Citrulline Pharmacological Perspectives

REV IEW

ART ICLE

Citrulline: pharmacological perspectives andits role as an emerging biomarker in future

Shilpa N. Kaorea*, Hanmant S. Amanea, Navinchandra M. KaorebaDepartment of Pharmacology, Peoples College of Medical Sciences, Bhanpur Road, Bhopal, Madhya Pradesh,

462037, IndiabDepartment of Microbiology, Peoples College of Medical Sciences, Bhanpur Road, Bhopal, Madhya Pradesh, 462037,

India

Keywords

biomarker,

citrulline,

nitric oxide,

rheumatoid,

T cell,

vasodilatation

Received 17 February 2012;

revised 18 May 2012;

accepted 8 June 2012

*Correspondence and reprints:

[email protected]

ABSTRACT

L-citrulline is a naturally occurring non-essential amino acid, an intermediate in

urea cycle and conditionally essential in intestinal pathology. It is a potent hydro-

xyl radical scavenger and much more effective precursor of arginine and nitric

oxide (NO) than arginine itself so exploited in therapeutics. Plasma citrulline con-

centration is used by clinicians to assess functional enterocyte mass in various

chronic and acute small bowel pathologies like short bowel syndrome that has

become an indication in clinical practice. Its supplementation is likely to be used

in conditions like erectile dysfunction, sickle cell anemia, short bowel syndrome (to

restore nitrogen balance), hyperlipidemia, cancer chemotherapy, hypercholestre-

mia, in hyperoxic lung damage, urea cycle disorders, Alzheimers disease, multi-

infarct dementia and as an immunomodulator. Its emerging role as a biomarker in

intestinal pathology and early diagnosis of Rheumatoid arthritis has spread consid-

erable interest. Antibody detection to Anti-cyclic citrullinated peptide (ACCP) anti-

bodies can be recommended for early detection of RA decreasing joint damage and

deformity, because these are detected well before the onset of disease manifesta-

tions of RA. The test is highly specific than RF (Rheumatoid factor), with moderate

sensitivity, but much useful in differentiating RA from other disorders. Further

studies and exploration is required in these areas.

INTRODUCT ION

L-citrulline is a naturally occurring non-essential amino

acid, present in mammals and also in each living

organism. It is produced by the body naturally and

also found in some foods like watermelons, cucum-

bers, pumpkins, muskmelons, bitter melons, squashes,

gourds. Its basic function is detoxification of ammonia

via conversion to urea. Citrullines name is actually

derived from the Latin word for watermelonCitrullusvulgarisbecause it was first isolated from it in 1930[1,2], unusually rich in citrulline (CIT) [3]. It may

also function as a potent hydroxyl radical scavenger

[4].

Citrulline, made from ornithine and carbamoyl phos-

phate, is a component of the urea cycle in the liver [5].

CIT is synthesized from arginine (ARG) and glutamine

in enterocytes. CIT entering into kidney, vascular endo-

thelium, and other tissues can be readily converted to

ARG and nitric oxide (NO) [6], and this ARG is made

available for utilization by peripheral tissues. Citrulline

is formed mainly in eneterocytes from glutamine, liver

contributing to minimal [7]. About 80% of CIT is con-

verted in the kidney to ARG [7,8], finally converting

ARG to CIT and NO and serving as a potent ARG pre-

cursor [911]. This is carried out via argininosuccinatesynthase (ASS) to l-argininosuccinate and subsequently

to ARG by argininosuccinate lyase (ASL) [12]. Evi-

dence suggest argininosuccinate pathway in pig [1315], converts CIT to ARG in cerebral vascular endothe-

lium, which is a constant, neuronal source of NO for

inducing cerebral vasodilatation [16]. CIT has very spe-

cific metabolism, it is not metabolized in liver/intestine

[6]. Hence, CIT is essential to make ARG, in turn, ARG

2012 The Authors Fundamental and Clinical Pharmacology 2012 Societe Francaise de Pharmacologie et de TherapeutiqueFundamental & Clinical Pharmacology 27 (2013) 3550 35

doi: 10.1111/j.1472-8206.2012.01059.x

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is needed to produce NO, that acts as an endogenous

vasodilator [17].

Nitric oxide is synthesized from ARG by tetrahydrobi-

opterin (BH4)-dependent NO synthase [18], dictating a

vital role of ARG in maintaining health and treating a

wide array of chronic diseases. NO improves endothe-

lial function by elevating cyclic guanosine monophos-

phate (cGMP) [19]. Decreased NO production and

endothelial dysfunction associated with ageing is corre-

lated with decreased NOS activity in elderly and with

progressive endothelial dysfunction [20].

There is growing evidence for the role of NO, regu-

lating insulin sensitivity, oxidation of energy substrates,

a key role in immune response [21], neurological func-

tion and hemodynamics in animals and humans [22].

NO released from endothelium is an important regula-

tor of vascular tone [23] inhibitor of platelet and leuco-

cyte aggregation and adhesion [23,24], as well as

inhibitor of cell proliferation [25,26], so plays a crucial

role in regulation of vascular disease undoubtedly.

However, as ARG supplementation was not as effective

and reliable as that of CIT, CIT may be a promising

and more successful therapy. Thus, supplementation

with CIT is a novel therapeutic approach in conditions

of ARG and NO deficiency [27].

Moreover, the other side of the coin is that lower lev-

els of NO protect cells from apoptosis, while excessive

NO is toxic [28] and may lead to apoptosis [29]. This

is because NO causes endoplasmic reticulum (ER) stress

that induces a transcription factor, CAAT/enhancer

binding protein (C/EBP), homologous protein (CHOP),

and leads to apoptosis [29]. So, ER calcium stores can

be the future targets of NO, and ER stress pathway is

an important mechanism of NO-mediated apoptosis in

various cells, including macrophages, microglia, and

pancreatic beta cells [29].

Previous clinical studies show ARG to improve flow-

mediated vasodilation (FMD) and thus endothelial func-

tion because of normalization of ARG/ADMA ratio

[20], where asymmetric dimethylarginine inhibition

(ADMA) is as an endogenous inhibitor of NO synthase

[30]. But it is now realized that both lack as well as

excess of NO production can have very important

implications in health and disease [27]. Hence, inhibi-

tion of excessive NO production is also a promising

future therapeutic target. Another essential AA, Gluta-

mine (GLn) inhibits nitric oxidergic neurovascular

transmission by undetermined mechanisms [31].

Citrulline is released by small intestine continuously,

taken up by kidney and metabolized into ARG, while

no uptake by liver protects from hepatic degradation of

CIT. Another means of sustaining protein homeostasis

is ARGCITARG cycle in kidney. Thus, plasma CITmay be a good marker of intestinal failure in short

bowel syndrome (SBS) where a decreased level of CIT is

directly proportional to severity of intestinal disease or

functional intestinal mass [32]. So, CIT may be a con-

ditionally essential amino acid (AA) in stress [33], or

when intestinal function is compromised [32]. This

notion is proved in SBS in rats where CIT is able to

restore nitrogen balance, increase ARG levels and

increase muscle protein content as well as muscle pro-

tein synthesis (+90%) in elderly malnourished rats[32]. Moreover, it also has a potential as a supplement

for total parenteral nutrition (TPN) in SBS patients

because it additionally prevents muscle atrophy too,

not seen with ARG supplementation [34].

Discovery of CIT as ARG and NO precursor, and the

close link between ARG and CIT, has urged consider-

able interest and is being explored further. Thus, CIT

supplementation can be an alternative to ARG supple-

mentation for clinical use [35], and even watermelon

can be used as a substitute for supplementing CIT to

increase plasma levels of ARG [2]. Hence, recently CIT

has raised tremendous interest in scientific community,

which was previously considered to be only a compo-

nent/intermediate of urea cycle [9].

The aim of this review is to summarize current

knowledge and future aspects in therapeutics and to

define its role as a biomarker. The values of plasma cit-

rulline can aid in diagnosis and severity of intestinal

failure and, possibly, for monitoring the bowel func-

tion. Its role in early diagnosis of rheumatoid arthritis

is emerging as a promising biomarker that needs fur-

ther exploration.

PHARMACOKINET ICS OF ORALC ITRULL INE

Citrulline supplementation shows significant increase

in plasma ARG and augmentation of NO-dependent

signalling mechanisms in a dose-dependent manner.

Oral CIT, increased AUC and C (max) of plasma ARG

concentration dose-dependently more effectively than

ARG supplementation (P < 0.01). The highest dose ofcitrulline (3 g bid) increased the C (min) of plasma

ARG and improved ARG/ADMA ratio significantly from

baseline (P < 0.01, 95% confidence interval (CI) 66,121). Moreover, urinary nitrate and cGMP were

increased significantly creatinine (P = 0.01, 95% CI 8,

2012 The Authors Fundamental and Clinical Pharmacology 2012 Societe Francaise de Pharmacologie et de TherapeutiqueFundamental & Clinical Pharmacology 27 (2013) 3550

36 S.N. Kaore & H.S. Amane

58) and from 38 3.3 to 50 6.7 nmol/mmol(1)creatinine (P = 0.04, 95% CI 0.4, 24), respectively.However, it failed to demonstrate improvement in flow-

mediated vasodilatation (FMD) over baseline, which is

in contradiction to pooled analysis of all FMD data that

documents a correlation between increased ARG/

ADMA ratio and improvement in FMD [36].

Citrulline is formed mostly in enterocytes; maximum

citrulline (80%) is converted in the kidney to ARG [7].

Chronic renal insufficiency results in decreased uptake

of CIT and release of ARG that are reduced by 6070%, and the study depicts linear relationship between

CIT extraction and ARG release in subjects with nor-

mal renal function [37]. Decreases in citrulline levels

are clinically relevant as they reflect a decrease in

functional mass of enterocytes [7,38]. CIT ingestion

decreases QT interval in healthy subjects which means

it shortens the time required for completion of myocar-

dial depolarization and repolarization [39], thus CIT

may be associated with cardiovascular risks [40].

ADVERSE DRUG REACT IONS

Short-term administration of CIT is safe and well toler-

ated. No adverse effects were observed in healthy sub-

jects that given different loading doses (2, 5, 10, or

15 g CIT) [9]. Further, CIT levels increased in plasma

but not in ARG with the highest dose of CIT that may

be due to saturation of renal conversion of CIT to ARG

[9]. Watermelon-induced citrullinemia may occur as

a result of consumption of large quantities of water-

melon, which comprises of elevated plasma citrulline

and, to a lesser extent, ARG, in the absence of orotic

or arginosuccinicaciduria or hyperammonemia. So,

physicians and laboratory persons should be aware of

this phenomenon and is also important in manage-

ment of urea cycle and related disorders [41]. There is

dearth of reliable scientific data regarding its use in

pregnancy and lactation.

DRUG INTERACT IONS

Currently no authentic scientific documentation

regarding the drug interactions was found.

PHARMACODYNAMICS

Studies with arginine

Arginine supplementation is found to reduce glucose

in chemically induced diabetic rats [4244], ZDF rats

(Zucker diabetic fatty rat) [45], reduce excess fat in

ZDF rats and patients with diabetes [46]and show

improvement in vascular reactivity in diabetes [47]

and hypercholesterolemic subjects [48]. No beneficial

effect was seen in terms of increase NO availability

and improving athletic performance in humans [49].

However, studies on ARG supplementation docu-

ments for improvement in NO production and is thus

helpful in CV diseases associated with endothelial

dysfunction such as hypertension, heart failure, athero-

sclerosis [50], diabetic vascular disease, and ischemia-

reperfusion injury, but the effects are not seen on

chronic ARG supplementation, so CIT supplementation

holds promise in this regard [6]. ARG in long term

improves hepatic and peripheral sensitivity to insulin

in type 2 diabetes (T2DM) patients [51].

To conclude, ARG supplementation has failed to pro-

vide consistent results in clinical trials reasons being

ARG metabolism in liver, ARG toxicity and role of

cofactor tetrahydrobiopterin in NO production and its

relation with ARG availability [27], all contributing to

arginine paradox [52]. In addition, oral ARG supple-

mentation is rendered ineffective because of extensive

presystemic and systemic elimination due to gut bacte-

ria and hepatic and gut arginase activity [19,36,53],

while CIT is subjected to systemic metabolism only and

not to presystemic one [19]. Additionally, ARG

enhance arginase expression and activity both, thus

reducing effectiveness of ARG, in contrast, and CIT not

only inhibits arginase activity but also does not induce

tissue arginase [6].

Arginine and glutamine studies

As discussed, GLn interferes with CIT-mediated NO pro-

duction [54], similarly found to modulate (increase)

the effects of ARG too. GLn supplementation is found

to reduce morbidity and mortality in critically ill

patients fed parenterally in selected patients [33]. GLn

regulates gut barrier function, antioxidant status and

immune-inflammatory response [33], improve nitrogen

balance so could be added to total parenteral nutrition

[55]. There is now an international consensus to

recommend GLN supplementation (0.20.4 g/kg/day ofL-glutamine) in critically ill patients receiving paren-

teral nutrition [56].

Arginine and GLn are non-essential AA, but both

become conditionally essential AA in stress with their

metabolism closely related [33,55]. GLn is an essential

AA and a possible precursor of ARG and thus for NO

synthesis in murine macrophages, and its consumption


Citrulline and its future perspectives 37

in sepsis probably represents NO production [57,58].

The simultaneous administration of ARG and GLn

resulted in additive or synergistic effects on gut barrier

function and inflammatory response, but the effects in

critically ill are not documented [33]. Thus, both needs

to be further explored and documented for beneficial/

detrimental effects. ARG and GLn consumption also

increases in response to high cholesterol diet, thus they

may have pharmacological implications in hypercholes-

terolemia [59].

Citrulline more efficacious than arginine

Citrulline is an effective substitute to restore NO pro-

duction in situations of limited ARG availability [54].

Studies in healthy volunteers support that oral CIT

supplementation raises blood ARG levels more effec-

tively than ARG supplementation itself [60]. Acute oral

CIT administration more efficiently raises the plasma

ARG levels vs. long-term ARG supplementation [6],

increasing plasma ARG levels to about 227% with

3.8 g CIT(within 4 h) compared to only 90% rise with

equivalent dose of ARG [6,61]. These facts make CIT, a

better substitute for ARG and NO supplementation,

than ARG itself [6,8].

Citrulline and studies on watermelon

Watermelon is found to accumulate CIT, ARG, and

glutamate in its leaves under drought condition [4].

Chronic consumption of watermelon increases plasma

ARG and NO concentration of in healthy subjects

[2,62], and thus watermelon pomace juice reduces

cardiovascular (CV) risk factors, improves glycemic con-

trol, ameliorates vascular dysfunction in obese animals

with type 2 diabetes mellitus (T2DM) [62] and in

genetic obese model of NIDDM (Zucker diabetic fatty,

i.e., ZDF rat) [62]. Results demonstrate increased plasma

ARG levels by CIT intake from watermelon [2,41].

Additionally, high sensitivity of ZDF rats to circulat-

ing ARG establishes a useful model to identify nutri-

tional treatments for metabolic syndrome in obesity

and diabetes [62]. The increased serum levels of both

ARG and NO in ZDF rats suggest ARG synthesis from

CIT and the subsequent conversion of ARG to NO [62].

STUDIES ON CITRULL INE

Citrulline in sickle cell anemia

Citrulline (oral) may be given in sickle cell disease as

ARG precursor required as a substrate in the argi-

nine-nitric oxide pathway for endogenous nitrovasodi-

lation and vasoprotection. The ARG-induced

vasoprotection is partly mediated by NO-induced inhi-

bition of endothelial damage and inhibition of adhe-

sion and activation of leukocytes, so is valuable in

palliative therapy of sickle cell disease (SCD) for which

it has cleared Phase II trials, decreasing complications

and increasing overall well-being of these patients

[10]. Further studies are indicated in this area

(Boxes 1 and 2; Figure 1).

Box 1

Citrulline natural sources

Sources Watermelon (Citrullis vulgaris), cucumbers,pumpkins, muskmelons, bitter melons, squashes,

gourds.

Chronic consumption:o improves glycemic control in NIDDM.o improves vascular dysfunction.

Consumption of large quantities of Watermelon resultsin Watermelon-induced citrullinemia.

Plants accumulate citrulline in leaves in drought condi-tions that works as antioxidant and helps to combat

stress, as in Kalahari desert.

Box 2

Citrulline supplementation major effects

A non-essential amino acid, donor of arginine & NO,and conditionally essential amino acid in intestinal

pathology.

Major pharmacological actions:o Antioxidanto Vasodilatationo Decrease leucocyte migrationo Restore nitrogen balance & increase muscle protein

content as well as muscle protein synthesis

o Improves endothelial dysfunctiono Increased arginosuccinate synthase (ASS) expres-

sion in cancer cells

o Preserve anti-inflammatory mediator response (insepsis)

Citrulline and cardiovascular effects

Impaired endothelial NO production is because of the

reduction in nitric oxide synthase (NOS), therefore,

increasing ARG availability either by ARG therapy or

by arginase inhibition may provide benefits in future in

hypertension [27].

Nitric oxide synthase exists in three isoforms: neuro-

nal NOS (nNOS, NOS 1), inducible NOS (iNOS, NOS 2),



and endothelial (eNOS, NOS 3) that convert ARG

to NO and CIT [63]. In addition, ADMA has very low

circulating levels in humans and is found to be an

endogenous inhibitor of all three isoforms of NOS [30].

The ARG/ADMA ratio is one of the determinants of NO

production [64], and NO activates soluble guanylyl

cyclase (sGC) in smooth muscles leading to increase

in intracellular cGMP causing vasodilation [65]; this

Figure 1 Schematic representation of synthesis of citrulline.



process being essential for endothelial function and dis-

turbed NO production in the human endothelium attri-

butes to endothelial dysfunction [63,63,66]. Under

ischemic conditions, NO is synthesized from nitrite, in

acidic conditions, via non-enzymatic pathway [67]

mainly in tissues [68].

Studies to analyze the cardiovascular effects with

CIT and ARG supplementation show improvement in

right ventricular function (RVF) by increasing RVF

ejection fraction [69], and probably decreasing sys-

tolic pulmonary artery pressure because of beneficial

effects on endothelial function [70] in heart failure

patients with preserved ejection fraction. CIT is a

safer way of delivering ARG for endothelial and

immune cells as well as can prevent excessive uncon-

trolled NO production [71] that exerts deleterious

effects of its own.

CIT sustainedrelease in cardiovascular diseasesFurther evaluation of newer formulation of CIT,

sustained release (SR) CIT, suggests SR to have more

sustained levels of CIT and ARG compared to immedi-

ate release CIT throughout the experimental study,

thus favouring SR use of SR formulation for beneficial

effects in cardiovascular diseases and atherosclerosis

[72]. This may further help in extending its beneficial

action to other clinical conditions too.

Citrulline in hypertension

Observations state that hypertensive patients had lower

arginine-to-citrulline ratio than normotensive patients

[26]. A known fact is that hypertension is often associ-

ated with NO deficiency, oxidative stress, increased

reactive oxygen species, and a disturbed constrictor-

dilator balance in the kidney [73]. Recently, melatonin

is found to exert antihypertensive effect in young spon-

taneously hypertensive rats (SHRs) owing to restora-

tion of the NO pathway by reduction in plasma ADMA,

preservation of renal ARG availability, attenuation of

oxidative stress [74]. This may be due to the defects in

the renal citrullinearginine pathway or ARG reabsorp-tion potentially reduce renal NO in prehypertensive

SHR [75]. It was also observed that if NO availability is

increased perinatally, it reduces blood pressure, so CIT

supplementation in SHR resulted in increased renal NO

significantly [75]. In addition, CIT supplementation

overcomes the early renal NO deficiency and its sequels

in male and female SHRs [75]. A recent study in rats

further evaluated the effect of maternal caloric restric-

tion in offspring and found less number of nephrons

and renal dysfunction and also developed hypertension

that were reversed with maternal CIT supplementation

with long-term antihypertensive effects [76]. Future

studies are required to support this notion.

Citrulline in hyperlipidemia

A recent analysis evaluated the role of CIT, and ARG

in hypercholesterolemic rats shows the reversal of

increased serum levels of aspartate aminotransferase

(AST), alanine aminotransferase (ALT), urea with con-

comitant increase in high density cholesterol (HDL-c)

and NO. The data indicate CIT as well as ARG good

efficacy as hypocholesterolemic and hypolipidemic

agents in rats [77].

Citrulline in diabetes

In addition to reduction in glucose levels, CIT may ben-

efit the underlying endothelial dysfunction [7881] indiabetes. In metabolic syndrome, one of the hallmarks

is decreased endothelial synthesis and bioavailability of

NO [47]. Previous studies demonstrate the activation of

the argininecitrulline cycle in macrophages in anautoimmune condition, increasing ARG synthesis from

CIT, sustaining increase in NO production, and proba-

bly related to autoimmune destruction of pancreatic

beta-cells in insulin-dependent diabetes mellitus (IDDM)

[82]. In IDDM, the destruction of beta-cells, which are

very much vulnerable to NO-induced apoptosis, is

found to be due to excessive NO production leading to

apoptosis [29], while lower levels of NO do not appar-

ently cause severe damage in mouse beta-cells [83].

A study on healthy volunteers revealed that CIT

may reduce NO-mediated pancreatic secretion or

increased insulin clearance, and does not elevate

plasma insulin in response to exercise in patients who

also received CIT supplementation [84]. But a recent

study contradicts to this, where citrulline maleate (CM)

supplementation significantly increased the insulin lev-

els in response to exercise returning to basal levels at

recovery [85].

Watermelon juice is shown to have beneficial effects

by increase in ARG availability and improve glycemic

control and vascular dysfunction in T2DM [62].

Citrulline in erectile dysfunction

In single blind, short-term study in men with erectile

dysfunction (ED) (erection hardness score of 3) L-citrul-

line was given 1.5 g/day for a month, which con-

cluded it to be a safe and psychologically well accepted

and reported as very satisfying by patients, although



less effective than phosphodiesterase type-5 enzyme

inhibitors, with improvement in erection hardness

score from 3 to 4 [86]. So, it may prove effective in

mild-to-moderate ED that requires further research in

this area.

Citrulline as immunomodulator

Citrulline production is significantly reduced in sepsis

because of diminished de novo conversion of ARG

and NO, reducing CIT and ARG availability in septic

patients and transgenic mice [8,27], in part due to

ADMA release [87]. Higher ADMA levels are corre-

lated with higher mortality rate in sepsis [87,88]. In

sepsis, there is a complex interplay between pro- and

anti-inflammatory cytokines and endothelial NO may

be a key factor in regulating this response. Further

observations found increased proteolysis in sepsis but

decreased ARG levels suggesting inadequate secondary

synthesis as a result of decreased levels of CIT too

[87].

L-citrulline (CIT) supplementation seems to be a valu-

able means of supplementing ARG, favourably

increases NO availability, and correlates with alteration

of the systemic response of mediators and cytokines

with varied degrees of sepsis. CIT preserved the anti-

inflammatory mediator response, decreased proinflam-

matory mediator response (IL-6 and resistin) without

impairing the secretion of anti-inflammatory mediators

(IL-10 and adiponectin), thus acting as an immuno-

modulator [52]. So, to conclude, CIT may be a safe

means of immunomodulation that preserves the anti-

inflammatory mediator response.

Citrulline: in arginase associated T-celldysfunction

Interestingly, T lymphocytes are found to depend on

ARG for their proliferation, zeta-chain peptide forma-

tion, T-cell receptor complex expression and develop-

ment of memory [89,90]. This makes obvious that

T-cell abnormalities like decreased proliferation and loss

of zeta chain are observed in cancer, after trauma [89]

and may provide new insights into T-cell dysfunction

[90].

Citrulline is found to have the potential as a substi-

tute for arginase-associated T-cell dysfunction. The

basis for this is the molecular capability of T cells to

increase CIT membrane transport and up-regulating

ASS expression and thus converting CIT to ARG in

invitro studies, thus escaping the ill effects of ARG

depletion [91]. Hence, CIT supplementation can pre-

serve T-cell proliferation and prevent the loss of CD3

zeta chain under conditions of low ARG [91]. So, new

drug targets as well as new insights may be explored

in future.

Citrulline: role in treatment for cancer

Decreased ASS expression is correlated with decreased

sensitivity of tumour cells, CIT is found to increase this

sensitivity because of concomitant increase in AS

expression. So, combination therapy of CIT supplemen-

tation with human recombinant arginase, that is,

HuArgI (Co)-PEG5000 (HuArgI) is selectively cytotoxic

to a fraction of human cancer cell lines in tissue cul-

ture, including some melanomas, mesotheliomas, acute

myeloid leukemias, hepatocellular carcinomas, pan-

creas adenocarcinomas, prostate adenocarcinomas,

lung adenocarcinomas, osteosarcomas, and small-cell

lung carcinomas. Unfortunately subsets of normal

human tissues are also found to be sensitive to HuArgI

with CIT supplementation, including umbilical endo-

thelial cells, bronchial epithelium, neurons, and renal

epithelial cells. Further, these invitro findings suggest

the combination therapy to be a therapeutic agent for

some ASS-deficient tumors [92].

Citrulline: role in dementia of Alzheimers diseaseand in multi-infarct dementia

The cerebrospinal fluid (CSF) levels of various amino

acids were examined in dementia, with the conclusion

that the CSF/serum levels of CIT were significantly

higher in comparison with control group in multi-

infarct dementia [93]. This is supported further by

elevated NOS activity found in microvessels of brain in

Alzheimers disease (AD), raising vascular NO produc-

tion, exerting neurotoxicity, and is responsible for sus-

ceptibility to neuronal injury and cell death in AD

[94]. Some studies contradict this notion and suggest

decreased NO production [95,96], to play a role in

neurodegenerative disorders.

Asymmetric dimethylarginine inhibition, which is

hydrolyzed by dimethylarginine dimethylaminohydro-

lase enzyme into CIT and dimethylamine, is also found

elevated specifically in neurons displaying cytoskeletal

abnormalities and oxidative stress in AD, but not in

early stages of AD [97]. So, as found in experimental

studies, pharmacological intervention using NO donors

and/or NO suppressors could delay or at least minimize

brain lesion development and further progression of

brain pathology and dementia [98,99]. Significantly

elevated CIT levels were found in multiple system atro-



phy [95]. Further research may prove or disprove the

use of CIT modulators in brain lesions in humans.

Citrulline: can it improve aerobic function/energyproduction?

Observations suggest high-intensity exercises increasing

ammonia levels in skeletal muscles [], ammonia in turn

activates phosphofructokinase and prevents oxidation

of pyruvate to acetyl CoA, thus leading to exhaustion

and exercise-induced fatigue [100,101]. Further, the

urea cycle being responsible for detoxification of ammo-

nia [102], while skeletal muscle buffers ammonia via

transamination reactions [103]. Experimental studies

prove that ARG, CIT, and ornithine supplementation

suppressed the accumulation of ammonia after exercise

and prolongs the time taken for exhaustion [103]. So,

there is possibility of improvement in treadmill time

with CIT, but in contrast, in humans, prior CIT inges-

tion was actually found to reduce it and became

exhausted [84].

A recent study in cyclists, also analysed the effect of

prior administration of CM on exercise, concluded that

there was a significant rise in plasma levels of CIT,

ARG, ornithine, urea, creatinine, and nitrite and signif-

icantly decreased the isoleucine concentration from

basal measures to after exercise, that is, essential

amino acids (AA) show significant decrease while non-

essential AA show significant rise after exercise. To

add, citrulline maleate (CM) can increase the produc-

tion of branched chain AA in response to exercise [85].

CM ingestion significantly reduced fatigue sensation,

because of increase in (by 34%) the rate of oxidative

ATP production during exercise and increase (20%) in

the rate of phosphocreatine recovery after exercise in a

clinical trial, suggesting larger contribution of oxidative

ATP synthesis to energy production [104]. This

improves aerobic energy production in muscles and

exercise tolerance, significantly enhancing aerobic per-

formance and prolongation of onset of muscular fati-

gue. CM also limits muscle fatigue or skeletal muscle

dysfunction induced by bacterial endotoxins [105,106].

Recent studies documented on effect on CIT supple-

mentation in healthy animals found to have ergogenic

effect associated with improvement in muscular con-

traction efficiency in healthy rats [107], and improve-

ment in athletic performance in mice may be probably

due to facilitation of detoxification of ammonia via the

urea cycle and inhibition of additional glycolysis [102].

A recent human trial supports the notion, where CM

single dose (8 g) increased athletic performance in

high-intensity anaerobic exercises with short rest times

and relieved postexercise muscle soreness that supports

athletes undergoing intensive preparation may gain

profits of CIT ingestion [108]. Thus, CIT may be used

as an agent to increase exercise capacity for various

reasons.

Citrulline for urea cycle disorders

Citrulline may be used for the detection of inborn

errors of metabolism of urea cycle, responsible for

removal of ammonia, that are fortunately quite rare.

The first step in the urea cycle is the reaction of ammo-

nia and bicarbonate using adenosine triphosphate

(ATP) and catalyzed by the enzyme carbamoyl phos-

phate synthetase (CPS) to produce carbamoyl phos-

phate. The second step is the reaction of carbamoyl

phosphate and L-ornithine to produce L-citrulline, cata-

lyzed by the enzyme ornithine transcarbamylase (OTC).

It is interesting to note that deficiencies of either of

these two enzymes lead to low serum levels of L-citrul-

line and are treated with oral L-citrulline. In the long-

term management, diet treatment is also a part of

management and treatment must be individualized

[109].

Citrulline: an antioxidant?

Citrulline in plants protects from stress situations, like

conditions of drought. A unique phenomenon

observed in watermelon is massive accumulations

amount of CIT in its leaves, making its photosynthetic

apparatus functional even in prolonged drought and

strong sunlight. Hence, CIT concentration in water-

melon, in Kalahari Desert, is higher. Under stressful

conditions, reactive oxygen species, including hydroxyl

radicals, increases oxidative damage to plants affecting

their nucleic acids, proteins, carbohydrates, and lipids

[3].

Studies suggest potent radical scavenging action of

CIT [4], affording protection from oxidative injuries

[4,110,111] and maintenance of water status [111].

This notion is supported by a study in healthy cyclists,

where prior CIT supplementation (6 g) reduced oxida-

tive damage by priming of polymorphonuclear neu-

trophils for oxidative burst without evidence of

oxidative damage and progressive decrease in reactive

oxygen species (ROS) production and markers of oxida-

tive damage [112]. Therefore, it is postulated that CIT,

probably a potent hydroxyl scavenger, tends to protect

DNA and metabolic enzymes against oxidative stress,

which needs further exploration.



Citrulline in intestinal pathology

Citrulline is easily taken up by gut by help of transport-

ers and is then taken up by enterocytes, and it has

been confirmed by pharmacokinetic studies (in rats)

that citrulline supplementation is found to be more effi-

cient than with ARG supplementation to provide ARG

[113]. CIT is found to increase protein synthesis during

refeeding in rodents with short bowel syndrome, aging,

malnutrition, and improves nitrogen balance in fed

healthy humans [114]. CIT pre-treatment improves

integrity of gut barrier, and preservation of ileum

mucosa was observed in mice thus reducing bacterial

translocation [115], similar effects are seen with ARG

too [116]. So CIT, rather than ARG is the limiting

amino acid in situations of intestinal failure which is

proved in rat model of SBS, since CIT supplementation

improved protein synthesis and ARG availability [71].

These studies suggest that CIT have a strong poten-

tial for total parenteral nutrition after massive intesti-

nal resection (SBS) [34] that cause increase in ARG

pool and restores nitrogen balance [117], whereas

long-term supplementation of ARG intraduodenally in

rats failed to show beneficial results in intestinal ische-

mia and reperfusion injury [118].

C ITRULL INE AS A B IOMARKER

Citrulline as a biomarker in intestinal disease

Serum citrulline is emerging as an innovative biomar-

ker candidate for the assessment of intestinal function

[119]. As CIT is not involved in protein synthesis or

nutrition product, it is now an established biomarker of

enterocyte functional metabolic mass in adults and

children [120,121]. It can assess the remnant length

of small bowel in intestinal diseases like short bowel,

extensive enteropathies, intestinal toxicity of chemo-

therapy and radiotherapy [120]. Normal plasma citrul-

line levels range between 30 and 50 lM independent ofnutritional status [120]. The levels

charge in ARG residues that alters the antigenicity of

self-proteins leading to autoimmunity and chronic

inflammation [131].

It is now revealed that autoantibodies in RA contain-

ing the unusual AA CIT are specifically found in serum

of RA patients [128]. Ab against citrullinated proteins/

peptides (filaggrin) [132], that is, detection of Anti-

cyclic citrullinatedpeptide (ACCP) antibodies are highly

specific [131,133137] biomarker in RA [130], otherbiomarkers for RA being rheumatic factor (RF) and anti-

perinuclear factor (APF) and anti-keratin antibodies

(AKA) [128,136]. Moreover, the frequency of false-posi-

tive results is lower with ACCP antibody detection [134]

than with RF [138], thus this modern biomarker of best

diagnostic value [139] needs further evaluation [140],

for early detection and preventing joint damage and

deformity. Further, it has similar diagnostic value in

patients with established or long duration disease, and is

a reliable marker of severe erosive disease and in RF

seronegative cases [139]. Another related biomarker,

equally sensitive as ACCP antibodies [136,141], that

needs to be analyzed further is antibodies to modified

citrullinized vimentin (AMCV), which is significantly

correlated with early detection of RA [140], and sub-

clinical atherosclerosis in RA [141].

As these CIT-containing antibodies (Ab) are detected

early, are specific, and detected well before the appear-

ance of other manifestations of the disease [128], that

may also indicate its role in the autoimmune response

[133] and pathogenesis [130,131]. Citrullinated anti-

gens (Ag) are expressed in inflamed joint and can be

detected early before the disease manifests [131].

ACCP-ELISA assays suggest diagnostic sensitivities

between 69.6% and 77.5% and specificities between

87.8% and 96.4% [142]. To conclude, estimation of

ACCP antibodies is highly specific [143] but not abso-

lutely specific in RA [135], can be exploited as a bio-

marker for RA, and be detected early in the course of

disease [128]. This may improve the prognostic value

in RA patients [140] and show new insights into etiol-

ogy, and pathogenesis of disease may be revealed

[128,129].

However, it also raises a future query whether ACCP

antibodies have better prognostic significance than RF

[144], will it really replace RF [145]? A recent study

has raised doubts because it failed to improve the per-

formance of American College of Rheumatology (ACR)

1987 criteria in diagnosing early RA in combination

with the detection of ACCP antibody [146]. Future

studies may have an answer to this, or the strategy

may be to use RF in combination with ACCP antibod-

ies for definitive diagnosis of RA [147], but the concur-

rent detection of ACCPAb and RF will surely increase

the chances of differentiating RA from other diseases

[132,148]. Further data collection may answer the

question and that whether the ACCP titers might prove

as predictors of efficacy of anti-TNF therapy [148].

Other effects of citrulline

Effect on proteins and hormones

In healthy subjects, effects on plasma amino acids and

hormonal patterns show that CIT, ornithine (ORN) and

ARG plasma concentrations were affected according to

CIT dose, while plasma insulin and growth hormone

were unaffected [9]. A recent double-blind study in

healthy volunteers did not show any correlation of CIT

supplementation with changes in protein metabolism

[114].

As discussed previously, citrulline increases protein

synthesis, during refeeding in rodents with SBS, aging

and malnutrition, and improves nitrogen balance in

fed healthy humans; its effect on proteins and hor-

mones was evaluated [114]. Data indicated that CIT

supplementation is able to restore nitrogen balance,

generate large amounts of ARG in rats with SBS and

increase muscle protein content (+20%) as well as

muscle protein synthesis (+90%) in elderly malnour-

ished rats [32, 113]. So, it may be concluded that CIT

increases ARG and ornithine concentrations [114] and

plays a crucial role in maintaining protein hemostasis,

improving muscle mass related to malnutrition [113]

and further understanding of molecular mechanisms

may help in developing new strategies in malnourished

patients with compromised intestinal functions [32].

Effects of amino acids on hair strength

Human studies report considerable amount of ARG

deposition on or in hair fibres from coloring agents

[150], while decreased amounts of ARG and CIT were

found in damaged hairs in users of Relaxers and

decreased CIT has been associated with inflammation

[151]. Study reports indicate that when coloring

agents were partially replaced with ARG, it decreased

the oxidative change in tensile strength of hair by pre-

venting the undesirable attack by hydrogen peroxide

on hair proteins and hair surface lipids [150]. So, pro-

spective studies need to be undertaken to know

whether or how relaxers induce inflammation [151]

and whether ARG or CIT can substantially reduce hair

damage and fragility.



CONCLUS ION

L-citrulline, a naturally occurring non-essential amino

acid, found in plants and animals has scope beyond

amino acid. Apart from its role in protein homeostasis,

and as an intermediate in urea cycle, L-citrulline is

useful in detecting inborn errors of urea cycle, also

found to be a potent hydroxyl radical scavenger and

much more effective precursor of ARG and NO than

ARG itself. CIT supplementation is likely to be highly

exploited in therapeutic conditions like erectile dysfunc-

tion, sickle cell anemia, SBS, hyperlipidemia, cancer

chemotherapy, sustained release preparation in cardio-

vascular diseases, and to increase protein content in

malnourished as well as elderly patients. Recently, its

role as a biomarker has been realized in intestinal

pathology, which may also be used for monitoring

patients with SBS receiving parenteral nutrition.

Recently, anti-cyclic citrullinated peptide (ACCP) anti-

bodies, ACCP-ELISA, can be recommended for early

detection of rheumatoid arthritis (RA) decreasing joint

damage and deformity as these are detected well before

the onset of manifestations of RA with high specificity

and moderate sensitivity much useful in differentiating

RA from other disorders. But future studies are

required for further analysis of biomarker as a sole

diagnostic entity. Human studies have supported that

CIT supplementation reduces oxidative stress after

cycling, so may be utilized as an antioxidant in

future. CIT alters the systemic response of mediators

and cytokines in sepsis and acts as an immunomod-

ulator. Moreover, multiple system atrophy is associ-

ated with elevated CIT levels, so role of CIT

modulators can be explored in certain brain lesions.

Further studies are needed to explore the pleotropic

effects of CIT supplementation in different therapeutic

conditions.

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