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Glossary, sports nutritionSuccess Chemistry Staff


Androstenedione (chemical name: 4-androstene-3,17-

dione) is a steroid hormone produced primarily in the reproductive

system and adrenal glands in men and women.

It circulates in the bloodstream and is the immediate precursor

to the potent anabolic/androgenic hormone testosterone

in the steroid synthesis pathway. Despite this well known

physiological classification, as well as a growing

body of evidence demonstrating that orally administered

androstenedione is converted to more potent steroid hormones,

the United States Food and Drug Administration

originally classified the hormone as a “dietary supplement.”

As such, it was available to the general public

without a prescription and for nearly a decade could be

easily purchased in health clubs, nutrition stores, and

over the Internet. This over-the-counter availability of

androstenedione came to an end when Food and Drug

Administration banned its sale in early 2004. The ban

was then codified with the passing of the 2004 Anabolic

Steroid Control Act. This law reclassified androstenedione

as an anabolic steroid and hence a controlled



The original and seemingly contradictory classification of

androstenedione as a dietary supplement was based on

the definition set forth in the 1994 Dietary Supplement

Health and Education Act (DSHEA). According to the

DSHEA, a substance was defined as a dietary supplement

if it was a “product (other than tobacco) intended

to supplement the diet that bears or contains one or more

of the following dietary ingredients: a vitamin, mineral,

amino acid, herb or other botanical. . . or a concentrate,

metabolite, constituent, extract, or combination of any ingredient

described above.” Hence, because androstenedione

could be synthesized from plant products, it fell

under that umbrella. Furthermore, the DSHEA specified

that the Department of Justice could not bring action to

remove a product unless it was proven to pose “a significant

or unreasonable risk of illness or injury” when

used as directed. Not surprisingly, after the passing of the

DSHEA, the use of dietary supplements increased dramatically.

In fact, by 1999, the dietary supplement industry

in the United States was generating annual sales of $12

billion (1).

Initially, androstenedione use was primarily confined

to athletes in strength and endurance-related sports,

an interest that seems to have sprung from reports of

its use in the official East German Olympic athlete doping

program. The event that most dramatically sparked

widespread curiosity in androstenedione, however, was

the media report that the St. Louis Cardinals baseball

player Mark McGwire had used androstenedione in the

1999 season (during which he broke the record for most

home runs in a season). The publicity that surrounded

this supplement also prompted an increased interest in

related “prohormones,” such as norandrostenedione and

androstenediol. This then led to a proliferation of claims

concerning the potential benefits of androstenedione use.

Manufacturers credited these products not only with

promoting muscle growth and improving athletic performance

but also with increasing energy, libido, sexual

performance, and general quality of life. Additionally,

androstenedione was often packaged in combination

with other substances as part of an intensive nutritional

approach to performance enhancement. An example

of such a combination is shown in Figure 1.

Clearly, the use of androstenedione and related compounds

during that time went well beyond the accumulation

of data that could provide a rational basis for

their use.


  • 4-Androstenedione: 100 mg

  • 19-Nor-5-Androstenedione: 50 mg

  • 5-Androstenediol: 50 mg

  • DHEA: 50 mg


  • L-Arginine Pyroglutamate: 2500 mg

  • L-Ornithine Alpha-Ketoglutarate: 1250 mg

  • Taurine: 750 mg

  • Colostrum: 250 mg


  • Tribulus: 250 mg

  • Acetyl-L-Carnitine: 250 mg

  • L-Carnitine: 100 mg


  • Saw Palmetto: 200 mg

  • Beta Sitosterol: 200 mg

  • Pygeum Africanum: 50 mg


  • Kudzu: 100 mg

  • Chrysin: 250 mg

  • 4-Androstenedione

  • Dehydroepiandrosterone

  • Estrone



  • 17β-HSD

  • CYP19 (aromatase)

  • CYP19 (aromatase)

  • 17β-HSD

  • 3β-HSD

  • Testosterone Estradiol-17β


Androstenedione is a steroid hormone that is produced

primarily in the adrenals, testes, and ovaries. It is classified

as a “weak androgen” because it binds to the body’s

receptor for androgen hormones in a much less potent

fashion than classic anabolic/androgenic steroids such

as testosterone (2). It is synthesized from the precursor

hormone dehydroepiandrosterone (itself a dietary supplement)

and is the direct precursor to testosterone. In normal

physiological circumstances, androstenedione can also be

converted to potent feminizing hormones such as estrone

and estradiol (both members of the “estrogen” class of hormones).

The relationship between androstenedione, other

steroid hormones, and the enzymes involved in the conversion

of androstenedione to testosterone and estrogens

is shown in Figure 2.

Importantly, the enzymes that convert androstenedione

to potent hormones such as testosterone and estradiol

are active not only in endocrine glands but also in

many peripheral body tissues such as muscle, bone, liver,

and brain (3). Thus, if orally administered androstenedione

has biological activity, it may act either directly or

by conversion to these more potent agents.


There were no precise data concerning the prevalence of

androstenedione use in the general population during the

time that it was widely available. Our best estimates were

based on industry sales figures and extrapolations from

data on classic anabolic/androgenic steroid use in specific

populations. For example, in 1997, it was estimated that

4.9% of male and 2.4% of female adolescents in the United

States had used illegal anabolic steroids (4). Because these

substances were so readily available, there was concern

that androstenedione use in this particularly susceptible

population may have greatly exceeded these numbers.

In fact, in a survey administered in five health clubs in

Boston, Massachusetts, in 2001, 18% of men and 3% of

women respondents admitted to using androstenedione

or other adrenal hormone dietary supplements at least

once. These percentages suggested that as many as 1.5

million U.S. health club members alone may have used

these substances (5).



Because so many of the claims that surrounded androstenedione

were based on the premise that oral administration

increases serum testosterone levels, it may

be surprising to some that prior to 1999, there was only a

single published study investigating the ability of orally

administered androstenedione to be converted to more

potent steroid hormones (6). In this study, two women

were given a single dose of androstenedione, and the

levels were subsequently measured over the next several

hours. Since 1999, however, numerous small studies

(mostly in men) have investigated the effects of the supplement

(6–16). In general, these studies report that serum

androstenedione levels increase dramatically after oral administration

and thus confirm that a significant portion of

the supplement is absorbed through the gastrointestinal

tract after ingestion. However, the answer to the more important

question, namely, whether it is then converted to

more potent steroid hormones such as testosterone and

estradiol, appears to be complex. In general, these studies

suggest that the ability of oral androstenedione to increase

estrogen and testosterone levels in men is dose dependent

and is possibly related to the age of the study population

as well. Specifically, the bulk of the research indicates that

when androstenedione is administered to men in individual

doses between 50 and 200 mg, serum estrogen levels

increase dramatically. However, larger individual doses

(e.g., 300 mg) are required to increase serum testosterone


For example, King and colleagues studied the effects

of a single 100-mg oral dose of androstenedione in 10 men

between the ages of 19 and 29 and reported that although

serum androstenedione and estradiol levels increased significantly,

testosterone levels did not change (13). These

investigators then specifically measured the portion of circulating

testosterone that is not bound to protein and considered

the “bioactive” portion (called “free testosterone”)

and similarly saw no effect of the supplement. In a separate

study, Leder and colleagues gave 0, 100, or 300 mg

of androstenedione to normal healthy men between the

ages of 20 and 40 for seven days and took frequent blood

samples on days 1 and 7 (14). As in the study by King, they

also found that men receiving both the 100- and 300-mg

doses of androstenedione experienced dramatic increases

in serum estradiol that were often well above the normal

male range.

Percentage change in serum testosterone and estradiol in healthy

men after a single androstenedione dose (as measured by eight hours of

frequent blood sampling). Source: Adapted from Ref. 14.

did not affect serum testosterone levels. As shown in

Figure 3, however, the novel finding of this study was

that 300 mg of androstenedione increased serum testosterone

levels significantly, even though by only a modest

amount (34%).

Leder and colleagues further observed that there

was a significant degree of variability among men with

regard to their serum testosterone response after androstenedione

ingestion. As shown in Figure 4, some subjects,

even in the 300-mg dose group, experienced relatively

little change in testosterone levels, whereas serum

testosterone levels doubled in other men. This finding

suggests that there may be individual differences in the

way androstenedione is metabolized that could impact

any one person’s physiological response to taking the


Brown and colleagues investigated the hormonal response

in a group of men between the ages of 30 and

Figure 4 Individual variability in the peak serum testosterone level

achieved after a single 300-mg dose of androstenedione in men. Each line

represents one study subject. Source: Adapted from Ref. 14.

56 (10). In this study, subjects consuming 100 mg of androstenedione

three times daily experienced increases in

serum estrogens but not in serum testosterone. However,

unlike in the study by King and colleagues discussed in the

previous text, free testosterone did increase significantly

(even though again by only a small amount).

Finally, several studies have compared the hormonal

effects of androstenedione with those of other

“prohormone” dietary supplements. Broeder and colleagues

studied the results of a 100-mg twice-daily dose

of oral androstenedione, androstenediol (a closely related

steroid hormone), or placebo in men between

the ages of 35 and 65 (7). They found that both compounds

increased estrogen levels but neither affected total

serum testosterone levels. Similarly, Wallace and colleagues

studied the effects of 50-mg twice-daily doses

of androstenedione and DHEA in normal men and reported

no increases in serum testosterone levels with

either (16).


The results of the studies discussed earlier suggest that

androstenedione use in men would be less likely to promote

the muscle building and performance-enhancing

effects associated with testosterone use and more likely

to induce the undesirable feminizing effects associated

with estrogens. Several studies have assessed the ability

of androstenedione (with or without exercise) to increase

muscle size and strength and have been uniformly

disappointing (7,9,13,15,16). For example, Broeder

and colleagues, in the study described earlier, also

measured changes in body composition and strength

in subjects taking 100 mg androstenedione twice daily

in combination with a 12-week intensive weight-training

program (7). Despite using sensitive methods that can detect

small changes in body composition, they found no

differences in muscle mass, fat mass, or strength in the

subjects receiving androstenedione compared with those

receiving a placebo tablet. Importantly, however, in this

study as well as all of these studies referenced earlier,

the supplement was given in doses that were not sufficient

to increase testosterone levels. It thus remains unknown

whether doses of androstenedione sufficient to increase

testosterone levels enhance muscle mass or athletic



One of the consistent findings of the various androstenedione

studies in men is the inefficiency of conversion of

the supplements to testosterone. Leder and colleagues explored

this issue further by investigating the pattern of

androstenedione metabolism in healthy men (17). Specifically,

they measured the concentration of inactive testosterone

metabolites (also called “conjugates”) in the urine

of subjects ingesting androstenedione and found an increase

of over 10-fold compared with their baseline levels.

This finding was in direct contrast to the much more

modest changes in serum testosterone they had observed.

It suggests that although much of the androstenedione

Figure 5 Serum testosterone levels during 12 hours of frequent blood

sampling in postmenopausal women. Circles represent control subjects receiving

no supplement, triangles those receiving 50 mg of androstenedione,

and squares those receiving 100 mg. Source: Adapted from Ref. 18.

that is absorbed after oral administration is converted to

testosterone, it is then immediately further metabolized

to inactive compounds in the liver. The investigators confirmed

this hypothesis by directly measuring the concentration

of one of these inactive metabolites (testosterone

glucuronide) in the serum of these subjects. As expected,

they found that testosterone glucuronide levels increased

by 500% to 1000% (as opposed to the 34% increase in biologically

active serum testosterone after a single 300-mg

dose of oral androstenedione). Together, these findings

demonstrate the effectiveness of the liver in inactivating

steroid molecules when taken orally.



Since the initial report of androstenedione administration

in two women in 1962 (6), research into the effects of the

supplement has focused largely on the hormonal response

to oral administration in young men. Between 2002 and

2003, however, two studies on women were published.

The first of these studies examined the effects of a single

dose of 0, 50, or 100 mg of androstenedione in postmenopausal

women (18). The findings of this study were

surprising. In contrast to the effects observed in men, even

these low doses increased testosterone levels significantly

in women (Fig. 5).

Also, unlike the results seen in men, estradiol levels

were unaffected by androstenedione administration.

In the other study, 100 mg of androstenedione was administered

to young, premenopausal, healthy women.

Similar to postmenopausal women, these subjects experienced

significant increases in serum testosterone levels

after androstenedione administration (estradiol was

not measured) (19). Importantly, in both of these studies,

the peak testosterone levels achieved by the older and

younger women taking androstenedione were often significantly

above the normal range. Together, these results

predict that the physiological effects of the supplement

may be different in men and women, as might their potential

toxicities. To date, however, there have been no

published reports investigating the long-term physiological

effects in women.


Ever since the publicity surrounding androstenedione exploded

in 1999, many reports in the lay press have focused

on the potential dangerous side effects. Nonetheless,

with the exception of a single case description of a

man who developed two episodes of priapism in the setting

of androstenedione ingestion (20), there have been no

published reports of androstenedione-associated serious

adverse events. This fact should be only partially reassuring,

however, because androstenedione’s prior classification

as a dietary supplement (as opposed to a drug)

allowed manufacturers to avoid responsibility for rigorously

monitoring any potential toxicity of their product.

It is well known that oral administration of certain

testosterone derivatives can cause severe liver diseases,

and anabolic steroid use in general is associated with

anecdotal reports of myocardial infarction, sudden cardiac

death, and psychiatric disturbances (“roid rage”).

Nonetheless, despite androstenedione close chemical

similarity to these substances, it is important to note that it

is not a potent anabolic steroid nor does it have a chemical

structure similar to those specific compounds that cause

liver problems. Thus, the potential of androstenedione to

cause these particular serious side effects appears to be

limited. Of more pressing concern to clinicians are the

possible long-term effects in specific populations. In clinical

trials, the supplement was generally well tolerated,

though several studies did report that it reduces high density

lipoprotein (or “good cholesterol”) levels in men.

Importantly, however, even the longest of these studies

lasted only several months. It thus remains quite possible

that androstenedione use, especially at high doses,

could cause subtle physiological changes over prolonged

periods that could directly lead to adverse health consequences.

In men, for example, the dramatic increase in

estradiol levels observed with androstenedione administration

could, over time, lead to gynecomastia (male breast

enlargement), infertility, and other signs of feminization.

In women, because the supplement increases testosterone

levels above the normal range, it could cause hirsutism

(excess body hair growth), menstrual irregularities, or

male-like changes in the external genitalia. In children,

increases in both testosterone and estrogen levels could

cause precocious puberty or premature closure of growth

plates in bone, thereby compromising final adult height.



During its period of over-the-counter availability, androstenedione

was available from multiple manufacturers

and could be purchased as a tablet, capsule, sublingual

tablet, or even a nasal spray. Often, it was combined

with other products that claimed to limit its potential side

effects (such as chrysin, for example, which is purported

to decrease androstenedione’s conversion to estrogens).

Because the manufacture of dietary supplements was not


Source: From Ref. 21.

subject to the same regulations as pharmaceuticals, the purity

and labeling of androstenedione-containing products

were often inaccurate. Catlin and colleagues, for example,

reported that urine samples from men treated with

androstenedione contained 19-norandrosterone, a substance

not associated with androstenedione metabolism

but rather with the use of a specific banned anabolic

steroid (21). Further investigation revealed that the androstenedione

product used contained a tiny amount of

the unlabeled steroid “19-norandrostenedione.” Though

the amount of 19-norandrostenedione was not physiologically

significant, it was enough to cause a “positive” urine

test for illegal anabolic steroid use when tested in the standard

fashion. In fact, it is precisely this type of contamination

that may have explained increases in positive tests

for 19-norandrosterone among competitive athletes in the

past decade. Additionally, it is now common for athletes

who test positive for norandrosterone or other androgenic

metabolites to point to dietary supplement contamination

as the potential explanation.

Catlin and colleagues also analyzed nine common

brands of androstenedione and showed that there was

considerable variation and mislabeling among products

in terms of both purity and content (Table 1).


Androstenedione was available over-the-counter from

1994 (when the DSHEA was passed) until it was reclassified

as an anabolic steroid by the Anabolic Steroid Control

Act in 2004. It is important to note that this reclassification

came without any evidence that androstenedione increased

muscle mass or strength, which was the previous

legal definition of an anabolic steroid. Virtually all sports

organizations, including the National Football League, the

National Collegiate Athletic Association, and the International

Olympic Committee, have banned androstenedione.

Despite these prohibitions, detection of androstenedione

has not been standardized. Specifically, the method

used most often to detect testosterone use, measurement of

the urinary testosterone-to-epitestosterone ratio, has not

proven to be reliable in establishing androstenedione use

(22). Further study is still needed to define novel testing

procedures that are able to detect androstenedione use



Androstenedione is a steroid hormone, which, until 2004,

was a popular over-the-counter dietary supplement. Since

then, however, it has been classified as an anabolic steroid,

and hence a controlled substance. It is purported to increase

strength, athletic performance, libido, sexual performance,

energy, and general quality of life. Studies indicate

that when taken orally by men, small doses are

converted to potent estrogens and larger doses to both

testosterone and estrogens. Comparatively, there appears

to be a much more physiologically important increase in

estrogens compared with testosterone in men. In women,

the effects are reversed. Studies have thus far failed to

confirm any effect on muscle size or strength, though the

dosing regimens were modest. Although documentation

of adverse side effects among users of androstenedione is

scarce, there is considerable concern over potential longterm

toxicity, especially in women and adolescents.


1. Anonymous. Herbal treatments: The promises and pitfalls.

Consum Rep 1999; 64:44–48.

2. Orth DN, Kovacs WJ. The adrenal cortex. In: Wilson D, Foster

DW, Kronenberg HM, et al., eds. Williams Textbook of

Endocrinology. Philadelphia, PA: W.B. Saunders Company,


3. Labrie F, Simard J, Luu-The V, et al. Structure, regulation

and role of 3 beta-hydroxysteroid dehydrogenase, 17 betahydroxysteroid

dehydrogenase and aromatase enzymes in

the formation of sex steroids in classical and peripheral

intracrine tissues. Baillieres Clin Endocrinol Metab 1994;


4. Yesalis CE, Barsukiewicz CK, Kopstein AN, et al. Trends

in anabolic-androgenic steroid use among adolescents. Arch

Pediatr Adolesc Med 1997; 151:1197–1206.

5. Kanayama G, Gruber AJ, Pope HG Jr, et al. Over-the-counter

drug use in gymnasiums: An underrecognized substance

abuse problem? Psychother Psychosom 2001; 70(3):137–140.

6. Mahesh VB, Greenblatt RB. The in vivo conversion of dehydroepiandrosterone

and androstenedione to testosterone in

the human. Acta Endocrinol 1962; 41:400–406.

7. Broeder CE, Quindry J, Brittingham K, et al. The Andro

Project: Physiological and hormonal influences of androstenedione

supplementation in men 35 to 65 years old

participating in a high-intensity resistance training program.

Arch Intern Med 2000; 160(20):3093–3104.

8. Brown GA, Vukovich MD, Martini ER, et al. Effects of

androstenedione-herbal supplementation on serum sex hormone

concentrations in 30- to 59-year-old men. Int J Vitam

Nutr Res 2001; 71(5):293–301.

9. Brown GA, Vukovich MD, Reifenrath TA, et al. Effects of anabolic

precursors on serum testosterone concentrations and

adaptations to resistance training in young men. Int J Sport

Nutr Exerc Metab 2000; 10(3):340–359.

10. Brown GA, Vukovich MD, Martini ER, et al. Endocrine responses

to chronic androstenedione intake in 30- to 56-yearold

men. J Clin Endocrinol Metab 2000; 85(11):4074–4080.

11. Earnest CP, Olson MA, Broeder CE, et al. In vivo 4-

androstene-3,17-dione and 4-androstene-3 beta,17 beta-diol

supplementation in young men. Eur J Appl Physiol 2000;


12. Ballantyne CS, Phillips SM, MacDonald JR, et al. The acute effects

of androstenedione supplementation in healthy young

males. Can J Appl Physiol 2000; 25(1):68–78.

20 Leder

13. King DS, Sharp RL, Vukovich MD, et al. Effect of oral androstenedione

on serum testosterone and adaptations to

resistance training in young men. J Am Med Assoc 1999;


14. Leder BZ, Longcope C, Catlin DH, et al. Oral androstenedione

administration and serum testosterone concentrations

in young men. J Am Med Assoc 2000; 283(6):


15. Rasmussen BB, Volpi E, Gore DC, et al. Androstenedione

does not stimulate muscle protein anabolism in

young healthy men. J Clin Endocrinol Metab 2000; 85(1):


16. Wallace MB, Lim J, Cutler A, et al. Effects of dehydroepiandrosterone

vs. androstenedione supplementation in

men. Med Sci Sports Exerc 1999; 31(12):1788–1792.

17. Leder BZ, Catlin DH, Longcope C, et al. Metabolism of

orally administered androstenedione in young men. J Clin

Endocrinol Metab 2001; 86(8):3654–3658.

18. Leder BZ, Leblanc KM, Longcope C, et al. Effects of oral

androstenedione administration on serum testosterone and

estradiol levels in postmenopausal women. J Clin Endocrinol

Metab 2002; 87(12):5449–5454.

19. Kicman AT, Bassindale T, Cowan DA, et al. Effect of androstenedione

ingestion on plasma testosterone in young

women: A dietary supplement with potential health risks.

Clin Chem 2003; 49(1):167–169.

20. Kachhi PN, Henderson SO. Priapism after androstenedione

intake for athletic performance enhancement. Ann Emerg

Med 2000; 35(4):391–393.

21. Catlin DH, Leder BZ, Ahrens B, et al. Trace contamination

of over-the-counter androstenedione and positive urine test

results for a nandrolone metabolite. J Am Med Assoc 2000;


22. Catlin DH, Leder BZ, Ahrens BD, et al. Effects of androstenedione

administration on epitestosterone metabolism in men.

Steroids 2002; 67(7):559–564.


Glossary, sports nutritionSuccess Chemistry Staff


Arginine was first isolated in 1895 from animal horn. It is

considered a nonessential amino acid under physiological

conditions; however, it may be classified as semi-essential

(or conditioned) in newborns, young children, or other

circumstances characterized by accelerated tissue growth

(e.g., infection, sepsis, trauma) when its production may

be too slow and not sufficient to meet the requirements

(1). Arginine is physiologically active in the L-form (L-Arg)

and participates in protein synthesis in cells and tissues.

It is essential for the synthesis of urea, creatine, creatinine,

and pyrimidine bases. It also strongly influences hormonal

release and has an important role in vascular dynamics,

participating in the synthesis of nitric oxide (NO).


Dietary arginine is particularly abundant in wheat

germ and flour, buckwheat, oatmeal, dairy products

(cottage cheese, ricotta cheese, nonfat dry milk, skimmed

yogurt), chocolate, beef (roasts, steaks), pork, nuts (coconut,

pecans, walnuts, almonds, hazel nuts, peanuts),

seeds (pumpkin, sesame, sunflower), poultry (chicken,

turkey), wild game (pheasant, quail), seafood (halibut,

lobster, salmon, shrimp, snails, tuna), chick peas, and

soybeans (2).

L-Arg, delivered via the gastrointestinal tract, is absorbed

in the jejunum and ileum of the small intestine. A

specific amino acid transport system facilitates this process

and participates also in the transport of the other

basic amino acids, L-lysine and L-histidine. About 60% of

the absorbed L-Arg is metabolized by the gastrointestinal

enterocytes, and only 40% remains intact reaching the

systemic circulation.

An insufficient arginine intake produces symptoms

of muscle weakness, similar to muscular dystrophy (3).

Arginine deficiency impairs insulin secretion, glucose production,

and liver lipid metabolism (4). Conditional deficiencies

of arginine or ornithine are associated with the

presence of excessive ammonia in the blood, excessive

lysine, rapid growth, pregnancy, trauma, or protein deficiency

and malnutrition. Arginine deficiency is also associated

with rash, hair loss and hair breakage, poor wound

healing, constipation, fatty liver, hepatic cirrhosis, and

hepatic coma (4).

Depending on nutritional status and developmental

stage, normal plasma arginine concentrations in humans

and animals range from 95 to 250 mol/L. Toxicity and

symptoms of high intake are rare, but symptoms of massive

dosages may include thickening and coarsening of

the skin, muscle weakness, diarrhea, and nausea.

The proximal renal tubule accounts for much of the

endogenous production of L-Arg from L-citrulline. In the

tubule, arginine reacts via the Krebs cycle with the toxic

ammonia formed from nitrogen metabolism, producing

the nontoxic and readily excretable urea (Fig. 1) (5). If this

mechanism does not efficiently handle metabolic byproducts

and if L-Arg intake is insufficient, ammonia rapidly

accumulates, resulting in hyperammonemia.

L-Arg undergoes different metabolic fates. NO,

L-citrulline, L-ornithine, L-proline, L-glutamate, and

polyamine-like putrescine are formed from L-Arg. Moreover,

the high-energy compound NO-creatinine phosphate,

which is essential for sustained skeletal muscle contraction,

is also formed from L-Arg (Fig. 2).

L-Arg, its precursors, and its metabolites are deeply

involved in the interaction of different metabolic pathways

and interorgan signaling. The amino acid influences

the internal environment in different ways: disposal

of protein metabolic waste; muscle metabolism; vascular

regulation; immune system function; healing and repair

of tissue; formation of collagen; and building of new bone

and tendons.

A leading role for arginine has been shown in the

endocrine system, vasculature, and immune response.

  • CO2 + NH4

  • +

  • NH2

  • NH2

  • C=O

  • 2ATP



  • 2ADP + Piz


  • NH4

  • +-GROUPS






  • ATP

  • AMP + Ppi + H2O

  • Figure 1 L-Arginine and Krebs cycle in the renal tubule.

  • 21

  • 22 Maccario et al.

  • Nitric Oxide

  • Nitric Oxide

  • Admatine

  • Aldehyde Agmatine

  • Agmatinase

  • Polyamines

  • Ornithine

  • Proline

  • Arginine

  • Group

  • Guanidine


  • HN

  • C NH

  • Protein

  • synthesis

  • Glycine

  • Guanidinoacetate

  • Pyrroline-5-carboxylate

  • Glutamyl-γ-′semialdehyde

  • Urea cycle Glutamine Glutamate

  • Creatine

  • Urea

  • Urea

  • NOS

  • NOS

  • Ca2+

  • ADC

  • ADC

  • OAT

  • Arginase-I

  • P-5-C

  • dehydrogenase

  • P-5-C

  • reductase

  • A-GAT

  • GMT

  • Glu synthase

  • DAO

  • NH3

Figure 2 L-Arginine metabolites. Abbreviations: ADC, arginine decarboxylase;

A:GAT, arginine:glycine amidinotransferase; DAO, diamine oxidase; Glu

synthase, glutamine synthase; GMT, guanidinoacetate-N-methyltransferase;

NOS, nitric oxide synthase; OAT, ornithine aminotransferase; P-5-C dehydrogenase,

pyrroline-5-carboxylate dehydrogenase; P-5-C reductase, pyrroline-

5-carboxylate reductase.


Endocrine Actions

L-Arg functions as a secretagogue of a number of important

hormones at the pituitary, pancreas, and adrenal levels.

The effects on growth hormone (GH), prolactin (PRL),

adrenocorticotropic hormone (ACTH), and insulin secretion

will be discussed in detail.

GH Secretion

Among the various factors modulating somatotropin

function, arginine is well known to play a primary stimulatory

influence. Arginine has been shown to increase

basal GH levels and to enhance the GH responsiveness

to growth hormone releasing hormone (GHRH) both in

animals and in humans throughout their life span (6–9);

its GH-stimulating activity occurs after both IV and oral

administration and is dose dependent; 0.1 and 0.5 g/kg

are the minimal and the maximal IV effective doses, respectively.

Moreover, a low orally administered arginine

dose has been shown to be as effective as a high IV dose

in enhancing the GH response to GHRH both in children

and in elderly subjects (10,11).

Arginine, directly or indirectly via NO, is likely

to act by inhibiting hypothalamic somatostatin (SS) release.

It has been shown that arginine—but not isosorbidedinitrate

and molsidomine, two NO donors—stimulates

GH secretion (12,13), suggesting that it does not exert its

effects through the generation of NO. Moreover, arginine

does not modify either basal or GHRH-induced GH increase

from rat anterior pituitary (14). On the contrary, it

potentiates the GH response to the maximal GHRH dose

in humans. Arginine can elicit a response even when the

response has been previously inhibited by a GHRH administration,

which induces an SS-mediated negative GH

auto feedback (7,8,15). Moreover, arginine counteracts the

GH-inhibiting effect of neuroactive substances that act by

stimulating SS release; it does not modify the GH-releasing

activity of stimuli acting via SS reduction (8). Again, in favor

of an SS-mediated mechanism is also the evidence

that ornithine, the active form of arginine, is unable to

modify plasma GHRH levels in humans (16). Moreover,

arginine fails to potentiate the increased spontaneous nocturnal

GH secretion, which is assumed to reflect circadian

SS hyposecretion and GHRH hypersecretion, respectively

(8). Arginine does not influence the strong GH-releasing

action of ghrelin, the natural ligand of GH secretagogue

receptors, which is supposed to act as a functional antagonist

of SS at both the pituitary and the hypothalamic levels


The GH-releasing activity of arginine is sex dependent

but not age dependent, being higher in females than

in males but similar in children, young, and elderly subjects

(8,19–23). Moreover, it has been clearly demonstrated

that arginine totally restores the low somatotroph responsiveness

to GHRH in aging, when a somatostatin ergic hyperactivity

is likely to occur (20–23). This evidence clearly

indicates that the maximal secretory capacity of somatotropic

cells does not vary with age and that the age related

decrease in GH secretion is due to a hypothalamic

impairment (20–23). This also points out the possible

clinical usefulness of this substance to rejuvenate the

GH/insulin-like growth factor-I (IGF-I) axis in aging. In

fact, the reduced function of the GH/IGF-I axis in aging

may account for the changes in body composition, structure,

and function. In agreement with this assumption, it

has been reported by some, but not all, authors that elderly

subjects could benefit from treatment with rhGH

to restore IGF-I levels within the young range (21,24). As

it has been demonstrated that the GH releasable pool in

the aged pituitary is basically preserved and that the age related

decline in GH secretion mostly reflects hypothalamic

dysfunction (21,23), the most appropriate, that is,

“physiological,” approach to restore somatotroph function

in aging would be a treatment with neuroactive substances

endowed with GH-releasing action. Among these

GH secretagogues, arginine received considerable attention.

In fact, the coadministration of arginine (even at

low oral doses) with GHRH (up to 15 days) enhanced

the GH responsiveness to the neurohormone in normal

aged subjects (11). However, the efficacy of long-term

treatment with oral arginine to restore the function of the

GH/IGF-I axis in aging has never been shown in elderly


Following the evidence that GHRH combined with

arginine becomes the most potent and reproducible stimulus

to diagnose GH deficiency throughout the lifespan

(25), GHRH + arginine is, at present, one of the two gold

standard tests for the diagnosis of GH deficiency (25,26).

In fact, the GH response to a GHRH + arginine test is

approximately threefold higher than the response to classical

tests and does not vary significantly with age (25,26).

Because of its good tolerability and its preserved effect in

aging, the GHRH + arginine test is currently considered

to be the best alternative choice to the insulin-induced

tolerance test (ITT) for the diagnosis of GH deficiency

throughout the lifespan (25).

L-Arginine 23

PRL Secretion

Among the endocrine actions of arginine, its PRL releasing

effect has been shown both in animals and in

humans after IV but not after oral administration (10,27).

The PRL response to arginine is markedly lower than

the response to the classical PRL secretagogues, such

as dopaminergic antagonists or thyrotropin-releasing

hormone (TRH) (6) but higher than that observed after

secretion of GH and other modulators of lactotrope

function (17).

The mechanisms underlying the stimulatory effect

of arginine on PRL secretion are largely unknown, but

there is evidence that this effect is not mediated by galanin,

a neuropeptide with PRL-releasing effect. In fact, galanin

has been shown to potentiate PRL response to arginine,

suggesting different mechanisms of action for the two substances


ACTH Secretion

Although some excitatory amino acids and their agonists

have been demonstrated to differently modulate

corticotropin-releasing hormone and arginine vasopressin

release in vitro and influence both sympathoadrenal and

hypothalamo-pituitary-adrenal (HPA) responses to hypoglycemia

in animals (29,30), little is known about arginine

influences on HPA axis in humans. Many studies have

shown that mainly food ingestion influences spontaneous

and stimulated ACTH/cortisol secretion in normal subjects

and that central 1-adrenergic-mediated mechanisms

are probably involved (31). In humans free fatty acids inhibit

spontaneous ACTH and cortisol secretion, but no

data exist regarding the effect of each nutrient component

on HPA function. Previous studies demonstrated that

arginine is unable to exert an ACTH-stimulatory effect in

humans via generation of NO (12) and our unpublished

preliminary data failed to demonstrate a significant effect

of arginine (30 g IV) on either ACTH or cortisol secretion

in normal subjects.

Insulin Secretion

Arginine is one of the most effective known insulin secretagogue

and it may be used with glucose potentiation

to determine a patient’s capacity to secrete insulin (32).

Arginine acts synergistically with glucose, and to a much

lesser extent with serum fatty acids, in stimulating insulin

release. A synergistic effect of arginine and glucose on insulin

secretion has been shown in humans (33,34), and the

combined administration of these two stimuli has been

studied in an attempt to test -cell secretory capacity in

diabetic patients (35).

A protein meal leads to a rapid increase in both

plasma insulin and glucagon levels (36). Administration

of arginine has a similar effect. An arginine transport system

is present in the -cell plasma membrane (37). When

arginine enters the cell, it causes ionic changes that depolarize

the cell and trigger Ca2+ uptake and exocytosis

of insulin-containing granules.

Several mechanisms for arginine-induced -cell

stimulation have been proposed. These include the

metabolism of L-Arg leading to the formation of ATP

(38,39), the generation of NO (40,41), and the direct depolarization

of the plasma membrane potential due to the

accumulation of the cationic amino acid (42–44).

A sustained Ca2+ influx is directly related to insulin

secretion following arginine uptake by cells. The

arginine-induced increase in Ca2+ concentration is inhibited

by the activation of ATP-sensitive potassium (K-ATP)

channels with diazoxide and seems dependent on the nutritional

status. These observations suggest that the K-ATP

channels, when fully open, act to prevent membrane depolarization

caused by arginine. The presence of a nutrient,

such as glucose, produces sufficient closure of K-ATP

channels to allow arginine-induced membrane depolarization

and activation of the voltage-activated Ca2+ channels


Nonendocrine Actions

Cardiovascular System

Increasing interest has been recently focused on NO. This

mediator, which is synthesized from L-Arg (45) by nitric

oxide synthases (NOS) (46), is a potent vasodilator

(47) and inhibitor of platelet adhesion and aggregation

(48). Three isoforms of NOS are described: neuronal NOS

(nNOS—NOS-1), inducible NOS (iNOS—NOS-2), and endothelial

NOS (eNOS—NOS-3). NOS-1 and NOS-3 are

expressed constitutively and they produce NO at low

rates (49). NOS-3 is responsible for a consistent vasodilator

tone and, although constitutive, can be regulated by

endothelial shear stress (50) and substances such as acetylcholine,

histamine, serotonin, thrombin, bradykinin, and

catecholamines. Calcium is required for NOS-3 activation

(51).NO production is mainly dependent on the availability

of arginine and NOS is responsible for the biochemical

conversion of L-Arg to NO and citrulline in the presence

of cofactors such as reduced nicotinamide adenine dinucleotide

phosphate (NADPH), tetrahydrobiopterin (BH4),

flavin mononucleotide, and flavin adenine nucleotide. Reduced production,

leading to vasoconstriction and increases

in adhesion molecule expression, platelet adhesion

and aggregation, and smooth muscle cell proliferation has

been demonstrated in atherosclerosis, diabetes mellitus,

and hypertension (52–54)—conditions known to be associated

with an increased mortality because of cardiovascular

disease. Taken together, these observations lead to the

concept that interventions designed to increase NO production

by supplemental L-Arg might have a therapeutic

value in the treatment and prevention of the endothelial

alterations of these diseases. Besides several actions exerted

mainly through NO production, arginine also has a

number of NO-independent properties, such as the ability

to regulate blood and cellular pH, and the effect on the

depolarization of endothelial cell membranes.

The daily consumption of arginine is normally about

5 g/day. Arginine supplementation is able to increase NO

production, although the Km for L-Arg is 2.9 mol and the

intracellular concentration of arginine is 0.8 to 2.0 mmol.

To explain this biochemical discrepancy, named “arginine

paradox,” there are theories that include low arginine levels

in some diseases (e.g., hypertension, diabetes mellitus,

and hypercholesterolemia), and/or the presence of enzymatic

inhibitors (55), and/or the activity of the enzyme

arginase (which converts arginine to ornithine and urea,

leading to low levels of arginine).

Recently attention has been given to the methylated

forms of L-Arg, generated by the proteolysis of

24 Maccario et al.

methylated proteins; they are represented by asymmetric

dimethylarginine (ADMA) and two symmetric dimethylated

derivatives: symmetric dimethylarginine (SDMA)

and monomethylarginine (MMA) (56). Only ADMA and

MMA, but not SDMA, exert inhibitory effects on NOS-3

activity (57). For this reason, ADMA is now recognized as

a new emerging cardiovascular risk marker and likely as

a causative factor for cardiovascular disease (58).

L-Arginine therapy in cardiovascular pathologies

showed contradictory results. However, it is now clear that

individual response to L-Argmaybe influenced by DMA.

In fact, no effects of L-arg therapy are demonstrated in patients

with low ADMA levels, whereas in patients with

high ADMA level, L-Arg normalizes the L-Arg to ADMA

ratio, thus normalizing the endothelial function (59).

Several studies demonstrated that L-Arg infusion

in normal subjects and patients with coronary heart disease

(60), hypercholesterolemia (61), and hypertension

(62) is able to improve the endothelial function, but the

results, although encouraging, are not conclusive because

of the short-term effects of IV arginine. However, arginine

does not affect endothelial function in patients with

diabetes mellitus. On the other hand, oral L-Arg has a

longer half-life and longer-term effects than L-Arg given

intra-arterially or intravenously (63). Thus, in the setting

of long-term health maintenance or symptom management,

the oral route would be preferred. Studies in animals

documented that oral L-Arg supplementation is able

to reduce the progression of atherosclerosis, preserving

endothelium function (64) and inhibiting circulating inflammatory

cells (65) and platelets (66) in animals with

hypercholesterolemia, and to decrease blood pressure and

wall thickness in animals with experimental hypertension

(67). On the other hand, studies in humans in vivo are

not so widely positive as the animal experimental data.

Actually, although the majority of the data is in normal

subjects, individuals with a history of cigarette smoking

and patients with hypercholesterolemia and claudication

demonstrate beneficial effects of oral L-Arg administration

on platelet adhesion and aggregation, monocyte adhesion,

and endothelium-dependent vasodilation (68,69).

Other studies do not show any benefit (70,71); therefore,

no definitive conclusions can be drawn. Taken together,

the studies show a major effect when L-Arg supplementation

was given in subjects with hypercholesterolemia,

probably because of an increase in NO production via reduction

of the ADMA intracellular concentration, which

is increased in the presence of LDL hypercholesterolemia.

In conclusion, despite several beneficial effects on

intermediate end points, particularly in hypercholesterolemic

patients, there is no evidence for a clinical

benefit in the treatment or prevention of cardiovascular

disease. More data, derived from large-scale prospective

studies evaluating the effect of long-term treatment with

L-Arg, are needed. Future perspectives of pharmacological

intervention are represented by the regulation of the enzyme

dimethylarginine dimethylaminohydrolase responsible

for the ADMA metabolism (57), the arginase (72),

and the endothelial cell L-Arg transporter (73).

Immune System

Many studies, in animals as well as in humans, have

shown that arginine is involved in immune modulation. In

fact, this amino acid is a component of most proteins and

the substrate for several nonprotein, nitrogen-containing

compounds acting as immune modulators.

There is clear evidence that arginine participates in

the cell-mediated immune responses of macrophages and

T lymphocytes in humans through the production of NO

by inducible nitric oxide synthase (iNOS-–NOS-2), which

occurs mostly in the macrophage (74,75), and through the

modulation of T-lymphocyte function and proliferation

(76,77). At intracellular levels, arginine is metabolized by

two different enzymatic pathways: the arginase pathway,

by which the guanidino nitrogen is converted into urea to

produce ornithine, and the NOS pathway, which results

in oxidation of the guanidino nitrogen to produce Land

other substances (78,79).

It has been shown that macrophage superoxide production,

phagocytosis, protein synthesis, and tumoricidal

activity are inhibited by high levels of arginine in vitro and

that sites of inflammation with prominent macrophage

infiltration, such as wounds and certain tumors, are deficient

in free arginine (80). In particular, a decrease in

arginine availability due to the activity of macrophage derived

arginase rather than the arginine/NO pathway

may contribute to the activation of macrophages migrating

at inflammatory sites (80). Arginine metabolism in the

macrophages is activity dependent. At rest, macrophages

exhibit minimal utilization of arginine and lower NOS-2

expression or arginase activity, whereas in activated cells,

arginine is transported into the cell, and NOS-2 expression

and arginase are induced by cytokines and other stimuli

(81). The types of stimuli that induce NOS-2 and arginase

are quite different. In vitro and in vivo studies demonstrated

that NOS-2 is induced by T-helper I cytokines (IL-

1, TNF, and -interferon) produced during activation of

the cellular immune response, such as severe infections or

sepsis (74,75), whereas arginases are induced by T-helper

II cytokines (IL-4, IL-10, and IL-13) and other immune regulators

aimed at inducing the humoral immune response

(82,83). Thus, in disease processes, where inflammatory

response predominates, NOS-2 expression and NO production

prevail. Under biological circumstances where Thelper

II cytokine expression is prevalent, arginase activity

and the production of ornithine and related metabolites

would predominate.

In vitro studies in animals demonstrated depressed

lymphocyte proliferation in cultures containing low levels

of arginine and maximal proliferation when arginine is

added at physiological plasma concentration (77,84), but

the molecular details have not been completely defined.

It has also been shown that supplemental arginine

increased thymic weight in rodents because of increased

numbers of total thymic T lymphocytes. On the other

hand, in athymic mice, supplemental arginine increased

the number of T cells and augmented delayed-type hypersensitivity

responses, indicating that it can exert its effects

on peripheral lymphocytes and not just on those within

the thymus (76).

The immunostimulatory effects of arginine in animal

studies have suggested that this amino acid could be

an effective therapy for many pathophysiological conditions

in humans, able to positively influence the immune

response under some circumstances by restoring cytokine

balance and reducing the incidence of infection.


In healthy humans, oral arginine supplementation

shows many effects on the immune system, including

increase in peripheral blood lymphocyte mitogenesis,

increase in the T-helper–T-cytotoxic cell ratio and, in

macrophages, activity against microorganisms and tumor

cells (85). Furthermore, the delayed-type hypersensitivity

response as well as the number of circulating natural

killer (NK) and lymphokine-activated killer cells are

increased (85–87). Therefore, it has been hypothesized

that arginine could be of benefit to patients undergoing

major surgery after trauma and sepsis and in cardiovascular

diseases, HIV infection, and cancer (88). In

fact, short-term arginine supplementation has been shown

to maintain the immune function during chemotherapy;

arginine supplementation (30 g/day for 3 days) reduced

chemotherapy-induced suppression of NK cell activity,

lymphokine-activated killer cell cytotoxicity, and lymphocyte

mitogenic reactivity in patients with locally advanced

breast cancer (89). It must be noted that chronic administration

of arginine has also been shown to promote cancer

growth by stimulating polyamine synthesis in both animal

and human studies (89). On the other hand, NO has

been shown to inhibit tumor growth. Thus, the real effect

on cancer processes depends on the relative activities of

NOS and arginase pathways that show variable expression,

depending on the stage of carcinogenesis (91).

These data clearly indicate the involvement of arginine

in immune responses in both animals and humans.

Large clinical trials are needed to clarify the clinical application

and efficacy of this amino acid in immunity and



The available form of supplemental L-Arg is represented

by the free base, the Cl− salt (L-Arg hydrochloride-–L-Arg-

HCl) and the aspartate salt of the amino acid (92).

L-Arg is stable under sterilization condition and its

administration is safe for mammals in an appropriate dose

and chemical form (91).

Oral L-Arg (up to 9 g of Arg-HCl per day for adults)

has no adverse effects on humans but higher doses can

lead to gastrointestinal toxicity, theoretically increasing

local production of NO and impairing intestinal absorption

of other basic amino acids (91). Moreover, the local

NOproduction may be particularly dangerous if intestinal

diseases are present (92).

Oral L-Arg supplement is commonly used to increase

GH release and consequentially physical performance;

moreover, it has been hypothesized that L-Arg

supplement could lead to improved muscular aerobic

metabolism and less lactate accumulation, enhancing NOmediated

muscle perfusion.

However, in a clinical trial, arginine supplement in

endurance-trained athletes did not show any difference

from placebo in endurance performance (maximal oxygen

consumption, time to exhaustion), endocrine (GH,

glucacon, cortisol, and testosterone concentrations), and

metabolic parameters (93).

In another study, the association “arginine plus exercise”

produced a GH response approximately 50% lower

than that observed with exercise alone, suggesting that

the acute use of oral L-Arg prior to exercise blunts the GH

response to subsequent exercise (94).

No effects on NO production, lactate and ammonia

metabolism, and physical performance in intermittent

anaerobic exercise were shown in well-trained male athletes

after short-term arginine supplementation (95). It has

been hypothesized thatNOproduction is not modified by

arginine supplementation in athletes because they may

have higher basal concentrations of NO than general population;

in fact, basal NO production can be increased by

regular exercise training, without any pharmacological intervention


There are many interesting clinical perspectives on

arginine supplementation therapy, especially in critical

care setting (96), treatment and prevention of pressure

ulcers (97), hypertension (59), and asthma and chronic obstructive

pulmonary disease (98), but further studies are

required to clarify which categories of patients may benefit

from this treatment (99).


From an endocrinological point of view, the simple classification

of arginine as an amino acid involved in peripheral

metabolism is no longer acceptable. In fact, besides other

nonendocrine actions, it has been clearly demonstrated

that arginine plays a major role in the neural control of

anterior pituitary function, particularly in the regulation

of somatotrophin secretion. One of the most important

concepts regarding arginine is the existence of an arginine

pathway at the CNS level, where this amino acid represents

the precursor of NO, a gaseous neurotransmitter of

major importance. On the other hand, NO does not necessarily

mediate all the neuroendocrine or the peripheral

arginine actions.

In the past years, new discoveries have led to a rapid

increase in our knowledge of the arginine/NO system,

from a neuroendocrine and nonendocrine point of view.

Up to now, there is no evidence for the utility of L-Arg

supplement for muscle strength or exercise performance

in humans. However, several other aspects still remain to

be clarified; the potential clinical implications for arginine

have also never been appropriately addressed and could

provide unexpected results both in the endocrine and in

the cardiovascular fields.


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Glossary, sports nutritionSuccess Chemistry Staff

L-Carnitine, Acetyl-L-Carnitine, and Propionyl-L-Carnitine


L-Carnitine [known chemically as R(−)--hydroxy--

(N,N,N-trimethylammonio)butyrate; molecular weight:

161.2 g/mol] is a water-soluble quaternary amine that facilitates

lipid metabolism.


Only the L isomer is biologically

active. Humans acquire varying amounts of L-carnitine

from dietary sources, but a dietary requirement has not

been established. The goal of this chapter is to survey the

literature on the clinical findings on L-carnitine and its esters

acetyl-L-carnitine and propionyl-L-carnitine. Due to

space constraints, this is not an exhaustive review. Readers

are directed to the references for more information.



The human body synthesizes L-carnitine from the essential

amino acids lysine and methionine in amounts that are

limited but adequate for the maintenance of normal health

(1). L-Carnitine participates in reversible transesterification

reactions, in which an acyl group is transferred from

coenzyme A to the hydroxyl group of L-carnitine (Fig. 1).

Acetyl-L-carnitine, propionyl-L-carnitine, and other esters

are biosynthesized in this manner. Carnitine and acetyl-Lcarnitine

(and lesser amounts of other esters of carnitine)

are also obtained from the diet (1).

Transfer of Long-Chain Fatty Acids from Cytoplasm

into Mitochondria

Long-chain fatty acids, as free acids or coenzyme A esters,

cannot cross the mitochondrial inner membrane.

In contrast, long-chain acylcarnitine esters rapidly cross

this membrane, facilitated by a carrier protein, carnitine acylcarnitine

translocase (CACT) (2). In the cytoplasm,

transesterification of long-chain fatty acids from coenzyme

A to L-carnitine is catalyzed by carnitine palmitoyltransferase

I (CPT I), an integral protein of the mitochondrial

outer membrane. This enzyme serves as the primary

regulator in partitioning fatty acids toward oxidation in

mitochondria or triglyceride synthesis, and its activity is

regulated principally through inhibition by malonyl-CoA

(3). On the matrix side of the mitochondrial inner membrane,

the acyl group of the carnitine ester is transferred to

intramitochondrial coenzyme A and carnitine is released

(2). This reaction is catalyzed by carnitine palmitoyltransferase

II (CPT II), an enzyme bound to the surface of the

membrane (2). L-Carnitine, either nonesterified or as a

short-chain acyl ester, may then exit the mitochondrion

via CACT

Acyl-coenzyme A ester



Reduced coenzyme A

Acyl-L-carnitine ester


Transesterification reaction between L-carnitine and coenzyme A.

Transfer of Chain-Shortened Fatty Acids from

Peroxisomes to Mitochondria

Very long-chain fatty acids are not metabolized in the mitochondria.

Instead, they enter peroxisomes and undergo

one or more -oxidation cycles, leading to the generation

of medium-chain acyl-CoA. These acyl groups are

then transesterified by carnitine octanoyltransferase for

export from the mitochondria (2). Medium-chain acylcarnitine

esters exported from peroxisomes are transported

into mitochondria by CACT, and the acyl moieties are

transesterified to coenzyme A and oxidized (Fig. 2) (2).

Modulation of the Acyl-CoA/CoA Ratio

in Cellular Compartments

Coenzyme A participates in many metabolic processes in

the cellular cytoplasm and organelles. However, neither

coenzyme A nor its thioesters can cross the membranes

separating these compartments. Thus, in each compartment,

sufficient nonesterified coenzyme A must be made

available to maintain metabolic activities in that part of

the cell. Because of its ability to be transported across

organelle membranes and undergo rapid transesterification

with coenzyme A, L-carnitine facilitates availability

of adequate amounts of the deacylated coenzyme. In mitochondria,

the amount of acetyl-CoA generated from

rapid -oxidation of fatty acids or carbohydrate utilization


L-Carnitine and acetyl-L-carnitine transport and function. Abbreviations: Carn, L-carnitine; AcCarn, acetyl-L-carnitine; OCTN2, organic cation transporter

2; UnTr, unidentified transporter(s); LCA-CoA, long-chain acyl-coenzyme A; MCA-CoA, medium-chain acyl-coenzyme A; VLCA-CoA, very long-chain acyl-coenzyme

A; LCA-Carn, long-chain acylcarnitine esters; MCA-Carn, medium-chain acylcarnitine esters; Ac-CoA, acetyl-coenzyme A; CPT I, carnitine palmitoyltransferase

I; CPT II, carnitine palmitoyltransferase II; CAT, carnitine acetyltransferase; COT, carnitine octanoyltransferase; CACT, carnitine-acylcarnitine translocase; PDH,

pyruvate dehydrogenase complex.

may exceed the capacity of the citric acid cycle to release

the coenzyme. Transesterification of acetyl units to

L-carnitine, catalyzed by carnitine acetyltransferase

(CAT), frees intramitochondrial coenzyme A for participation

in subsequent cycles of substrate utilization (3,4).

Lowering of the acetyl-CoA/CoA ratio stimulates pyruvate

oxidation, secondary to an increase in pyruvate dehydrogenase

complex activity (4). Acetyl-L-carnitine can

be removed from the mitochondrion via CACT for use in

the cytoplasm, for export and use in other cells or tissues,

or for excretion (3,4). This function has long been viewed

primarily as a means to dispose of acetyl units from mitochondria.

However, export of acetyl-L-carnitine fromsome

cells and tissues (e.g., liver and kidney) may be important

for supply of this metabolite to other tissues (e.g., brain),

where it may have specific functions in addition to its

use as a substrate for energy production. Moreover, by

modulating the intramitochondrial acetyl-CoA/CoA ratio,

L-carnitine plays a significant role in regulating glucose

metabolism in skeletal muscle and heart (4).

Membrane Phospholipid Remodeling

L-Carnitine and extramitochondrial CPT are important

modulators of long-chain fatty acid utilization for

membrane phospholipid biosynthesis and remodeling.

L-Carnitine acts as a reservoir of long-chain fatty acids

for incorporation into erythrocyte membrane phospholipids

during repair after oxidative insult (2) and for

use in the synthesis of dipalmitoylphosphatidylcholine,

the major component of surfactant, in lung alveolar

cells (2).

Other Reported Actions of L-Carnitine and/or Its Acetyl

and Proponyl Esters

L-Carnitine may mimic some of the actions of glucocorticoids

in vivo. In HeLa cells, L-carnitine reduces

glucocorticoid receptor- affinity for its steroid ligand and

triggers nuclear translocation of the receptor (5). It suppresses

glucocorticoid receptor–mediated tumor necrosis

factor- and interleukin-12 release by human primary

L-Carnitine, Acetyl-L-Carnitine, and Propionyl-L-Carnitine 109

monocytes stimulated with lipopolysaccharide ex vivo

(5). All of these effects of L-carnitine are concentration

dependent. In rats and mice, L-carnitine is found to

markedly suppress liposaccharide-induced cytokine production,

improving their survival during cachexia and

septic shock (5). In humans, L-carnitine supplementation

of surgical and AIDS patients decreased serum tumor

necrosis factor- concentration (5). Glucocorticoids also

increase the expression and activity of urea cycle enzymes.

Hyperammonemia associated with chronic valproic acid

therapy and with several inborn errors of metabolism,

including CACT deficiency and medium-chain acyl-CoA

dehydrogenase deficiency, is attenuated by L-carnitine

administration. It has been experimentally found that

L-carnitine supplementation protects against lethal ammonia

intoxication in mice (6). One suggested mechanism

for these effects of L-carnitine is increased synthesis and

activity of urea cycle enzymes, a process also responsive to


L-Carnitine is a peripheral antagonist of thyroid hormone

action in some tissues (7). It inhibits thyroid hormone

entry into cell nuclei. In a controlled clinical trial,

L-carnitine was shown to reverse or prevent some symptoms

of hyperthyroidism (7).

Acetyl-L-carnitine may directly or indirectly reverse

age-associated mitochondrial decay (8). It acts as a chaperone

to protect macromolecules, including CAT, from

structural alteration and/or loss of function. Acetyl-Lcarnitine

partially reverses age-associated loss of mitochondrial

membrane potential and decline in membrane

cardiolipin concentration, and protects against oxidative

damage to mitochondrial DNA (8).

L-Carnitine inhibits arachidonic acid incorporation

into platelet phospholipids, agonist-induced arachidonic

acid release, and arachidonic acid–inducedNADPH

(nicotinamide adenine dinucleotide phosphate)-oxidase

activation (9). It has been proposed and some evidence

has been obtained to support this notion that acetyl-

L-carnitine stimulates or upregulates expression of heat

shock proteins, redox-sensitive transcription factors, and

sirtuins that protect against oxidative cellular damage (10).

These actions may be particularly important in modulating

the aging process and in slowing the progression of

neurodegenerative diseases.



Dietary Intake and Biosynthesis

Meat, fish, chicken, and dairy products are rich sources of

dietary L-carnitine (1). Plant-derived foods contain very

small amounts of the substance. Most commercially available

infant formulas contain L-carnitine, either provided

from the milk component or supplemented, as in the

case of soy-protein-based formulas. There is no recommended

dietary allowance or dietary reference intake for


In mammals, L-carnitine is synthesized from

ε-N-trimethyllysine, which is derived from posttranslationally

methylated lysine residues in proteins, and

protein turnover (1). In normal humans, the rate of synthesis

is estimated to be approximately 1.2 mol/kg body

weight/day (1). The rate of L-carnitine biosynthesis is regulated

by the availability of ε-N-trimethyllysine. Thus,

conditions that increase protein methylation and/or protein

turnover may increase the rate of L-carnitine biosynthesis



Dietary L-carnitine may be absorbed through active or

passive mechanisms. Evidence from several in vivo and

in vitro studies indicates that L-carnitine is actively transported

from the small intestinal lumen into enterocytes

(11). However, the preponderance of data suggests that

intracellular L-carnitine in the intestinal mucosa does not

cross serosal membranes by an active transport mechanism.

Absorption of dietary L-carnitine and L-carnitine

supplements appears to occur primarily by passive diffusion

(11). In humans, approximately 54% to 87% of dietary

L-carnitine is absorbed, depending on the amount

in the diet. The bioavailability of dietary supplements

(0.6–4 g/day) is 15% to 20% (11,12). Unabsorbed

L-carnitine is degraded by microorganisms in the large intestine.

Major metabolites identified are trimethylamine

oxide in urine and -butyrobetaine in feces (11,12).

Bioavailability of oral acetyl-L-carnitine has not been studied

in normal healthy humans.

Distribution in Tissues, Fluids, and Cells

L-Carnitine and acylcarnitine esters are present in all tissues.

In most tissues and cells, they are present in higher

concentration than in the circulation. For example, in human

skeletal muscle and liver, respectively, nonesterified

L-carnitine is concentrated 76-fold and 50-fold from that

in serum (estimated from data in Ref. 13).


Tissue Accumulation

L-Carnitine and acetyl-L-carnitine are concentrated in

most tissues via high-affinity, Na+-dependent organic

cation transporter OCTN2 (14). Kt for L-carnitine binding

is 3 to 5 M; OCTN2 binds acetyl-L-carnitine and

propionyl-L-carnitine with comparable affinity. This protein

is highly expressed in heart, placenta, skeletal muscle,

kidney, pancreas, testis, and epididymis and weakly

expressed in brain, lung, and liver. L-Carnitine entry

into the liver occurs via a low-affinity (Kt = 5 mM)

transporter, probably distinct from OCTN2. Several other

L-carnitine transporters have been identified, including

OCTN1, OCTN3, and ATB0,+, and Oat9S (2,15). Specific

roles for these transporters in carnitine metabolism in humans

have not been determined.

Homeostasis, Renal Reabsorption, and Excretion

Circulating L-carnitine concentrations are maintained

at a fairly constant level of around 50 M, predominantly

through efficient reabsorption by the kidney

(11). At a filtered load of 50 mol/L, the efficiency of

L-carnitine and acylcarnitine ester reabsorption is 90% to

98%. However, as the filtered load of L-carnitine increases,

for example, after consumption of a dietary supplement

or after intravenous infusion, the efficiency of reabsorption

declines rapidly. Physiologically, the efficiency of

L-carnitine reabsorption is sensitive to the amount in the

diet as well as the differences in the macronutrient content

of the diet. Clearance of acylcarnitine esters is often

higher than that of nonesterified L-carnitine. Experimental

110 Rebouche

studies have shown that in rats and humans, kidneys

are able to synthesize acetyl-L-carnitine from L-carnitine

and either acetoacetate or -hydroxybutyrate, and that

L-carnitine, acetyl-L-carnitine, and -butyrobetaine (also

synthesized in human kidneys) are secreted from mucosal

cells into the tubular lumen (11). Because the kinetics of

transport of these metabolites by the sodium-dependent

L-carnitine transporter are not different, the relative proportions

appearing in urine reflect not only those in the

glomerular filtrate but also those in the renal tubular epithelium

that are secreted into the lumen. Thus, under

conditions of rapid intracellular synthesis of acylcarnitine

esters or direct accumulation from the circulation, secretion

of these species will lead to a higher proportion of

acylcarnitine esters in urine compared to that in the circulation.

By inference, kidneys may be substantially involved

in the regulation of circulating acylcarnitine ester

concentrations (11).



L-Carnitine deficiency is defined biochemically as abnormally

low concentration (<20 M) of nonesterified

L-carnitine in plasma (2). A concentration ratio of acylcarnitine

esters/nonesterified L-carnitine of 0.4 or greater

in plasma is also considered abnormal. Nutritional

L-carnitine deficiency has not been shown to occur in the

absence of other mitigating factors (2).

Primary L-carnitine deficiency occurs as a result of

defects in the gene coding for the plasma membrane

L-carnitine transporter OCTN2 (16). Characteristic features

of this disease are cardiomyopathy, hypoketotic hypoglycemia,

and muscle weakness. Secondary carnitine

deficiency occurs in association with genetic diseases of

organic acid metabolism and in genetic diseases involving

defects in fatty acid oxidation. Secondary carnitine deficiency

has been described in patients with end-stage renal

disease requiring maintenance hemodialysis. Secondary

carnitine deficiency has been observed during chronic use

of drugs, including valproic acid, cisplatin, ifosfamide, zidovudine,

and pivalic acid-containing prodrugs (17–20).



L-Carnitine, acetyl-L-carnitine, and/or propionyl-Lcarnitine

may be used for replacement therapy to

restore normal carnitine concentrations and/or abnormal

nonesterified-to-esterified carnitine ratio. They may be

used as supplements to increase the carnitine load of

the body and/or increase the flux of carnitine among

compartments. In some conditions, both replacement

therapy and supplementation are appropriate. For primary

and some secondary carnitine deficiencies (see

earlier), L-carnitine is used for replacement therapy.

L-Carnitine Supplementation in Neonatal Nutrition

L-Carnitine has been described as a conditionally essential

nutrient for infants. For the last 25 years, commercial

enteral infant formula products have included L-carnitine,

where necessary, to achieve an L-carnitine concentration

similar to that in human milk (∼60 mol/L). Bovine milkbased

formulas typically contain a higher concentration

of L-carnitine than does human milk. On the other hand,

premature infant formulas, both enteral and parenteral,

typically do not include L-carnitine at the time of manufacture,

but are sometimes supplemented at the time of

use. The rationale for supplementation is twofold: Infants

utilize lipids as a primary source for energy and growth after

birth, requiring a high rate of mitochondrial oxidation,

and the concentration of L-carnitine in infant circulation

and tissues typically is lower without supplementation

than in infants fed human milk or formulas containing

carnitine. Studies in premature infants typically have focused

on the effect of L-carnitine supplements on growth

rate and morbidity, with mixed results. In one doubleblind,

placebo-controlled, randomized trial of L-carnitine

supplementation in premature infants 28 to 34 gestational

weeks of age at birth, no differences in two-week

weight gain over 8 weeks were observed between supplemented

and nonsupplemented infants (21). More recently,

in another prospective, randomized, placebo-controlled,

double-blinded study, 29 premature neonates received

carnitine supplementation (20 mg/kg body weight/day)

or placebo for up to eight weeks. Supplemented neonates

regained their birth weight more rapidly than placebo

group neonates, indicating that L-carnitine supplementation

may promote more rapid catch-up growth (22). An

extensive review of these and many more relevant studies

has been published (23). For infants expected to be on

parenteral nutrition for seven days or longer, supplementation

with L-carnitine 10 to 20 mg/kg body weight/day,

given intravenously or orally, is recommended (23).

L-Carnitine Replacement Therapy and Supplementation

in End-Stage Renal Disease

Regular L-carnitine supplementation in hemodialysis patients

can improve lipid metabolism, antioxidant status,

and anemia requiring erythropoietin and may reduce incidence

of intradialytic muscle cramps, hypotension, asthenia,

muscle weakness, and cardiomyopathy (24,25). The

recommended dosage is 50 mg/kg body weight/day, to a

maximum of 3 g/day.

L-Carnitine and Propionyl-L-Carnitine Supplementation

for Cardiac Ischemia, Congestive Heart Failure,

Cardiomyopathy, and Peripheral Arterial Disease

Experimental studies have shown L-carnitine to be an

effective antianginal agent that reduces ST-segment depression

and left ventricular end-diastolic pressure during

stress in patients with coronary artery disease (25). Cardioprotective

effects of L-carnitine have been observed following

aortocoronary bypass grafting and following acute

myocardial infarction. Carnitine administration initiated

early after acute myocardial infarction attenuated left ventricular

dilation and resulted in smaller left ventricular

volumes (25).

L-Carnitine deficiency syndromes sometimes

present with dilated cardiomyopathy and are often

effectively treated with L-carnitine (26). Thus, it was suggested

that cardiomyopathy progressing to congestive

heart failure but not associated with inherited L-carnitine

deficiency might respond to L-carnitine supplementation.

A large-scale clinical trial of L-carnitine supplementation

L-Carnitine, Acetyl-L-Carnitine, and Propionyl-L-Carnitine 111

versus placebo in 574 patients with heart failure produced

promising results with regard to improvement in maximum

duration of exercise, but other endpoints, including

death and hospital admissions during the follow-up

period, were not different between treatment groups

(26). Utility of L-carnitine supplements for congestive

heart failure not associated with inherited L-carnitine

deficiency remains debatable (26,27).

Peripheral arterial disease is a common manifestation

of atherosclerosis and is associated with reduced arterial

circulation in the lower extremities. In five large, randomized,

double-blind, placebo-controlled studies, with

long duration of therapy, and three short-duration studies,

propionyl-L-carnitine supplementation (1–3 g/day,

orally) for up to one year improved initial claudication

distance (distance walked before muscular symptoms appeared)

and absolute claudication distance (distance at

which patient stopped walking due to muscular cramps)

(28). Quality of life outcomes were also improved with

propionyl-L-carnitine treatment relative to placebo. Experimental

and clinical studies suggest that the improvements

observed in clinical trials are due to protection

by propionyl-L-carnitine from the effects of oxidative

stress and inflammation in ischemic tissue endothelium

(28), as well as due to increased blood flow resulting

from endothelium-dependent vasodilation elicited by

propionyl-L-carnitine (29).

L-Carnitine Supplementation for Exercise Performance

and Weight Reduction

L-Carnitine supplementation has been suggested to improve

exercise performance in healthy humans. Proposed

mechanisms include enhanced muscle fatty acid oxidation,

altered glucose homeostasis, enhanced acylcarnitine

production, modification of training responses, and altered

muscle fatigue resistance (30).Areview of published

studies has led to the conclusion that L-carnitine supplements

do not improve exercise performance in healthy humans

(30–33). On the other hand, in conditions where the

nonesterified L-carnitine concentration of skeletal muscle

may be significantly reduced, such as in peripheral arterial

disease and end-stage renal disease, L-carnitine supplementation

has afforded some benefit to muscle function

and exercise performance (33).

In a double-blind, placebo-controlled, crossover design

clinical trial, oral administration of 4.5 g of propionyl-

L-carnitine (as glycine propionyl-L-carnitine) to trained

athletes significantly enhanced peak power production

in resistance-trained males with significantly lower lactate

accumulation (34). The incremental differences in

power production were modest and were observed only

after repeated short-durationWingate cycle sprints. However,

it is noted that, particularly for elite athletes, modest

enhancement in performance can be highly significant

in outcomes in competitive sports. Propionyl-L-carnitine

may be more effective in enhancement of exercise performance

than L-carnitine, because of its ability to increase

nitric oxide production and vasodilation.Moreover,

propionyl-L-carnitine may enhance citric acid cycle activity

by providing propionyl units that can be converted to


Because of its role in facilitating fatty acid oxidation,

L-carnitine has been suggested to aid in weight loss

regimes. Two facts argue against this. First, there is no

evidence that it facilitates, directly or indirectly, mobilization

of fatty acids from adipose tissue. Second, in normal

humans, the intracellular concentration of L-carnitine is

not rate-limiting for transesterification of fatty acids by

CPT I. Adding an increment of L-carnitine will not increase

the rate at which this reaction occurs. There is no

scientific evidence that L-carnitine supplements facilitate

weight loss in humans.

L-Carnitine and Acetyl-L-Carnitine Replacement Therapy

and Supplementation for Chronic Fatigue

L-Carnitine may improve symptoms of fatigue in humans.

Use of the cancer chemotherapeutic agents cisplatin and

ifosfamide is associated with fatigue. In a prospective,

open-label study, improvement of symptoms of fatigue

was observed in 50 nonanemic patients following

L-carnitine supplementation to the chemotherapeutic

regimen of cisplatin or ifosfamide (35). However, in a subsequent

randomized, double-blind, placebo-controlled

trial, no improvement in measures of fatigue was observed

as a result of L-carnitine supplementation (36).

The study included an open-label phase, in which fatigue

symptoms did show some improvement with L-carnitine


Chronic fatigue syndrome (CFS) in humans was

found to be associated with low circulating acetyl-Lcarnitine

concentration and decreased accumulation in

several brain regions (37). It has been suggested that

acetyl-L-carnitine helps maintain neuronal metabolic activity

by promoting glucose and lactate uptake and utilization

through its role as a precursor of glutamate in

neurons (38). In a randomized, open-label study of 30

patients with CFS, acetyl-L-carnitine and propionyl-Lcarnitine

showed beneficial effects on fatigue and attention

concentration (39).

Acetyl-L-Carnitine Supplementation for Depression and

Cognitive Function in the Elderly

Acetyl-L-carnitine appears to have specific and perhaps

unique roles in brain metabolism. Animal studies and

in vitro experiments suggest that this agent has promise

in slowing or reversing memory and cognition decline

as well as the decline in physical performance that normally

occurs in the process of aging. In studies of the

elderly, patients with depressive syndrome scored significantly

lower on the Hamilton Rating Scale for Depression

(modified for the elderly) following supplementation with

acetyl-L-carnitine (40). Older subjects with mild mental

impairment had improved scores on cognitive performance

tests following such supplementation (41).Ametaanalysis

of the efficacy of acetyl-L-carnitine in mild cognitive

impairment and mild Alzheimer disease included

all identified double-blind, placebo-controlled, prospective,

parallel-group studies using treatment doses of 1.5

to 3.0 g/day of acetyl-L-carnitine that were conducted between

1983 and 2000 (42). This analysis showed a significant

advantage for acetyl-L-carnitine compared to placebo,

with beneficial effects observed on both clinical scales and

psychometric tests. The benefit of supplement use was

112 Rebouche

observed for three months, and it was found to increase

over time. The typical usage recommended by vendors is

1 to 3 g/day.

L-Carnitine Supplementation in Liver Dysfunction with


Hyperammonemia occurs in some inborn errors of

metabolism and as a result of drug- or toxicant-induced

hepatotoxicity. Mortality and metabolic consequences

of acute ammonium intoxication in mice are reduced

by pharmacologic administration of L-carnitine (6). The

mechanism for this effect may have two components.

L-Carnitine administration normalizes the redox state

of the brain (perhaps by increasing the availability of

-hydroxybutyrate and/or acetyl-L-carnitine to the brain),

and it increases the rate of urea synthesis in the liver,

perhaps, in part, by activation of the glucocorticoid receptor.

At least part of the protective effect is associated

with flux through the carnitine acyltransferases, as

analogs of L-carnitine that are competitive inhibitors of

carnitine acyltransferases enhance the toxicity of acute

ammonium administration (6). Thus, it has been proposed

that L-carnitine increases urea synthesis in the liver by facilitating

fatty acid entry into mitochondria, leading to increased

flux through the -oxidation pathway, an increase

of intramitochondrial reducing equivalents, and enhancement

of ATP production (6). Carnitine supplementation

may benefit individuals with hepatic dysfunction due to

inborn errors of metabolism or chemical intoxication.

L-Carnitine and Acetyl-L-Carnitine Replacement Therapy

and Supplementation in Diabetes

L-Carnitine infusion improves insulin sensitivity in

insulin-resistant diabetic patients (43). Glucose oxidation

is increased during L-carnitine administration, concurrent

with lower plasma concentration of lactate. These

observations suggest that L-carnitine activates normally

depressed pyruvate dehydrogenase activity in insulinresistant

patients (43). Intravenous administration of

acetyl-L-carnitine increases glucose disposal in Type 2 diabetic

patients (44). Such administration appears to promote

storage of glucose as glycogen, rather than increase

in glucose oxidation (44).

L-Carnitine and Acetyl-L-Carnitine Replacement Therapy

and Supplementation in HIV Infection

L-Carnitine and acetyl-L-carnitine ester concentrations are

below normal in some human immunodeficiency virus

(HIV)-infected patients undergoing antiretroviral therapy

(45). L-Carnitine administration as part of antiretroviral

therapy with either zidovudine or didanosine reduced

lymphocyte apoptosis and oxidant stress compared to the

antiretroviral regimens without L-carnitine (46).

L-Carnitine and Acetyl-L-Carnitine Supplementation in


Male Reproductive Dysfunction

L-Carnitine and/or acetyl-L-carnitine supplementation

may be beneficial in men with oligoasthenospermia, a condition

in which low sperm count is associated with low

sperm motility. Epididymal fluid contains the highest concentration

of L-carnitine in the human body. L-Carnitine is

secreted from the epithelium into epididymal plasma via

a testis-specific carnitine transporter (47). The very high

concentration of L-carnitine in epididymal fluid provides

for passive diffusion of L-carnitine into spermatozoa during

transit and maturation through the epididymis. Mature

spermatozoa acetylate L-carnitine to generate a pool

of intracellular acetyl-L-carnitine (48). In semen obtained

from 101 men with normal or abnormal spermiograms,

concentrations of L-carnitine and acetyl-L-carnitine correlated

positively with the number of spermatozoa, the

percentage of motile spermatozoa, and the percentage of

normal cells (49). A meta-analysis comparing L-carnitine

and/or acetyl-L-carnitine to placebo treatment and including

nine randomized, controlled clinical trials revealed

significant improvements in pregnancy rate, total sperm

motility, forward sperm motility, and presence of atypical

sperm cells (50). The benefits of L-carnitine and acetyl-Lcarnitine

may be due to increased mitochondrial fatty acid

oxidation, resulting in improvement in motility epididymal

sperm, as well as due to the putative antiapoptotic

effect(s) of carnitine in the testes (51).


Transient diarrhea, nausea, vomiting, abdominal cramps,

and/or “fish-odor syndrome” have been noted in rare

cases after consumption of 2 to 6 g of L-carnitine (52).


L-Carnitine is approved as a pharmaceutical by the U.S.

Food and Drug Administration for treatment of primary

systemic carnitine deficiency, as well as for acute

and chronic treatment of patients with inborn errors

of metabolism that result in secondary carnitine deficiency

(e.g., medium-chain acyl-CoA dehydrogenase deficiency,

glutaric aciduria, Type 2 diabetes, methylmalonic

aciduria, and propionic acidemia) (52). L-Carnitine is also

approved, by the U.S. Food and Drug Administration, as

a pharmaceutical for the prevention and treatment of carnitine

deficiency in patients with end-stage renal disease

who are undergoing dialysis (52).



L-Carnitine and esters of L-carnitine have a proven, essential

role in cellular fatty acid metabolism. Beyond that,

numerous other functions have been suggested by physiological

and pharmacological studies in experimental animals

and in humans. These include, but are not limited

to, antioxidant and antiapoptotic properties, a role

in membrane lipid remodeling, and modulation of gene

expression. Identification of and evidence for these putative

functions have led to hypotheses concerning a role for

L-carnitine and its esters in promotion of physiological

function (e.g., exercise performance), in prevention,

slowing, or attenuation of progressive alteration or loss

of physiological function (e.g., cognitive function in aging,

cardiac or liver dysfunction, and male reproductive

dysfunction). In some cases, establishment of a positive

role for L-carnitine supplementation through blinded,

L-Carnitine, Acetyl-L-Carnitine, and Propionyl-L-Carnitine 113

randomized clinical trials has been hampered by lack

of accessibility of definitive, quantitative endpoint(s),

and/or by the requirement for large sample sizes to detect

small but meaningful physiological or pathological

differences in a heterogeneous population. Nevertheless,

it seems likely that new data will emerge from welldesigned

clinical trials to provide definitive answers regarding

the efficacy of L-carnitine and/or its esters supplementation

to promote better health and function in the

human population.



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GlossarySuccess Chemistry Staff

Conjugated linoleic acid (CLA) consists of a group of positional

and geometric fatty acid (FA) isomers of linoleic

acid (C18:2; cis-9, cis-12 octadecadienoic acid).

CLA isomers are found naturally in ruminant meats and dairy

products due to biohydrogenation of linoleic or linolenic

acids in the rumen of these animals. Larger quantities of

CLA are chemically synthesized for use in dietary supplements

or fortified foods. Initially identified as a potential

anti carcinogen, CLA has been reported to prevent obesity,

diabetes, or atherosclerosis in different animal and

cell models, depending on the doses, isomers, and models

used. Potential mechanisms for preventing these diseases

include inducing cancer cell apoptosis, increasing

energy expenditure and delipidating adipocytes, increasing

insulin sensitivity, or reducing aortic lesions. However,

unequivocal evidence in human participants is

still lacking. Ironically, potential side effects of CLA

supplementation include chronic inflammation, insulin

resistance, and lipodystrophy. Long-term, well-controlled

clinical trials and more mechanistic studies are needed to

better understand the true potential health benefits versus

risks of consuming CLA isomers and their mechanisms

of action.


Natural Synthesis of CLA Isomers

CLA isomers are produced naturally in the rumen of ruminant

animals by fermentative bacteria Butyrivibrio fibrisolvens,

which isomerize linoleic acid into CLA isomers. A second pathway of CLA synthesis in ruminants

is in the mammary gland via -9-desaturase of trans-11, octadecanoic

acid (1). Thus, natural food sources of CLA are

dairy products including milk, cheese, butter, yogurt, and

ice cream and ruminant meats such as beef, veal, lamb, and

goat meat (2–4). The cis-9, trans-10 (9,11) isomer

(i.e., rumenic acid) is the predominating CLA isomer in

these products (∼80%), whereas the trans-10, cis-12 (10,12)

isomer represents approximately 10%. Although several

other isoforms of CLA have been identified, the 9,11 and

10,12 isomers appear to be the most biologically active

(5). Levels of CLA isomers in ruminant meats or milk can

be augmented by dietary manipulation, including feeding

cattle on fresh pasture (6) or by adding oils rich in linoleic

acid (e.g., safflower oil) or ingredients that alter biohydrogenation

of linoleic acid (e.g., ionophores) to their diet (7).

Structures of linoleic acid, cis-9, trans-11 CLA, and trans-10,

cis-12 CLA.

Chemical Synthesis of CLA Isomers

Because of the relatively low levels of CLA isomers in

naturally occurring foods that are high in fat content, the

chemical synthesis of CLA has been developed for making

supplements and for fortifying foods. CLA can be

synthesized from linoleic acid found in safflower or sunflower

oils under alkaline conditions, yielding a CLA mixture

containing approximately 40% of the 9,11 isomer and

44% of the 10,12 isomer (reviewed in Ref. 8). Commercial

preparations also contain approximately 4% to 10% trans-

9, trans-11 CLA and trans-10, trans-12 CLA, as well as trace

amounts of other isomers.

Conjugated Linoleic Acid

 CLA Content of Various Foods

Food mg/g fat Food mg/g fat

Meats/fish Dairy

  • Corned beef 6.6 Condensed milk 7.0

  • Lamb 5.8 Colby cheese 6.1

  • Fresh ground beef 4.3 Butter fat 6.1

  • Salami 4.2 Ricotta 5.6

  • Beef smoked sausage 3.8 Homogenized milk 5.5

  • Knackwurst 3.7 Cultured buttermilk 5.4

  • Smoked ham 2.9 American processed cheese 5.0

  • Veal 2.7 Mozzarella 4.9

  • Smoked turkey 2.4 Plain yogurt 4.8

  • Fresh ground turkey 2.6 Butter 4.7

  • Chicken 0.9 Sour cream 4.6

  • Pork 0.6 Cottage cheese 4.5

  • Egg yolk 0.6 Low fat yogurt 4.4

  • Salmon 0.3 2% milk 4.1

  • Vegetable oils Medium cheddar 4.1

  • Safflower oil 0.7 Ice cream 3.6

  • Sunflower oil 0.4 Parmesan 3.0

  • Peanut 0.2 Frozen yogurt 2.8

  • Sources: Based on values reported in Refs. 2–4; and the University of Wisconsin

Food Research Institute (Dr. Pariza, Director).


Human and Animal Studies

As with other long chain unsaturated fatty acids (FA)s,

CLA is absorbed primarily in the small intestine, packaged

into chylomicrons, and distributed to extrahepatic tissues

having lipoprotein lipase (LPL) activity or returned to

the liver via chylomicron remnants or other lipoproteins.

The average daily intake of CLA is approximately 152 to

212 mg for nonvegetarian women and men, respectively

(9), and human serum levels range from 10 to 70 mol/L

after supplementation (10,11).

One major discrepancy between animal and human

studies is the dose of CLA administered (i.e., equal levels

of 9,11 and 10,12 isomers—referred to as a CLA mixture),

when expressed per unit body weight. For example,

most adult human studies provide 3 to 6 g/day of

a CLA mixture, whereas rodent studies provide 0.5% to

1.5% of a CLA mixture (w/w) in the diet. When expressed

per unit of body weight, humans receive approximately

0.05 g CLA/kg body weight, whereas mice received

1.07 g CLA/kg body weight, which is 20 times the human

dose based on body weight. Thus, part of the discrepancy

in results obtained from human and animal studies

is likely due to this large difference in the dose of CLA

administered. Supplementing humans with higher, or animals

with lower, doses of CLA would address this issue.

Other discrepancies in experimental designs include using

CLA isomer mixtures versus single isomers, duration

of CLA supplementation, and the age, weight, gender, and

metabolic status of the subjects or animals.

Cell Studies

In vitro studies have been conducted in a variety of cells

types, primarily using an equal mixture of 9,11 and 10,12

CLA, or each isomer individually. Doses used in cell

studies generally range between 1 to 100 M, reflecting

the concentration found in human participants following

supplementation. Results from these studies suggest

that these isomers are readily taken up by cells. For example,

we found that 10,12 CLA is readily incorporated

into neutral and phospholipid fractions of the primary

human adipocyte cultures and reduced lipid and glucose

metabolism (12). Similar to in vivo studies, 9,11 CLA acted

more like the linoleic acid controls.


CLA Reduces Tumor Growth

Pariza’s group initially discovered that CLA isomers in

fried ground beef acted as anticarcinogens (13). Subsequently,

numerous investigators have shown that CLA

mixtures or individual isomers decrease tumor cell growth

or increase cancer cell death in in vitro and in vivo models

of mammary, gastric, or skin cancer (reviewed in Ref. 14).

For example, feeding 0.8% to 1.0% individual CLA isomers

or mixtures block the initiation or progression of chemically

induced carcinogenesis in several rodent models

(15–17). A 5 M CLA mixture prevented cell growth and

cytokine production in transformed human keratinocyte like

cells (18). Proposed anticarcinogenic mechanisms

for CLA include decreasing nuclear factor (NF) B and

cyclooxygenase (COX) activity, thereby suppressing the

levels of prostaglandin (PG)E2, an inflammatory PG that

promotes the progression of certain forms of cancer and induces

human epidermal growth factor receptor 2 (HER2)

oncogene expression (19).

CLA Induces Apoptosis of Cancer Cells

Several groups have reported that CLA isomers cause

apoptosis or programmed cell death in cancer cells (reviewed

in Ref. 11). For example, 32 to 128 M CLA mixture

prevented rat mammary cancer cell growth through

apoptosis and decreased DNA synthesis in rat mammary

cancer cells (20). Moreover, 40 to 80 M 10,12 CLA induces

apoptosis in breast cancer cells (19,21,22). Proposed

proapoptotic mechanisms of CLA include inducing atypical

endoplasmic reticulum (ER) stress, leading to caspase-

12 activation (22).

In contrast to the cell and animal studies cited in

the preceding text, a recent prospective cohort study conducted

in Sweden found no evidence to support a protective

effect of CLA consumption on the development

of breast cancer in women (23). Furthermore, some studies

show that 10,12 CLA enhances the risk of developing

certain types of cancer (24). Thus, clinical studies examining

the effects of purified CLA isomers on preventing or

treating cancer, and safety issues, are needed.


Due to the substantial rise in obesity over the past 30 years,

there is a great deal of interest in CLA as a weight loss

treatment, as it has been shown to decrease body weight

and body fat mass (BFM).

Conjugated Linoleic Acid weight loss

supplementation with a CLA mixture (i.e., 10,12 + 9,11 isomers in equal

concentrations) or the 10,12 isomer alone decreases BFM

in many animal and some human studies (reviewed in

Refs. 25 and 26). Of the two major isomers of CLA, the Martinez et al isomer is responsible for the antiobesity properties (27–31).

CLA Decreases Body Weight and Body Fat Mass

Park et al. (32) were one of the first groups to demonstrate

that CLA modulated body composition. Compared

with controls, male and female mice supplemented with a

0.5% (w/w) CLA mixture had 57% and 60% less BFM, respectively.

Since these findings, researchers have demonstrated

that CLA supplementation consistently reduces

BFM in mice, rats, and pigs.

For example, dietary supplementation with 1% (w/w) CLA mixture for 28 days

decreased body weight and periuterine white adipose tissue

(WAT) mass in C57BL/6J mice (36).

In humans, some studies show that CLA decreases

BFM and increases lean body mass (LBM), whereas others

show no such effects. For example, supplementation of 3

to 4 g/day of a CLA mixture for 24 weeks decreased BFM

and increased LBM in overweight and obese people (37).

On the other hand, supplementation of 3.76 g/day of a

CLA mixture in yogurt for 14 weeks in healthy adults had

no effect on body composition (38). Supplementation with

3.2 g/day of aCLAmixture decreased totalBFMand trunk

fat compared with placebo in overweight participants, but

not obese participants (39). These contradictory findings

among human studies may be due to the following differences

in experimental design: (i) mixed versus individual

CLA isomers, (ii) CLA dose and duration of treatment,

and (iii) gender, weight, age and metabolic status of the


These antiobesity effects of CLA do not appear to

be solely due to reductions in food intake in animals (40–

42) or humans (43,44). Several mechanisms by which CLA

decreases BFM will now be examined.

CLA Increases LBM

A recent meta-analysis of 18 human, placebo-controlled

CLA studies found that consuming a CLA mixture increased

fat-free mass (FFM) by 0.3 kg, regardless of the

duration or dose (45). When these same 18 studies were

examined for reductions in BFM, it was shown that CLA

supplementation decreased BFM by 0.05 kg/week for up

to one year (25). The average CLA mixture dose for these

studies was 3.2 g/day. Collectively, these meta-analyses

studies suggest that CLA supplementation of humans results

in a rather small but rapid increase in FFM or LBM,

and a much larger decrease in BFM over an extended period

of time. The effects of CLA on FFM orLBMin humans

mayvary depending on baseline body mass index, gender,

age, and exercise status of the participants.

Two proposed mechanisms by which CLA increases

LBM are via increasing bone or muscle mass. 10,12 CLA

supplementation for 10 weeks with a 0.5% (w/w) CLA

mixture increased bone mineral density (BMD) and muscle

mass in C57BL/6 female mice (46). CLA supplementation

has been proposed to increase BMD via increasing

osteogenic gene expression and decreasing osteoclast activity

(46,47). Furthermore, CLA supplementation alone

or with exercise increased BMD compared with control

mice (48). An alternative mechanism could be that CLA

decreases adipogenesis of pluripotent mesenchymal stem

cells (MSC) in bone marrow, and instead promotes their

commitment to become bone cells. Indeed, 10,12 CLA has

been shown to decrease the differentiation of MSC into

adipocytes and increase calcium deposition and markers

of osteoblasts (49). In contrast, 9,11 CLA increased

adipocyte differentiation and decreased osteoblast differentiation.

Consistent with these in vitro data, CLA mixture

supplementation of rats treated with corticosteroids prevented

reductions in LBM, BMD, and bone mineral content

(50). Increasing LBM is directly linked to an increase

in basal metabolic rate (BMR).

In addition to its effects on BMD, recent evidence

supports a role of CLA in increasing endurance and muscle

strength. For example, maximum swimming time until

fatigue was higher in CLA fed versus control mice

(51). Aging mice supplemented with a CLA mixture and

10,12 CLA had higher muscle weight compared with

9,11 CLA and corn oil controls (52). In addition, CLA

isomers increased levels of antioxidant enzyme activity,

ATP, and enhanced mitochondrial potential, indicating a

protective effect against age-associated muscle loss (52).

In humans, CLA increased bench-press strength in men

supplemented with 5 g/day for seven weeks who underwent

resistance training three days per week (53).

Furthermore, supplementation with CLA combined with

creatine monohydrate (C) and whey protein (P) led to

greater increases in bench-press and leg-press strength

than supplementation with C+P or P alone (54). Although

preliminary, these data suggest that CLA may enhance

exercise-induced muscle strength or prevent sarcopenia

or age-related muscle loss.

CLA Increases Energy Expenditure

CLA has been proposed to reduce adiposity by elevating

energy expenditure via increasing BMR, thermogenesis,

or lipid oxidation in animals (27,42,55). In BALB/c male

mice fed mixed isomers of CLA for six weeks, body fat

was decreased by 50% and was accompanied by increased

BMR compared with controls (42). Enhanced thermogenesis

may be associated with increased uncoupling of mitochondria

via uncoupling protein (UCP)s, which facilitate

proton transport over the inner mitochondrial membrane

thereby leading to dissipation of energy as heat instead

of ATP synthesis. UCP1 is highly expressed in brown adipose

tissue (BAT), and in WAT at lower levels. UCP3 is

expressed in muscle and in a number of other tissues,

whereas UCP2 is the form expressed at the highest level

across most tissues. Supplementation with a CLA mixture

or 10,12 CLA in rodents induced UCP2 mRNA expression

in WAT (29,56). Recently, it was demonstrated that CLA

increased mRNA and protein expression of UCP1 inWAT

(57). Similarly, CLA supplementation induced UCP gene

expression and elevated -oxidation in muscle and liver


CLA Increases Fat Oxidation

CLA has been shown to regulate the gene expression

or activity of proteins associated with FA oxidation in

adipose tissue, muscle, and liver. For example, CLA induced

the expression of carnitine palmitoyl transferase 1

(CPT1) in WAT of obese Zucker fa/fa rats (63). Additionally,

10,12 CLA increased the expression of peroxisome

proliferator-activated receptor (PPAR) coactivator-1

Conjugated Linoleic Acid 169

(PGC1) in WAT of mice (57). Consistent with these

in vivo findings, 10,12 CLA increased -oxidation in differentiating

3T3-L1 preadipocytes (64). Furthermore, 10,12

CLA treatment increased AMP kinase (AMPK) activity

and increased phospho-acetyl-CoA carboxylase (ACC)

levels in 3T3-L1 adipocytes, suggesting an increase in FA

oxidation and a decrease in FAesterification to triglyceride

(TG) (65).

In muscle, 10,12 CLA increased CPT1 expression in

hamsters fed an atherogenic diet (60). Supplementation of

a CLA mixture in high fat fed hamsters led to increased

CPT1 activity in muscle (66). A CLA mixture increased

CPT1b, UCP3, acetyl-CoA oxidase (ACO) 2, and PPAR

mRNA levels in skeletal muscle of Zucker rats (67). Consistent

with these data, 10,12 CLA increased mRNA levels

(63) and activity (68) of CPT1 in the liver. Additionally,

10,12 CLA increased hepatic peroxisomal fatty COactivity

(68), suggesting increased peroxisomal -oxidation in

addition to mitochondrial oxidation. These findings suggest

CLA may reduce adiposity through increased energy

expenditure via increased mitochondrial uncoupling and

FA oxidation in WAT, muscle, and liver.

At least one report demonstrates that CLA increases

FA oxidation in human participants (69). In this study,

overweight adults supplemented with 4 g/day of a CLA

mixture for six months had a lower respiratory quotient

(RQ), indicating an increase in FA oxidation compared

with placebo controls. However, others have shown no

effect of CLA on energy expenditure or fat oxidation in

humans (70,71). These discrepancies may be due to the

length of treatment, time period of measurement, and

time at which measurements are taken. For instance, CLA

treatment for four to eight weeks had no effect on energy

expenditure or FA oxidation, based on a 20-minute measurement

during resting and walking (70). In contrast, the

study by Close et al. (69) administered CLA for six months

and measured FA oxidation over a 24-hour period and

found that CLA increased FA oxidation and energy expenditure.

Thus, discrepancies in this area may be due to

insufficient duration of CLA treatment or measurements

of energy expenditure or FA oxidation.

CLA Decreases Adipocyte Size

Lipolysis is the process by which stored TG is mobilized,

releasing free fatty acids (FFAs) and glycerol for use by

metabolically active tissues. C57BL/6J mice fed 10,12 CLA

for three days had increased mRNA levels of hormone sensitive

lipase (HSL), a key enzyme for TG hydrolysis

(56). Consistent with these data, acute treatment withCLA

mixture or 10,12 CLA alone increased lipolysis in 3T3-L1

(32,72) and newly differentiated human adipocytes (73).

In vitro, a CLA mixture and to a greater extent 10,12 CLA

decreased TG content, adipocyte size, and lipid locule size

in adipocytes (74). Similarly, mice fed 1% CLA displayed

increased numbers of small adipocytes with a reduction in

the number of large adipocytes (75). Furthermore, a CLA

mixture reduced adipocyte size rather than cell number

in Sprague Dawley (40) and fa/fa Zucker rats (76). Thus,

CLA may reduce adipocyte size by increasing lipolysis.

CLA Decreases Adipocyte Differentiation

The conversion of preadipocytes to adipocytes involves

the activation of key transcription factors such as

PPAR and CAAT/enhancer-binding proteins (C/EBPs).

There is much evidence showing that CLA suppresses

preadipocyte differentiation in animal (77–79) and human

(12,80) preadipocytes treated with a CLA mixture or 10,12

CLA alone. 10,12 CLA treatment has been reported to decrease

the expression of PPAR, C/EBP, sterol regulatory

element-binding protein-1c (SREBP-1c), liver X receptor

(LXR), and adipocyte FA-binding protein (aP2),

thereby reducing adipogenesis and lipogenesis (12,29,79).

In rodents, supplementation of 10,12 CLA decreased

the expression of PPAR and its target genes (79,81–83).

In contrast, humans supplemented with a CLA mixture

had higher mRNA levels of PPAR in WAT, but no difference

in body weight or BFM (38). In mature, in vitrodifferentiated

primary human adipocytes or in mature

3T3-L1 adipocytes, 10,12 CLA treatment leads to a substantial

decrease in the expression and activity of PPAR

(82,83), and a decrease in PPAR target genes and lipid

content (80). This shows that 10,12 is not only able to inhibit,

but also to reverse the adipogenic process and indicates

that this may be mediated by suppression of PPAR

activity. In addition to its effect on PPAR, 10,12 CLA may

also directly impact the activity of other transcription factors

involved in adipogenesis and lipogenesis (i.e., LXR,

C/EBPs, SREBP-1c), which could contribute to CLA’s antiobesity


CLA Decreases Glucose and FA Uptake and TG Synthesis

Conversion of glucose and FAs to TG is a major function

of adipocytes. Genes involved in lipogenesis, such

as a LPL, ACC, fatty acid synthase (FAS), and stearoyl-

CoA desaturase (SCD), were decreased following supplementation

with mixed isomers of CLA or 10,12 CLA

alone (12,56,72,80). PPAR is a major activator of many

lipogenic genes including glycerol-3-phosphate dehydrogenase

(GPDH), LPL, and lipin as well as many genes encoding

lipid droplet-associated proteins, such as perilipin,

adipocyte differentiation-related protein (ADRP), and cell

death–inducing DNA fragmentation factor of apoptosislike

effector c (CIDEC) (84). Thus, the antilipogenic action

of 10,12 CLA may be explained by inhibition of PPAR activity.

In addition,CLArepression of expression of SREBP-

1 and its target genes may play an important role in delipidation.

Finally, CLA suppression of insulin signaling may

also impair insulin’s ability to activate or increase the

abundance of a number of lipogenic proteins including

LPL, ACC, FAS, SCD-1, and the insulin-dependent glucose

transporter GLUT4.

CLA Decreases Adipocyte Number

Apoptosis is another mechanism by which CLA may reduce

BFM. Apoptosis can occur through activation of the

death receptor pathway, ER stress, or the mitochondrial

pathway. A number of in vivo and in vitro studies have

reported apoptosis in adipocytes supplemented with a

CLA mixture or 10,12 CLA alone (56,64,85,86). For example,

supplementation of C57BL/6J mice with 1% (w/w)

CLA mixture reduced BFM and increased apoptosis in

WAT (75). Mice fed a high-fat diet containing 1.5% (w/w)

CLA mixture had an increased ratio of BAX, an inducer of

apoptosis relative to Bcl2, a suppressor of apoptosis (87).

170 Martinez et al.

Figure 2 Reported mechanisms by which 10,12 CLA decreases adipose

tissue mass and obesity.

Reported mechanisms by which CLA reduces adiposity

are shown in Figure 2.


Feeding obese ob/ob C57BL/6 mice 0.6% 9,11 CLA for

six weeks improved plasma levels of glucose, TG, and

insulin and reduced the expression of markers of inflammation

and insulin resistance in WAT (88). Furthermore,

these authors demonstrated that 50 M 9,11 CLA prevented

tumor necrosis factor (TNF)-mediated insulin resistance

in 3T3-L1 murine adipocytes. Their data suggest

that 9,11 CLA improves insulin sensitivity by elevating

GLUT4 levels or translocation to the plasma membrane,

which are adversely affected by inflammation, thereby

facilitating glucose disposal. Similarly, Wistar rats fed a

high-fat diet supplemented with a 0.75% to 3.0% CLA

mixture for 12 weeks had lower plasma levels of glucose,

TG, and insulin compared with high-fat fed control rats

(89). The CLA mixture enhanced the expression of PPAR

target genes in WAT, which was proposed to be responsible

for the improvement in insulin sensitivity. Consistent

with these data, adiponectin, a WAT-specific, PPAR target

gene that reduces blood glucose by enhancing its oxidation

in liver and muscle, was increased in the plasma

of Zucker diabetic fatty (ZDF) rats fed a 1% CLA mixture

for eight weeks (55). Similarly, feeding 0.5% 9,11 CLA to

insulin resistant C57BL/6J mice improved insulin sensitivity

without affecting BFM (90). Conversely, these authors

found that feeding 0.5% 10,12 CLA lowered BFM

and increased LBM in these mice, but caused insulin resistance.

Other studies have also reported that 10,12 CLA

causes insulin resistance, especially in mice (81,99). Taken

together, these data suggest that 9,11 and 10,12 CLA have

opposite effects on insulin sensitivity, most likely due

to their opposing effects on the activity of PPAR, visa-

vis 9,11 CLA activates PPAR and 10,12 CLA inhibits



CLA has been reported to decrease risk factors of

atherosclerosis in several important animal models (reviewed

in Ref. 91). For example, feeding 0.5% mixed or

individual isomers of CLA to New Zealand White rabbits

fed a high saturated fat and cholesterol-rich diet reduced

blood lipids and atherosclerotic lesion area (92). Syrian

Golden hamsters fed a high saturated fat and cholesterolrich

diet containing 1.0% mixed CLA isomers (93), 0.9%

9,11 CLA (94) or 1.0% 10,12 CLA (95), had decreased aortic

lipid accumulation or fewer fatty aortic streaks compared

with controls. In apoE−/− deficient mice, feeding a 1.0%

CLA mixture decreased aortic lesion area, and reduced

macrophage infiltration and inflammatory gene expression

in the lesions (96). In contrast to these animal studies,

other animal and clinical trials with CLA mixtures have

yet to show beneficial effects on reducing risk factors for

atherosclerosis (reviewed in Ref. 97).


Adverse side effects have been reported for CLA supplementation

such as elevated levels of inflammatory

markers, lipodystrophy, steatosis, and insulin resistance.

Most adverse side effects are due to the 10,12 CLA


CLA Increases Markers of Inflammation

Treatment with 10,12 CLA increases the expression or

secretion of inflammatory makers such as TNF, interleukin

(IL)-1, IL-6, and IL-8 from adipocyte cultures

(56,73,80,81,83). Moreover, CLA increases the expression

of COX-2, an enzyme involved in the synthesis of PGs,

and the secretion of PGF2 (79,98). These inflammatory

proteins are known to antagonize PPAR activity and insulin

sensitivity (87,98–100).

Consistent with these in vitro data, 10,12 CLA

supplementation increases the levels of inflammatory

cytokines and PGs in humans (101,102). For example,

women supplemented with 5.5 g/day of a CLA mixture

for 16 weeks had higher levels of C-reactive protein

in serum and 8-iso-PGF2 in urine (44). 10,12 CLA

supplementation in mice resulted in macrophage recruitment

in WAT (81). In contrast, 9,11 CLA exhibits antiinflammatory

actions (6).

CLA Causes Insulin Resistance

Insulin resistance has been reported in vivo (56,102–104)

and in vitro (12,73,79,98) following supplementation with

a CLA mixture or 10,12 CLA alone. For example, 10,12

CLA supplementation of 3.4 g/day for 12 weeks in obese

men with metabolic syndrome increased serum glucose

and insulin levels and decreased insulin sensitivity (103).

Supplementation with a CLA mixture in type-2 diabetics

increased fasting plasma glucose levels and reduced

insulin sensitivity (102). Mice fed 1% (w/w) 10,12 CLA

displayed elevated fasted and feeding plasma insulin

levels and had reduced insulin sensitivity (75). Consistent

with these data, the mRNA levels of adiponectin,

a key adipokine associated with insulin sensitivity, decrease

following supplementation with 10,12 CLA in vivo

(36,81,100) and in vitro (79,82,105,106).

CLA Causes Lipodystrophy

The combination of inflammation and insulin resistance

results in reduced FA and glucose uptake in WAT,

leading to ectopic lipid accumulation in the blood (hyperlipidemia),

liver (steatosis), or muscle. CLA-mediated

hyperlipidemia and steatosis has been reported in several

animal studies (36,76,107). For example, 1% (w/w)

CLA time-dependently increased insulin levels and led

Conjugated Linoleic Acid 171

Figure 3 Reported mechanisms by which CLA reduces the risk of cancer,

obesity, diabetes, and atherosclerosis.

to increased liver weight and liver lipid accumulation in

C57BL/6J mice (36). Aging C57BL/6J mice fed 0.5% 10,12

CLA displayed increased insulin resistance and liver hypertrophy


US Regulatory Status

Recently, the FDA approved CLA as GRAS (generally recognized

as safe) for use in foods and beverages (not to

exceed 1.5 g/serving) due its potential favorable effects.

However, the use ofCLAas a dietary supplement or ingredient

should be cautioned based on the aforementioned

safety issues.


There is an abundance of evidence in animals suggesting

that CLA consumption may reduce the incidence or risk

of developing cancer, obesity, diabetes, or atherosclerosis,

depending on the type and abundance of CLAisomer consumed

and the physiological status of the animal model

(Fig. 3). Data on the antiobesity properties of 10,12 CLA

in animals, especially mice, are the most reproducible.

However, these potential benefits are not without risks,

as the 10,12 isomer is associated with increased levels of

inflammatory markers, lipodystrophy, and insulin resistance.

More clinical studies are needed to determine the

efficacy of CLA isomers in humans, and more mechanistic

animal and cell studies are needed to determine the precise,

isomer-specific mechanisms of action of CLA, and

potential side effects.


This work was supported by NIH NIDDK R15 DK 059289,

NIH NIDDK/ODS R01DK063070, USDA-NRI 199903513,

and NCARS 06771 awards to Michael McIntosh, NRSA

NIH Fellowships to Kristina Martinez (F31DK084812)

and Arion Kennedy (F31DK076208), and a United Negro

College Fund-Merck predoctoral Fellowship to Arion



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Glossary, sports nutritionSuccess Chemistry Staff

Creatine (Cr)—methylguanidino acetic acid is a naturally

occurring compound that was first described by

Chevreul in 1832. Its name is derived from the Greek

word kreas (flesh).

Creatine is found in abundance in skeletal muscle (red meat) and fish.

It is essential in energy transmission and storage via creatine kinase (CK). The

daily Cr dosage is obtained by both endogenous synthesis

and via nutritional intake, followed by absorption in

the intestine (1). Creatine supplementation is widespread

among sportspersons because of its documented and/or

presumed ergogenic effects (2–4). In addition, supplementation

with Cr has proven to be instrumental for the treatment

of rare inborn errors of metabolism due to defects in

Cr biosynthesis enzymes (5–8).

Creatine is stored in high concentrations in skeletal

and heart muscles and to a lesser extent in the

brain. It exists in both free and phosphorylated form

[phosphocreatine (PCr)] and is important for maintaining

high ratios between adenosine triphosphate (ATP) and

adenosine diphosphate (ADP). Upon increases in workload,

ATP hydrolysis is initially buffered by PCr via the

CKreaction. During high-intensity exercise, PCr in muscle

is depleted within several seconds. Whether de novo Cr

biosynthesis occurs in the brain or whether Cr is taken up

into the brain through the blood–brain barrier, is currently

a matter of debate.


Patients with Cr deficiency syndromes (CDS), that is, patients

with a Cr biosynthesis defect or a Cr transporter

defect, have developmental delay and mental retardation

(MR), indicating that Cr is crucial for proper brain function.

Surprisingly, however, CDS patients do not suffer

from muscular or heart problems. Those with a Cr biosynthesis

defect, in contrast to Cr transporter-deficient subjects,

can partly restore their Cr pool in brain upon Cr

treatment (5–10).

Creatine supplementation, due to its ergogenic effects,

has become a multimillion dollar business (3). In the

Western world, Cr has received wide public interest. A

simple search on “creatine” in the World Wide Web using

common database search engines results in more than

500,000 entries. Besides the use by sportspersons, Cr supplementation

is explored in several animal models of neuromuscular

disease (i.e., Huntington and Parkinson disease,

amyotrophic lateral sclerosis) and in human disease

(3,6,11,12). A recent study suggests that Cr supplementation

increases intelligence and memory performance tasks


The goal of this entry is to provide an overview on Cr

and its metabolism in health and disease. The functions of

Cr and PCr, Cr biosynthesis, its degradation, tissue distribution,

transport and molecular aspects, as well as the benefits

and risks of Cr supplementation are discussed. (For

in-depth reviews, see Refs. 2, 3, 6 and references therein.)


Creatine Structure

Creatine is a naturally occurring guanidino compound.

Its chemical structure is depicted in Figure 1. Creatine is a

hydrophilic, polar molecule. Phosphocreatine is zwitterionic,

with negatively charged phosphate and carboxylate

groups and a positively charged guanidino group.


Creatine Synthesis Biosynthesis

The transfer of the amidino group of arginine to glycine

yielding L-ornithine and guanidinoacetic acid (GAA) represents

the first step in the biosynthesis of Cr and is performed

by L-arginine:glycine amidinotransferase (AGAT;

EC This reaction is reversible and occurs in mitochondria,

into which arginine has to be taken up for guanidinoacetate

biosynthesis. The human AGAT mRNA encodes

a 423-amino acid polypeptide including a 37-amino

acid mitochondrial targeting sequence. The AGAT gene is

located on chromosome 15q15.3, is approximately 17 kb

long, and consists of 9 exons.

The second step involves the methylation of GAA

at the amidino group by (S)-adenosyl-L-methionine:Nguanidinoacetate

methyltransferase (GAMT; EC,

whereby Cr is formed. The methyl group is provided

by (S)-adenosylmethionine. The human GAMT mRNA

encodes a 236-amino acid polypeptide. The gene is located

on chromosome 19p13.3, is approximately 12 kb long, and

consists of 6 exons.

Chemical synthesis

Creatine is produced by chemical synthesis, mostly from

sarcosine and cyanamide. This reaction is prone to generation

of contaminants such as dicyandiamide, dihydrotriazines,

or Crn (14). Some manufacturers may fail to separate

these contaminants from Cr. The toxicological profiles

of these contaminants are often not known. Dicyandiamide

liberates hydrocyanic acid (HCN) when exposed

to strongly acidic conditions (such as in the stomach). For

human consumption, only pure preparations of Cr should

thus be allowed. Unfortunately, no generally accepted and

Schematic representation of the creatine kinase (CK) reaction,

and chemical structures of creatine (Cr) and phosphocreatine (PCr).

meaningful quality labels are yet in place that would allow

a consumer to judge the origin and quality of Cr in

a given commercial product. Moreover, for most studies

published so far, it is not possible to correlate the presence

or lack of ergogenic, preventive, or adverse side effects

with the quality of the many Cr preparations used.

Creatine Function (CK Reaction)

Creatine is involved in ATP regeneration via the CK reaction.

The phosphate group of PCr is transferred to ADP

to yield Cr and ATP, the “universal energy currency” in

all living cells. The CK reaction serves as an energy and

pH buffer and has a transport/shuttle function for high energy


Several CK subunits exist that are expressed in a

tissue- and/or spatial-specific manner. In mammals, four

CK isoforms exist: the cytosolic M-CK (M for muscle) and

B-CK (B for brain) subunits form dimeric molecules, that

is, the MM-, MB-, and BB-CK isoenzymes. The two mitochondrial

CK isoforms, ubiquitous Mi-CK and sarcomeric

Mi-CK, are located in the mitochondrial intermembrane

space and form both homodimeric and homo-octameric

interconvertible molecules.

In fast-twitch skeletal muscles, a sizeable pool of PCr

is available for immediate regeneration of ATP, which is

hydrolyzed during short periods of intense work. In these

muscles, the cytosolic CK activity is high and “buffers”

the cytosolic phosphorylation potential that seems to be

crucial for the proper functioning of a variety of reactions

driven by ATP. Slow-twitch skeletal muscles, the heart,

and spermatozoa depend on a more continuous delivery

of high-energy phosphates to the sites of ATP utilization.

In these tissues, distinct CK isoenzymes are associated

with sites of ATP production (e.g., Mi-CK in the mitochondrial

intermembrane space) and ATP consumption

[e.g., cytosolic CK bound to the myofibrillar M line, the

sarcoplasmic reticulum , or the plasma membrane] and

fulfill the function of a “transport device” for high-energy

phosphates. The -phosphate group of ATP, synthesized

within the mitochondrial matrix, is transferred by Mi-CK

in the mitochondrial intermembrane space to Cr to yield

ADP and PCr. ADP may directly be transported back to

the matrix where it is phosphorylated to ATP. Phosphocreatine

leaves the mitochondria and diffuses through the

cytosol to the sites of ATP consumption. There, cytosolic

CK isoenzymes locally regenerate ATP and thus warrant

a high phosphorylation potential in the vicinity of the respective

ATPases. Subsequently, Cr diffuses back to the

mitochondria, thereby closing the cycle. According to this

hypothesis, transport of high-energy phosphates between

sites ofATP production andATP consumption is achieved

mainly by PCr and Cr. The CK system is required to allow

most efficient high-energy phosphate transport, especially

if diffusion of adenine nucleotides across the outer mitochondrial

membrane is limited.


Tissue Distribution of Creatine and of Its Biosynthesis Enzymes

In a 70-kg man, the total body creatine pool amounts to

approximately 120 g (1). Creatine and PCr are found in

tissues with high and fluctuating energy demands such

as skeletal muscle, heart, brain, spermatozoa, and retina.

In skeletal and cardiac muscle, approximately 95% of the

total bodily Cr is stored, and the concentration of total

creatine may reach up to 35 mM. Intermediate levels are

present in brain, brown adipose tissue, intestine, seminal

vesicles and fluid, endothelial cells, and macrophages.

Low levels are found in lung, spleen, kidney, liver, white

adipose tissue, blood cells, and serum (25–100 M) (2).

Until recently, GAA biosynthesis was presumed to

occur mainly in the kidney (and pancreas), where AGAT

is highly expressed, followed by its transport via the blood

and uptake of GAA into the liver, the presumed major site

of the second reaction, the methylation of GAA by GAMT.

Current knowledge suggests that AGAT and GAMT expression

is not limited to these organs. Synthesis outside

of these organs may allow local supply of Cr (e.g., in brain;

see creatine biosynthesis in mammalian brain) and may,

to a minor extent, contribute to the total Cr content in the


Creatine Accumulation: Transporter-Mediated

Creatine Uptake

Cellular transport is of fundamental importance for creatine

homeostasis in tissues devoid of Cr biosynthesis. Creatine

needs to be taken up against a steep concentration

gradient [muscle (mM), serum (M)]. The Cr transporter

gene (SLC6A8) (MIM300036) has been mapped to chromosome

Xq28. Northern blots indicated that this gene is expressed

in most tissues, with the highest levels in skeletal

muscle and kidney, and somewhat lower levels in colon,

brain, heart, testis, and prostate. The SLC6A8 gene product

is a member of a superfamily of proteins, which includes

the Na+-dependent and Cl−-dependent transporters responsible

for uptake of certain neurotransmitters. The Cr

transporter gene spans approximately 8.4 kb, consists of

13 exons, and encodes a protein of 635-amino acids.

Creatine/Creatinine Clearance

Creatine can be cleared from the blood via either uptake

into different organs by the Cr transporter or by excretion

via the kidney. There is evidence that tissue uptake

204 Salomons et al.

of Cr may be influenced by carbohydrates, insulin, caffeine,

and exercise and that transporter molecules located

in kidney are able to reabsorb Cr. Nevertheless, Cr is found

under normal conditions in urine in various amounts. The

main route for clearance of Cr is via creatinine excretion.

Creatine and PCr are nonenzymatically converted to creatinine.

The rate of creatinine formation, which mainly

occurs intracellularly, is almost constant (∼1.7% per day

of the Cr pool). Because muscle is the major site of creatinine

production, the rate of creatinine formation is mostly

a reflection of the total muscle mass. Creatinine enters the

circulation most likely by passive transport or diffusion

through the plasma membrane, followed by filtration in

kidney glomeruli and excretion in urine.

Creatine Deficiency Syndromes

Both AGAT and GAMT deficiencies are autosomal recessive

inborn errors of metabolism. This is in contrast to

the third disorder of Cr metabolism, which is an X-linked

inborn error due to a defect in the Cr transporter (Table 1).

GAMT Deficiency

The first inborn error of Cr biosynthesis,GAMTdeficiency

(MIM601240), was identified in 1994. The absence of a

Cr signal in the proton magnetic resonance spectroscopy

(1H-MR) spectrum of brain, the low amounts of urinary

creatinine, and the increased levels of GAA in plasma and

urine led to the diagnosis of this disease. In addition to creatinine,

Cr is also reduced in body fluids. Clinical symptoms

are usually noted within the first eights months of

life. Possibly Cr is provided in high amounts in utero via

the umbilical cord and in newborns via the mother’s milk,

thereby delaying the clinical signs. All patients identified

so far have developmental delay, MR to various degrees,

expressive speech and language delay, epilepsy, autistiform

behavior, and very mild-to-severe involuntary extrapyramidal

movements. The disorder has a highly heterogeneous

presentation, varying from very mild signs to

severe MR, accompanied by self-injurious behavior.

AGAT Deficiency

In 2001, the first family with AGAT deficiency

(MIM602360) was identified. The two sisters, four and six

years old presented with MR, developmental delay from

the age of eight months, and speech delay. GAMT deficiency

was ruled out because GAA was not increased in

urine and plasma. Creatine supplementation (400 mg/kg

body weight per day) increased the Cr content in the

brain to 40% and 80% of controls within three and nine

months, respectively. A homozygous nonsense mutation

in the AGAT gene, predicting a truncated dysfunctional

enzyme, was finally identified. Lymphoblasts and fibroblasts

of the patients indicated impaired AGAT activity. A

third related patient was identified with similar clinical

presentation. The biochemical hints to detect this disorder

are reduced levels ofGAA(and creatinine) in plasma, cerebrospinal

fluid (CSF) and possibly urine, together with

reduced undetectable levels of Cr in the brain.

SLC6A8 Deficiency (Creatine Transporter Deficiency)

Like AGAT deficiency, the X-linked Cr transporter defect

was unraveled in 2001. An X-linked Cr transporter

(MIM300352) defect was presumed because of: (i) the

absence of Cr in the brain as indicated by proton magnetic

resonance spectroscopy (MRS); (ii) elevated Cr levels in

urine and normal GAA levels in plasma, ruling out a Cr

biosynthesis defect; (iii) the absence of an improvement

on Cr supplementation; and (iv) the fact that the pedigree

suggested an X-linked disease. The hypothesis was

proven by the presence of a hemizygous nonsense mutation

in the male index patient and by impaired Cr uptake

by cultured fibroblasts. The hallmarks of this disorder are

MR, expressive speech and language delay, epilepsy, developmental

delay, and autistiform behavior.

Unfavorable skewed X-inactivation is likely the cause of the difference

in severity of the clinical signs in females.

Intriguing Questions Linked to CDS

Does a Muscle-Specific Creatine Transporter Exist?

It is noteworthy that the SLC6A8-deficient patients do not

seem to suffer from muscle and/or cardiac failure. This

could indicate sufficient endogenous Cr biosynthesis in

muscle. Alternatively, Cr uptake is taken over by other

transporters, or a yet unknown Cr transporter exists that

is specifically expressed in skeletal and cardiac muscle.

Creatine Biosynthesis in Mammalian Brain

It is a matter of debate whether Cr biosynthesis occurs in

mammalian brain. The following findings suggest that it

actually does: (i) In rat brain, AGAT and GAMT mRNA

and protein were detected (16), (ii) The Cr content in brain

of mice treated with guanidinopropionic acid, an inhibitor

of the Cr transporter, was—in contrast to muscle tissues—

hardly decreased. (iii) In contrast to skeletal muscle, Cr

supplementation in AGAT- and GAMT-deficient patients

requires months to result in an increment in Cr concentration

in the brain. These findings make it unlikely that the

brain is entirely dependent on Cr biosynthesis in the liver

or on its nutritional intake, followed by transport through

the blood–brain barrier into the brain.

However, why do Cr transporter deficient patients

also reveal Cr deficiency in the brain? One explanation

could be that Cr synthesis in the brain, although present,

is too low to be relevant physiologically. Alternatively, the

expression of AGAT and GAMT may be separated spatially

(i.e., AGAT and GAMT molecules may be found

in the same or different cell types, but may not be expressed

in one and the same cell). This is in line with

data of Braissant et al. (17) showing such spatial separation

in rat brain at both the mRNA and protein level.

These findings suggest thatGAAneeds to be taken up into

the appropriate cells prior to GAA methylation, which in

case of the transporter defect is not feasible. This would

explain the incapability to synthesize Cr in the brain of

SLC6A8-deficient patients. Clearly, more thorough investigations

are needed to study these discrepancies toward

a better understanding of Cr metabolism in the human


Significance of CDS/relevance for Health Care

Mental retardation occurs at a frequency of 2% to 3% in

the Western population. In 25% of MR cases, a genetic

cause is suspected, of which Down syndrome and fragile

X syndrome are the most common. Mutations in the

SLC6A8 gene may be, together with other X-linked MR

genes, partly responsible for the skewed ratio in sex distribution

in MR, autism, and individuals with learning

disabilities. SLC6A8 deficiency appears to be a relatively

common cause of X-linked MR, though not as common as

fragile X. Creatine biosynthesis defects may be less common.

Because the damage incurred in these three diseases

is irreversible to a large part and an effective treatment

is available at least for the Cr biosynthesis defects, early

diagnosis of these patients is highly important.

To date, the clinical phenotype appears to be nonspecific

and suggests that allMRpatients should be tested

in diagnostic centers by 1H-MRS, metabolite screening,

and/or sequence analysis of the SLC6A8 gene. In the case

of X-linked MR or X-linked autism due to a genetic, but

unknown, cause, the parents are confronted with a risk of

recurrence (50% chance that the mother passes the mutant

allele on to her child). The diagnosis of SLC6A8 deficiency

or a Cr biosynthesis defect allows prenatal diagnosis for

subsequent pregnancies.

Creatine Supplementation/Therapeutic Use

Creatine Sources

Creatine is present in high amounts in meat

(4.5 g/kg in beef, 5 g/kg in pork) and fish (10 g/kg in herring, 4.5 g/kg

in salmon), which are the main exogenous Cr sources in

the human diet. Low amounts of Cr can be found in milk

(0.1 g/kg) and cranberries (0.02 g/kg) (17). As discussed

earlier, Cr is also synthesized endogenously, which supplies

around 50% of the daily requirement of approximately

2 g. This suggests that in vegetarians, who have a

low intake of Cr, the bodily Cr content is reduced, unless

its endogenous biosynthesis is largely increased. Indeed,

in vegetarians, the Cr concentration in muscle biopsies

was reported to be reduced (18).

Dosing as an Ergogenic Aid

Creatine can be obtained as nutritional supplement in the

form of various over-the-counter creatine monohydrate

products, which are supplied by many manufacturers.

Commercial Cr is chemically produced. The majority of

consumers are sportspersons, due to Cr’s documented

and/or presumed ergogenic and muscle mass increasing

effects. Usually, a loading phase of five to seven days of

20 g/day (in four portions of 5 g) is recommended, followed

by a maintenance phase with 3–5 g Cr per day.


Benefits in Sportspersons

Creatine supplementation is common among cyclists,

mountain bikers, rowers, ski jumpers and tennis, handball,

football, rugby, and ice hockey players.


While there is a large body of evidence supporting the ergogenic effects

of Cr in high-intensity, intermittent exercise, the situation

is more controversial in sports involving single bouts of

high-intensity exercise, such as sprint running or swimming

(2,19). In endurance exercise, there is currently no

reason to believe that Cr supplementation has any benefit.

There is a widespread contention that Cr supplementation,

by accelerating recovery between exercise bouts, may

allow more intensive training sessions. Similarly, supplementation

seems to enhance recovery after injury.

In most studies, a significant weight gain has been

noted upon Cr supplementation. The underlying basis for

this weight gain is still not entirely clear, and may be due

to stimulation of muscle protein synthesis or increased

water retention. The proportion of fat tends to decrease.

Most likely, the increase in body weight reflects a corresponding

increase in actual muscle mass and/or volume.

Therefore, it is not surprising that Cr use is popular among

206 Salomons et al.

bodybuilders and wrestlers. On the other hand, in masssensitive

sports like swimming and running, weight gain

due to Cr supplementation may impede the performance,

or may at least counteract the ergogenic effects of Cr.

Creatine supplementation may improve muscle performance,

especially during high-intensity, intermittent

exercise, in four different ways by: (i) increasing PCr

stores, which is the most important energy source for immediate

regeneration of ATP in the first few seconds of

intense exercise; (ii) accelerating PCr resynthesis during

recovery periods; (iii) depressing the degradation of adenine

nucleotides and possibly also the accumulation of

lactate; and (iv) enhancing glycogen storage in skeletal



Benefits in Neuromuscular Disease

Besides its ergogenic effects, supplementary Cr has a neuroprotective

function in several animal models of neurological

disease, such as Huntington disease, Parkinson

disease, and amyotrophic lateral sclerosis (ALS) (2,3,6,11).

The rationale could be that these disorders, due to different

causes, hamper cellular energy metabolism in the

brain. In animal studies, Cr also protected against hypoxic

and hypoxic-ischemic events. Therefore, Cr may be

useful in the treatment of a number of diseases, for example,

mitochondrial disorders, neuromuscular diseases,

myopathies, and cardiopathies. Currently, the first clinical

studies with Cr supplementation in neuromuscular

disease are emerging. In two studies on patients with mitochondrial

myopathies or other neuromuscular diseases,

Tarnopolsky’s group showed increased muscle strength

upon Cr supplementation (11). A randomized, doubleblind,

placebo-controlled trial to determine the efficacy

of creatine supplementation did not show a significant

beneficial effect on survival and disease progression in a

group of 175 ALS patients. These data are in contrast to

what was suggested from animal models of ALS and tissue

specimens of ALS patients (12). Studies on single subjects

and small groups of neuromuscular disease patients

have been reported to show both the presence and absence

of beneficial effects of Cr supplementation. Recent publications

on Cr supplementation in Huntington disease

showed difficulty in proving the effect of Cr on the deterioration

of cognitive function (20,21). In Duchenne muscular

dystrophy, enhanced muscle strength upon treatment

was shown; whereas, for example, in myotonic dystrophy

type 2/proximal myotonic myopathy, no significant

results were seen (22,23). Future studies with enough statistical

power are warranted to unravel the relevance of

Cr supplementation in these disorders. Clinical trials of

patients with ALS, Parkinson, and other neurological diseases

are currently ongoing (

Benefits in Creatine Biosynthesis Disorders

Oral supplementation with 350 mg to 2 g/kg body weight

per day has been used in patients with GAMT and AGAT

deficiencies. In these patients, the Cr concentration in

their brains increased over a period of several months (5).

In GAMT deficiency, the GAA concentration in plasma,

urine, and CSF decreased with Cr supplementation, but

still remained highly elevated. Guanidinoacetic acid was

found to be toxic in animals and may be partly responsible

for some of the clinical signs (i.e., involuntary extrapyramidal

movements). Combination therapy of Cr plus

ornithine supplementation with protein (arginine) restriction

reduced GAA in CSF, plasma, and urine, and almost

completely suppressed epileptic seizures (7). In general,

all patients with a Cr biosynthesis defect who were treated

with Cr alone or in combination therapy showed improvements.

Clearly, younger patients will experience the

largest benefits, because less irreversible damage is to be

expected. However, even older patients showed remarkable

improvements (7).


Adverse Effects

Weight gain is the only consistent side effect reported.

Gastrointestinal distress, muscle cramps, dehydration,

and heat intolerance have been reported repeatedly.

Most of these complaints may be due to water retention

in muscle during the loading phase of Cr supplementation.

Although a causal relationship with fluid

intake has not been proven yet, subjects should take

care to hydrate properly to prevent these side effects.

The French Agency of Medical Security of Food

( released a

statement in January 2001 that the health risk associated

with oral Cr supplementation is not sufficiently evaluated,

and that Cr may be a potential carcinogen. Because

at present there is no scientific basis for the assertion (both

Cr and Cr analogs were actually reported to display anticancer

activity), this in turn has resulted in a wave of

protest from suppliers and defenders of oral Cr supplementation.

In fact, based on the current scientific knowledge

in healthy individuals, Cr supplementation at the

recommended dosages (see dosing as an ergogenic aid)

should be considered safe. Unfortunately, almost nothing

is known about the use of Cr in pregnancy, nor are

appropriate studies in children available. Furthermore, a

potential health hazard is the possible presence of contaminants

in some commercial Cr preparations (see chemical



Oral Cr supplementation is known or presumed to have

a number of favorable effects. For example, it prevents or

ameliorates clinical symptoms associated with inherited

Cr biosynthesis defects, it may protect against neurological

and atherosclerotic disease, (2,6) and it increases sports

performance, particularly in high-intensity, intermittent

exercise. Despite widespread use of Cr as an ergogenic aid

and the significant public interest, the majority of studies

on the properties, metabolism, and function of Cr have

focused on physiological questions rather than on pharmacokinetics.

As yet, the pharmacokinetics is difficult to

interpret due to different (and incomplete) study designs.

Currently, therefore, it is not adequately known whether

Cr supplementation causes any long-term harmful effects.

Some precaution is warranted based on the fact that the

daily recommended dosage for ergogenic effects (i.e., 20 g

during the loading phase, 3–5 g during the maintenance

phase) cannot be met by normal food intake.