerns about possible adulteration and

mislabeling. There is also an ortho isomer of synephrine

(o-synephrine), whose content in C. aurantium is unknown.

p-, m-, and o-synephrine can each exist in D or L forms.

p-Synephrine, an undisputed component of C. aurantium,

is typically referred to simply as synephrine (13).

It is an -adrenergic agonist (14) that also has some -

adrenergic properties (15). p-Synephrine occurs naturally

in the human body in small quantities and might act as

a neurotransmitter (16). Under the name oxedrine, it has

been used since 1927 (17) in eyedrops. p-Synephrine is

thought to be the ingredient in C. aurantium primarily

responsible for weight loss. However, neither this nor

whether C. aurantium actually produces weight loss in

humans is firmly established.

m-Synephrine, often referred to as phenylephrine,

is an isomer of p-synephrine. To the best of our knowledge,

m-synephrine is not contained naturally in C. aurantium.

m-Synephrine is also an -adrenergic agonist that has

some -adrenergic agonist properties. It has been studied

more extensively than p-synephrine and is one of the

two most widely used OTC decongestants today (Fig. 1)

(13). p-Synephrine and m-synephrine have similar structure

to ephedrine, as well as other substances that have

some effects on reducing food intake and/or body weight

such as epinephrine and norepinephrine (Fig. 1), supporting

the conjecture that, to the extent that function follows

structure, p-synephrine and m-synephrine, may also reduce

food intake and or body weight.

The -adrenergic sympathomimetic amine, octopamine,

is also present in C. aurantium, though possibly

at appreciable levels (2). Like both forms of

synephrine, it is an -adrenergic agonist with some

Chemical structures of (A) p-synephrine, (B) m-synephrine,

(C) ephedrine, (D) epinephrine, and (E) norepinephrine.

-adrenergic properties. It is used to treat hypertension

and as a cardiotonic (13) and has also been examined for

its potential role in promoting weight loss (18).

Because of their similar properties and the overlap

of their inclusion in supplements, we will refer to these

substances collectively as synephrine alkaloids (SAs). SAs

are used clinically as decongestants (1), during surgical

procedures as a vasopressor (19), for acute treatment of

priapism (20), and in ophthalmological examinations for

pupil dilation (21). Products that contain C. aurantium or

its derivatives, including OTC weight loss supplements,

will be referred to as C. aurantium products (CAPs).

Regulatory oversight for dietary supplements is

much less rigorous than for pharmaceuticals, and extensive

evidence is not required prior to release of a product

on the public market. While a phase of requirements for

meeting good manufacturing practices is currently underway,

this may help to explain why the quality and quantity

of the evidence we have available to evaluate the safety

and efficacy of C. aurantium is minimal.


As sympathomimetic agents with both - and -

adrenergic receptor agonist properties, SAs might increase

energy expenditure and/or decrease food intake (22). In

addition, there is some evidence that adrenergic agonists,

including SAs, decrease gastric motility (23). Similar to

compounds such as cholecystokinin and other gut peptides

which both decrease gastric motility and food intake

(24), one might conjecture that SAs may also decrease food

intake via reducing gut motility. Activation of lipolysis is

a known -adrenergic activity (25) that may be fueled by

these components of C. aurantium.


Bitter Orange Weight Loss

SAs reduce food intake in rodents (26), and some studies

indicate that SAs can reduce rodent body weight

(13,26). SAs have also been shown to promote lipolysis

in adipocytes through -adrenergic stimulation (27)

and to increase lipoprotein lipase activity in the parametrial

fat pad of female hamsters (28). However, among

monosodium glutamate–treated obese mice, SAs reduced

weight gain but had no effect on body fat percent (29).

Toxicity and Mortality

Data suggest that m-synephrine (not present in bitter orange)

may prolong life in rodents. A 2-year study by the

National Toxicology Program (13) evaluated the effects

of m-synephrine on spontaneous food intake of rats and

mice. At 2 years, there were no significant differences in

survival among mice or female rats. However, for male

rats, there was a significant reduction in mortality rate,

although there was increased mortality in the early phase

of the study at the highest dose. It should be noted that

too few deaths occurred during the 2-year trial to provide

the degree of precision and power desired for a rigorous

longevity study (30). Nonetheless, similar results have

been reported for ephedrine, another sympathomimetic

amine (31).

Arbo et al. (32) conducted a subchronic toxicity

study in mice and the effects of p-synephrine and C. aurantium

L. extract on oxidative stress biomarkers that are

believed to be indicators of cell membrane injury (malondialdehyde)

and (glutathione and the enzyme glutathione

peroxidase) indicative of amphetamine-induced toxicity.

The study evaluated adult male CF1 mice treated with

400, 2000, or 4000 mg/kg C. aurantium dried extract and

p-synephrine 30 or 300 mg/kg over the course of 28 days.

Results showed a reduction in glutathione in mice treated

with C. aurantium 400 mg/kg and p-synephrine 30 and

300 mg/kg. Inhibition of glutathione peroxidase activity

occurred within mice treated with C. aurantium 400 and

2000 mg/kg and p-synephrine 30 and 300 mg/kg; however,

no change occurred within malondialdehyde levels.

These two findings suggest the possibility of subchronic

toxicity. No significant change in weight occurred in any

of the groups, suggesting on the positive side a lack of

severe toxicity, and on the negative side a lack of efficacy

in producing weight loss.

With regard to adverse effects, a study (33) of male

Sprague-Dawley rats reported what was believed to be

evidence of cardiotoxicity when C. aurantium fruit extracts

standardized to 4% and 6% SAs were administered. Increased

mortality has been observed among CAPs-treated

rats (33) as well as a strain of mice selected to be uniquely

susceptible to the effects of adrenergic stimulation (34).

CLINICAL TRIALS Bitter Orange Weight Loss

Few clinical trials have examined the effects of CAPs alone

or in combination with other ingredients on body weight

and/or body composition (Table 1). It should be kept in

mind that these trials are of short duration and the sample

sizes are frequently quite small. Nonetheless, these

trials suggest that body weight and/or fat loss may be

enhanced by CAPs or SAs. The mechanisms involved are

unclear but may be partially due to a suppressing effect

of appetite and/or a moderate increase in resting energy


Armstrong et al. (37) evaluated exercise and herbal

preparation containing Ma Huang, bitter orange (5 mg

SAs), and guarana over 6 weeks in a randomized, controlled

trial. Compared with controls, the intervention

group obtained significant reductions in fat mass and a

nearly significant reduction in body mass index (kg/m2)

54 Haaz et al.

Table 1 Summary of Clinical Weight Loss Trials

Reference Treatment Design Sample size Duration Results Comments

Colker et al. (10) 975 mg Citrus

aurantium, +528

caffeine and 900 mg

St. John’s wort;

placebo (with pill) and

control (no pill)

Blinded parallel

groups RCT

Supplement n = 9;

placebo, n = 7;

control group (no

pills), n = 4

6 wk Supplement group lost

more fat (3.1 kg; P <

0.05) than other

groups and increased

RMR (2–3%)

Citrus aurantium may

assist individuals in

losing body fat, due to

increased energy and

reduced energy intake

expenditure. No

adverse events were


Kalman et al. (36) Ephedrine and

synephrine alkaloids

(SAs) (5 mg twice

daily) based product

vs. placebo with

exercise and diet



double blind

30 overweight

subjects; BMI > 27

8 wk 3.4 kg weight loss in

experimental group vs.

2.05 kg in placebo

(P < 0.05)

No adverse events;

findings indicate

apparent short-term

safety and efficacy of

ephedrine and



Armstrong et al.


Exercise program with

assignment to drug

(Ma Huang, bitter

orange, and guarana)

or placebo. Bitter

orange standardized

for 5 mg synephrine


trial—unclear if

study is blinded

Five overweight

males/14 females

44 days Supplement increased

fat loss (2.5 kg; P =

0.033) more than

placebo (0.5 kg))

Low statistical power,

no marked side effects

Greenway et al.

(38): Pilot 1

Two capsules


pantothenic acid,

40 mg; green tea leaf

extract, 200 mg;

guarana extract,

550 mg; bitter orange,

150 mg; white willow

bark extract, 50 mg;

ginger root, 10 mg;

proprietary charge


(L-tyrosine, L-carnitine,

naringin), 375 mg



double blind

Eight subjects (1:1

ratio) between

supplement group and

placebo group

8 wk Supplement group

gained more weight

(1.04 °æ 0.27 kg; P <

0.04) than placebo

and increased RMR

(but not at 8 wk)

CAP was not

efficacious for weight


Greenway et al.

(38): Pilot 2

m-Synephrine 20 mg Prospective,


double blind

Twenty subjects (1:1

ratio) between

supplement group and

placebo group

8 wk Supplement group lost

weight (0.8 °æ 3.4 kg;

not significant) in 8

wk, and increased

RMR in 8 wk. No

control group was

used (Greenway,



November 1, 2009)

m-Synephrine was not

efficacious for weight


Abbreviations: BMI, body mass index; CAP, Citrus aurantium product; RCT, randomized, controlled trial; RMR, resting metabolic rate.

and fat percentage. No significant changes were noted in

resting energy expenditure, blood chemistries, or dietary

intake between the placebo and experimental groups.

In a double-blind, placebo-controlled, randomized

trial, Colker et al. found that subjects receiving a

combination of C. aurantium, caffeine, and St. John’s wort,

along with diet and exercise protocols, lost a statistically

significant amount of body weight. Analysis comparing

changes in this group with those in placebo or control

groups on the same diet and exercise regimen did not

show significant differences, though loss of fat mass was

significantly greater in the experimental group (35). BP,

heart rate, electrocardiographic, blood, and urine analyses

were not significantly different between the groups.

Another randomized trial (36) of 30 overweight

adults investigated the effects of supplementation, along

with a cross-training exercise regimen and dietary education

program compared with exercise and dietary

education alone on body composition. Supplementation

included ephedrine, SAs, caffeine, and calicine.

Greater weight and fat loss occurred for the supplement

group compared with the exercise–diet only


Overall, studies indicate a weight loss of 2.4–3.4 kg

among participants using SAs, while placebo groups lost

0.94–2.05 kg, suggesting the plausibility of some weight

loss benefit from SA supplementation, beyond diet and

exercise alone. However, these studies do not separate

Bitter Orange 55

the effects of C. aurantium or SAs from other ingredients,

particularly ephedrine and caffeine.

Metabolic Rate and Cardiovascular Effects

Several studies have evaluated the effects of acute administration

of SAs on cardiovascular indicators. Kalman et al.

(39) tested a product containing 335 mg Ma Huang standardized

for 20 mg ephedrine alkaloids, 910 mg guarana

standardized for 200 mg caffeine, and 85 mg bitter orange

standardized for 5 mg SAs per two capsules. Twentyseven

overweight adults were randomized to treatment

or placebo for 14 days. BP, heart rate, electrocardiogram,

and Doppler echocardiograms were evaluated before and

after treatment. Ingestion of this commercial weight loss

supplement did not produce any detectable cardiovascular

side effects.

Penzak et al. (10) examined cardiovascular outcomes

in 12 normotensive individuals who were administered

8 oz of Seville orange juice (containing 13–14 mg SAs)

and water in a crossover fashion, followed by a repeat

ingestion 8 hours later. No changes in cardiovascular indices

(BP, maximal arterial pressure, and heart rate) were


Thomas et al. (40) evaluated the cardiovascular effects

of 10 mg oral SAs in healthy volunteers over a 4-hour

period on impedance cardiography and forearm plethysmography.

Elevation in total peripheral resistance was observed

30–60 minutes after dosing, although other hemodynamic

indexes were not affected.

Hemodynamic effects were observed in a crossover

design, placebo-controlled study (41) with the administration

of Xenadrine, a CAP that contains a variety of other

potentially bioactive substances, including green tea extract,

cocoa extract, yerba mate, ginger root, grape seed

extract, and others. However, these increases in heart rate,

and systolic and diastolic BP were not observed with administration

of Advantra Z, which contains C. aurantium

alone, even at an eightfold higher dose.

Haller et al. (42) evaluated a dietary supplement

[Ripped Fuel Extreme Cut, containing synephrine from

C. aurantium (presumably p-synephrine) and caffeine] in

10 healthy adults (three women) aged 20–31 years. Each

subject was given one dose of the dietary supplement

under three conditions: (i) resting conditions (without

placebo); (ii) moderately intense exercise; and (iii) placebo

plus moderately intense exercise in a three-arm, randomized,

crossover study. Greater postexercise diastolic BP

was seen with the dietary supplement plus exercise than

with placebo plus exercise. There were no obvious supplement

effects on postexercise HR, systolic BP, or body


Bui et al. (43) reported the effect on BP (systolic

and diastolic) and heart rate over 6 hours after one

dose of a CAP (Nature’s Way Bitter Orange) on 15

young, healthy adults in this prospective, randomized,

double-blind, placebo-controlled, crossover study. Systolic

and diastolic BP increased significantly within the

1–5 hours time period in comparison with the placebo

group with the peak being 7.3 °æ 4.6 mm Hg, while the

4–5 hours time period increase was 2.6 °æ 3.8 mm Hg after

consumption in comparison with the placebo group

with the peak being 4.2 °æ 4.5 beats/minute, while

heart rate was significantly elevated 2–5 hours after


In one study of obese adults, increases in resting

metabolic rate (RMR) were observed with C. aurantium,

both alone and with food, beyond the thermic effect of

food (TEF) alone (44). (RMR is a measure of the energy

required to maintain basic physiological function while

the body is at rest.) However, another recent investigation

(45) found that the thermic response to CAPs increased in

women only, who had lower TEF than men at baseline.

After the intervention, TEF did not differ by gender. BP

and pulse rate were not affected, but epinephrine secretion

increased. In normal weight adults, an increase in RMR

was also found when the extract was taken with a meal

(46).Noadverse changes in pulse rate or BP were reported.

Finally, the effects of two dietary supplement formulas

onRMRand other metabolic indicators were evaluated

(47). When compared with placebo, Formula A (containing

ephedra, guarana, green tea, yohimbe, and quercetin)

and Formula B (containing C. aurantium, jing jie, fang feng,

guarana, green tea, yohimbe, and quercetin) resulted in increased

total RMR, decreased respiratory exchange ratio

toward fat burning, and increased body core temperature.

Heart rate and RMR increased at each 15-minute interval

with Formula A only. BP increased with both, but to a

greater extent with Formula A.



Nykamp et al. (48) describe a case of acute lateral-wall

myocardial infarction co-occurring with consumption of

CAPs in a 55-year-old woman with undetected coronary

vascular disease. She reported taking a multicomponent

dietary weight loss supplement containing 300 mg of bitter

orange over the preceding year.

Consumer Reports article (49) describes a 21-yearold

woman who took ephedra-free Xenadrine EFX (which

contains C. aurantium). After 3 weeks on the supplement,

she suffered a seizure. Her neurologist believes the bitter

orange in the supplement was the most likely the cause,

though the basis for this conclusion is unknown.

Nasir et al. (50) described exercise-induced syncope

in a healthy 22-year-old woman that occurred 1 hour after

a second dose of Xenadrine EFX, a weight loss supplement

that contains, among other compounds, ephedrine and

synephrine. The electrocardiography revealed prolongation

of the QT interval, which resolved in 24 hours.

Bouchard et al. (51) report a case of a 38-year-old

male patient with ischemic stroke that occurred after taking

a CAP for 1 week. The patient reportedly had no relevant

medical history or major atherosclerotic risk factors

and took no other medications.

Gray andWoolf (52) reported a case of CAPs use by

an adolescent with anorexia nervosa and raised concerns

that the SAs may have masked bradycardia and hypotension

while exacerbating her weight loss. Firenzuoli et al.

(53) report a case of a 52-year-old woman that had an

allergic reaction after taking a CAPs product.

Sultan et al. (54) reported a case of a 52-year-old

woman with ischemic colitis that occurred 1 week after

consumption of a CAP (Natural Max Skinny Fast,

containing bitter orange). She reported no known drug

56 Haaz et al.

Table 2 Summary of Effects, Safety, and Efficacy of Citrus aurantium

Physiological effects Effects on weight Effects on body composition Safety

Variable changes in BP in animals;

generally stable BP, heart rate, pulse

rate, blood and urine measures in

humans; inconsistent changes to

resting metabolic rate

Weight loss documented in rodents;

weakly supported in humans, as

studies used multiple supplements or

did not find significant difference

from controls

Limited support for loss of fat mass in

human studies, noting a trend or

using multiple supplements; for

animals, some increased lipase


Inconsistent mortality data in

rodents; some evidence of

elevated BP. Results not

consistent from study to study,

but this may be a function of

small sample sizes used in most

studies. Several case reports of

serious adverse events

allergies and took no other medications. Symptoms resolved

over 24–48 hours with conservative management

after the supplement was discontinued.

Health Canada reported that fromJanuary 1, 1998, to

February 28, 2004, it received 16 reports in which products

containing bitter orange or synephrine were suspected

of being associated with cardiovascular events, including

tachycardia, cardiac arrest, ventricular fibrillation, transient

collapse, and blackout. All cases were considered

serious (55).

Adverse events from CAPs are currently fairly rare

in scientific literature. As CAPs are used more widely in

place of ephedrine-containing products, any potentially

harmful effects may be clarified over time.


The Safety of CAPs

Some have hailed the potential therapeutic value of CAPs

(1), while others have warned about possible safety concerns

(33). The safety concerns pertain primarily to adverse

cardiovascular and cerebrovascular effects. Information

on the safety of CAPs comes from the three sources

described above: animal studies, clinical trials, and case

reports. To date, no large epidemiologic (case control or

cohort) studies have evaluated the safety of CAPs.

Of course, one cannot extrapolate the safety of CAPs

from short-term studies used for one indication (e.g., several

days for relief of nasal congestion among the general

population) to long-term studies use for another indication

(e.g., several months or years for weight loss

among obese individuals). Although substantial safetyrelated

data exist for CAPs (13,56), there is no published

human weight loss trial of CAPs with more than 20 participants

or for a duration of more than 7 weeks.

It is important to note that the majority of

studies evaluating the safety of CAPs are performed

with normotensive subjects. However, because hypertension

is a common comorbidity associated with overweight/

obesity, studies that evaluate the effects of CAPs

on BP should also be conducted with obese hypertensive


While C. aurantium extracts have been used in a variety

of cultures for thousands of years, they have not been

traditionally utilized for long periods of time, or specifically

for weight loss (1). As such, there is little, if any, basis

for making definitive statements about the intermediate

or long-term safety/risk of CAPs used for weight loss.

Table 2 summarizes the physiological effects, safety, and

weight loss efficacy of C. aurantium.



Given the dearth of weight loss trials, the optimal dose

(if one exists) of C. aurantium or its SA constituents for

weight loss is unknown. Table 3 highlights some relevant

dosage information. Although generalizing across species

and compounds is difficult and can only provide a limited

basis for conjecture, the following comparisons with

ephedrine can be made. We analyzed data (12) in which

ephedrine or SAs was given to mice. Regression of weight

and food intake on dose of ephedrine or SAs yielded

slopes (in absolute value) that were approximately four

to six times greater for ephedrine than for SAs. Based on

linear projections, it would take four to six times the dose

of SAs (in these mice) to achieve equivalent reduction in

intake and body weight as for ephedrine. In human studies

of ephedrine, doses of about 50 mg per day begin to be

effective (57). Although an extrapolation, this might suggest

a useful clinical dose for SAs as high as 240–360 mg

Table 3 Dosage Information on Citrus aurantium or Synephrine Alkaloids (SAs)


5–14 mg/day Citrus aurantium extract with SAs has been used (34–36) and no serious adverse events were reported. These doses purportedly

showed efficacy, but products tested included substances beyond C. aurantium, notably ephedrine which we know to be effective for

weight loss. We believe that these doses of SAs are very unlikely to be effective when used without ephedrine

32 mg/day The nasal decongestant Endal (60) contains 20 mg of m-s per tablet and two tablets per dose twice per day are recommended

120 mg/day Via C. aurantium extract, SAs are marketed in over-the-counter (OTC) products for weight loss. In products, such as Nutres Lipo 6

(61), the directions suggest that for “extreme fat loss” a recommended dosage is two capsules three times per day. The SA content

per capsule is 20 mg; this provided a maximal recommended dose of 120 mg/day

300 mg/day According to Clarke’s Analysis of Drugs and Poisons (62), oxedrine (p-synephrine) is used clinically at ∼300 mg/day

1000 mg/day Minimum adult lethal dose of m-s (63)

Bitter Orange 57

per day. From a safety point of view, SAs (per equal

weight) have lower potential to raise BP than ephedrine;

however, nearly all commercial preparations of SAs also

contain caffeine, which might compound any cardiovascular

effects. In the absence of caffeine, human studies

suggest that 15–30 times the dose of SAs are required to

elevate BP to the same degree as ephedrine (58,59). This

suggests that such high doses might be well tolerated,

but clearly more data are needed, particularly regarding

potential synergistic effects of CAPs components.

SAs appear to be readily absorbed after oral administration

(63). About 80% of oral doses are excreted in

the urine within 24 hours. After single oral doses, peak

plasma concentrations are typically reached in 1–2 hours.

Plasma half-life is ∼2–3 hours. Sympathomimetic drugs

for weight loss are typically given TID before meals (64)

reducing the evening dose if sleep problems arise.


Topical application (as with aromatherapy or antifungal

uses) of CAPs may result in photosensitivity for fair skinned

individuals (65) (possibly due to photosensitizing

furanocoumarins that occur in the rinds of certain

citrus species, especially immature fruits). Although

rare, this has also occurred after oral ingestion. To reduce

this risk, exposure to ultraviolet light can be minimized.

Caution is recommended for use in children, as it

may conceivably produce toxic effects (66). Some sources

advise that CAPs should be avoided by women who are

pregnant or breastfeeding (7,67), while others claim that

CAPs can be used safely during pregnancy (66). While

effects on BP are unclear, those with hypertension, tachyarrhythmia,

or narrow-angled glaucoma may consider

refraining from use of CAPs until further evidence confirms

their safety (67).CAPs could also possibly exacerbate

symptoms for those with stomach or intestinal ulcers (68).


Because CAPs may increase stomach acid, they could potentially

reduce the efficacy of acid-lowering drugs, such

as antacids and ulcer medications (69). Although a speculative

precaution, those taking medications containing

SAs, including some cold medications and monoamine oxidase

inhibitors (MAOIs), should consider the combined

dose of these products with the SAs present in CAPs formulations

and possible multiplicative effects (68,69). It has

been suggested that CAPs could interfere with the activity

of drugs that are metabolized by the liver enzyme cytochrome

P450-3A, CYP3A (70,71). A recent comment in

Experimental Biology and Medicine noted that some research

on drug effects have utilized parts of the plant or

methods of administration that may not be applicable to

oral consumption of currently marketed dietary supplements


The safety and efficacy of CAPs and SAs for weight

loss are not well established. While existing literature

demonstrates plausibility for reducing weight, previous

trials were not designed to rigorously evaluate safety and

efficacy. Doing so will require better-designed randomized

clinical trials with large sample sizes, reliable well established

outcome measures, and active surveillance of

side effects and adverse events. To better understand the

effects of CAPs or SAs specifically, studies will need to test

these components without combining them with other ingredients

postulated to have antiobesity effects. It would

also be worthwhile to examine differences between the

types of synephrine-containing compounds that are derived

from various sources and how this influences the

consistency and potency of supplements.


The writing of this entry was supported in part

by NIH grant nos. P30DK056336, AR49720–01A1, and

T32HL072757. The opinions expressed are solely the responsibility

of the authors and do not necessarily represent

the official views of the NIH or any other organization

with which the authors are affiliated.

Disclosure: Dr. Allison has received grants, honoraria,

consulting fees, and donations from numerous

companies, government agencies, and nonprofit organizations

with interests in obesity in general and dietary

supplements in particular, including organizations litigating

cases involving C. aurantium.


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