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
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
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
A leading role for arginine has been shown in the
endocrine system, vasculature, and immune response.
CO2 + NH4
2ADP + Piz
AMP + Ppi + H2O
Figure 1 L-Arginine and Krebs cycle in the renal tubule.
22 Maccario et al.
Urea cycle Glutamine Glutamate
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-
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.
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).
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
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
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.
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
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).
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
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
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
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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