<<
nutrients
Review
L-Citrulline Supplementation: Impact on
Cardiometabolic Health
Timothy D. Allerton
1
, David N. Proctor
2
, Jacqueline M. Stephens
1
, Tammy R. Dugas
3
,
Guillaume Spielmann
1,4
and Brian A. Irving
1,4,
*
ID
1
Pennington Biomedical Research Center, Baton Rouge, LA 70808, USA; Timothy.Allerton@pbrc.edu (T.D.A.);
jsteph1@lsu.edu (J.M.S.); gspielmann@lsu.edu (G.S.)
2
Department of Kinesiology, Pennsylvania State University, University Park, PA 16802, USA; dnp3@psu.edu
3
Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University,
Baton Rouge, LA 70803, USA; tammydugas@lsu.edu
4
Department of Kinesiology, Louisiana State University, Baton Rouge, LA 70803, USA
* Correspondence: brianairving@lsu.edu; Tel.: +1-225-578-7179; Fax: 225-578-3680
Received: 20 June 2018; Accepted: 16 July 2018; Published: 19 July 2018
Abstract:
Diminished bioavailability of nitric oxide (NO), the gaseous signaling molecule involved in
the regulation of numerous vital biological functions, contributes to the development and progression
of multiple age- and lifestyle-related diseases. While L-arginine is the precursor for the synthesis
of NO by endothelial-nitric oxide synthase (eNOS), oral L-arginine supplementation is largely
ineffective at increasing NO synthesis and/or bioavailability for a variety of reasons. L-citrulline,
found in high concentrations in watermelon, is a neutral alpha-amino acid formed by enzymes
in the mitochondria that also serves as a substrate for recycling L-arginine. Unlike L-arginine,
L-citrulline is not quantitatively extracted from the gastrointestinal tract (i.e., enterocytes) or
liver and its supplementation is therefore more effective at increasing L-arginine levels and NO
synthesis. Supplementation with L-citrulline has shown promise as a blood pressure lowering
intervention (both resting and stress-induced) in adults with pre-/hypertension, with pre-clinical
(animal) evidence for atherogenic-endothelial protection. Preliminary evidence is also available
for L-citrulline-induced benefits to muscle and metabolic health (via vascular and non-vascular
pathways) in susceptible/older populations. In this review, we examine the impact of supplementing
this important urea cycle intermediate on cardiovascular and metabolic health outcomes and identify
future directions for investigating its therapeutic impact on cardiometabolic health.
Keywords:
supplements; therapeutics; interventions; watermelon; nitric oxide; arginine; endothelial
function; flow mediated dilation; mitochondria; enterocytes; liver; adipocytes; muscle; immune cells;
obesity; aging; hypertension; inflammation; insulin resistance; diabetes; cardiovascular disease
1. Introduction
Diminished bioavailability of nitric oxide (NO), the gaseous signaling molecule involved in the
regulation of numerous vital biological functions, contributes to the development of multiple age- and
lifestyle-related risk factors and diseases including hypertension, atherosclerosis, insulin resistance,
type 2 diabetes (T2D), and cardiovascular disease [
1
–
4
]. In endothelial cells, NO is synthesized from
L-arginine (precursor) by endothelial-nitric oxide synthase (eNOS) generating NO and L-citrulline
(products) [
5
–
7
]. In addition to reductions in NO synthesis, elevations in reactive oxygen species
(ROS), especially superoxide (O
2
−
), can reduce the bioavailability of NO through the generation of
peroxynitrite (ONOO
−
), which further promotes endothelial dysfunction that is commonly associated
with cardiometabolic diseases [
6
]. Thus, augmenting L-arginine levels in the circulation may represent a
Nutrients 2018, 10, 921; doi:10.3390/nu10070921 www.mdpi.com/journal/nutrients
Nutrients 2018, 10, 921 2 of 24
potential therapeutic mechanism to increase NO synthesis and bioavailability. However, oral L-arginine
supplementation is largely ineffective due to gastrointestinal and hepatic extraction of L-arginine [
8
]
(Figure 1), as well as a dose-dependent presentation of gastrointestinal distress [
9
]. Alternatively, oral
L-citrulline supplementation consistently increases plasma and tissue levels of L-arginine and NO
bioavailability [10–12].
Nutrients 2018, 10, x FOR PEER REVIEW 2 of 23
commonly associated with cardiometabolic diseases [6]. Thus, augmenting L-arginine levels in the
circulation may represent a potential therapeutic mechanism to increase NO synthesis and
bioavailability. However, oral
L-arginine supplementation is largely ineffective due to
gastrointestinal and hepatic extraction of
L-arginine [8] (Figure 1), as well as a dose-dependent
presentation of gastrointestinal distress [9]. Alternatively, oral
L-citrulline supplementation
consistently increases plasma and tissue levels of
L-arginine and NO bioavailability [10–12].
Figure 1. Comparison of oral L-citrulline (via pharmaceutical/nutraceutical grade L-citrulline or
watermelon products) versus oral
L-arginine. The activity of the arginase enzyme located in the
enterocytes of intestines and liver (first-pass extraction) substantially reduces the availability of
oral
L-arginine, instead yielding increased urea and L-ornithine production. L-citrulline is not
acted on by arginase enzyme or first-pass extraction but is converted to
L-arginine by
argininosuccinate lyase in the kidneys. Increased circulating
L-arginine serves a substrate for the
eNOS to produce nitric oxide (NO) and increase smooth muscle vasodilation.
L-citrulline may
directly activate inducible nitric oxide synthase (iNOS) in skeletal muscle and increase protein
synthesis via mTOR activation.
L-citrulline may indirectly activate neuronal nitric oxide
synthase (nNOS) in skeletal muscle leading to increases in NO and stimulation of mitochondrial
biogenesis.
L-citrulline has reported actions on adipose tissue to increase lipolysis, fatty acid
oxidation, and uncoupling protein 1 (UCP1) expression.
L-citrulline has also been reported to
indirectly activate iNOS in activated macrophages and increase NO production.
L-citrulline’s
Figure 1.
Comparison of oral L-citrulline (via pharmaceutical/nutraceutical grade L-citrulline or
watermelon products) versus oral L-arginine. The activity of the arginase enzyme located in the
enterocytes of intestines and liver (first-pass extraction) substantially reduces the availability of oral
L-arginine, instead yielding increased urea and L-ornithine production. L-citrulline is not acted on by
arginase enzyme or first-pass extraction but is converted to L-arginine by argininosuccinate lyase in
the kidneys. Increased circulating L-arginine serves a substrate for the eNOS to produce nitric oxide
(NO) and increase smooth muscle vasodilation. L-citrulline may directly activate inducible nitric oxide
synthase (iNOS) in skeletal muscle and increase protein synthesis via mTOR activation. L-citrulline
may indirectly activate neuronal nitric oxide synthase (nNOS) in skeletal muscle leading to increases in
NO and stimulation of mitochondrial biogenesis. L-citrulline has reported actions on adipose tissue to
increase lipolysis, fatty acid oxidation, and uncoupling protein 1 (UCP1) expression. L-citrulline has
also been reported to indirectly activate iNOS in activated macrophages and increase NO production.
Nutrients 2018, 10, 921 3 of 24
L-citrulline’s systemic effects positively impact hypertension, atherosclerosis, inflammation, insulin
resistance, type 2 diabetes, and cardiovascular disease. Emerging evidence also suggests that L
-citrulline itself can positively impact skeletal muscle and adipose tissue to improve metabolic syndrome.
This figure was partially modified from Irving and Spielmann (2016) [13].
L-Citrulline is a neutral, non-essential [
14
] alpha-amino acid that is an important component
of the urea cycle in the liver and kidneys [
15
]. As a non-protein amino acid, L-citrulline is rarely
found in food, but is highly concentrated in watermelon [
16
]. The concentration of L-citrulline in
watermelon grown in the United States can range from 1.6 to 3.5 g/kg of fresh
watermelon [16–18]
.
As such, consumption of approximately 1–1.5 kg/day (2.2–3.3 lbs/day) of fresh watermelon would
be needed to achieve the minimum effective dose of L-citrulline (3 g/day) and 3.3–5.0 kg/day
(7.3–16.5 lbs/day) of fresh watermelon would be needed to achieve the maximum effective dose
of L-citrulline (10 g/day) [
16
–
18
]. Given the growing evidence that endothelial dysfunction has its
origins in deficient L-arginine-NO metabolism and given the relative ineffectiveness of L-arginine
supplementation on NO metabolism, researchers have begun to explore the potential therapeutic
benefits of L-citrulline. L-citrulline is typically supplemented using pharmaceutical/nutraceutical grade
L-citrulline, L-citrulline conjugated with malate (1:1 ratio), or as watermelon extract. L-citrulline, as with
many other NO-boosting supplements, has received much interest for its potential cardiovascular and
anti-hypertensive capabilities [
19
]. Although recent reviews have eloquently reviewed the impact of
citrulline supplementation in health and disease [
14
,
20
,
21
], the present review focuses on L-citrulline’s
NO-dependent and NO-independent effects on cardiometabolic outcomes. Moreover, we specifically
summarize current literature regarding the benefits of both pharmaceutical/nutraceutical grade
L-citrulline and watermelon supplements (juice, water, extract, etc.) on vascular and metabolic
physiology and their potential therapeutic impact on cardiometabolic health. We also discuss recent
studies that have begun to examine L-citrulline’s direct and indirect effects on clinically relevant aspects
of skeletal muscle and adipose tissue metabolism, which are key mediators for the development of
cardiometabolic disorders.
2. Health Applications
The health-related applications of L-citrulline supplementation are largely predicated on the
capacity for L-citrulline to increase L-arginine availability for NO production. NO released from the
endothelium as a gas initiates a signaling cascade involving the activation of soluble guanylate cyclase
(sGC) to increase cyclic guanosine monophosphate (cGMP) synthesis [
22
] (Figure 1). Increased levels
of cGMP acts as a second messenger to, in the case of NO, increase vasodilation by relaxing the
smooth muscle cells of the conduit and resistance arteries [
22
]. Reduced eNOS expression and NO
production/bioavailability has been reported in patients with essential hypertension, healthy older
individuals, and heart failure patients [
2
,
23
,
24
]. Moreover, reduced NO bioavailability has both direct
and indirect effects on skeletal muscle metabolism that likely contribute to the development of insulin
resistance and type 2 diabetes as well as age-related muscle wasting [25,26].
3. Pharmacokinetics, Transport and Metabolism
Historically, researchers have considered L-glutamine within enterocytes as the major precursor
for the synthesis of L-citrulline and subsequent release into systemic circulation [
27
]. However, recent
data suggest that L-glutamine’s contribution to L-citrulline biosynthesis may be overestimated [
28
].
On the other hand, there is evidence that enterocytes take up orally ingested L-citrulline and effectively
transport the neutral amino acid through the gastrointestinal tract to the portal circulation, likely using
the Na
+
-dependent, neutral amino acid, including the ASC or B
0,+
-amino acid transporters located in
the enterocytes within the jejunum and ileum [
15
,
29
,
30
]. Quantitatively, L-citrulline is not extracted
by the gastrointestinal tract or liver (net uptake~0) [
31
], which likely facilitates greater down-stream
production of NO through the recycling of L-citrulline to the NO precursor L-arginine (Figure 1).
Nutrients 2018, 10, 921 4 of 24
In contrast, orally ingested L-arginine is subject to moderate-to-high rates of first-pass extraction
both in intestine and liver, likely due to their high expression of arginase [
32
–
34
], increasing arginine
catabolism and limiting systemic increases in circulating levels following its ingestion [8].
To date only a few studies have systematically investigated the pharmacokinetics of L-citrulline
supplementation [
11
,
12
]. Following oral L-citrulline ingestion, circulating L-arginine concentrations
peak after ~1–2 h [
11
,
12
]. As has been shown for both L-arginine and L-ornithine, circulating
concentrations of L-citrulline return to baseline within 8 h [
11
]). The higher activity and bioavailability
of L-citrulline, compared to L-arginine, is due to several factors. For example, 0.75 grams of L-citrulline
ingested twice daily (1.5 g total) increased the L-arginine area under the curve to a similar degree as
ingesting 1.6 g of L-arginine twice daily (3.2 g total) (271 vs. 289
µ
mol
·
h
·
L
−1
) [
12
]. Orally ingested
L-citrulline is absorbed by the enterocytes of the small intestine. However, the lack of gastrointestinal
distress from L-citrulline compared to L-arginine supplementation may suggest that L-citrulline uptake
utilizes a differing transport system. L-arginine is mainly transported across the intestinal membrane
through Na+-independent cationic amino acid transporters (CAT-1, 2 and 3) [
35
]. L-citrulline transport
has been demonstrated in enterocytes, macrophages, glial cells, and aortic smooth muscle, with the
highest K
m
(4.1
±
0.9 mM) reported in the enterocytes [
19
,
30
,
36
]. While a specific L-citrulline
transporter has not been identified, the B
0
-transporters have been suggested to play a role in the
Na
+
dependent transport of L-citrulline across the enterocytes [30].
Clinical dose ranging and tolerability studies have also been conducted for L-citrulline
supplementation. One such human study demonstrated a tolerance of up to 15 g L-citrulline per day in
healthy volunteers [
11
]. By comparison, high-doses of L-arginine (~13 g) can induce significant
gastrointestinal complications [
9
,
21
]. However, at 15 g doses of L-citrulline, a lower fractional
absorption rate and plasma retention of L-citrulline was observed, potentially due to saturation of its
transporters (e.g., ASC or B
0,+
-amino acid transporters) or reduced renal conversion of L-citrulline to
L-arginine. As such, the authors suggested a dose of 10 g L-citrulline for clinical use [
11
]. However, for
increasing circulating L-arginine concentrations, doses of L-citrulline as low as 3 g have been shown to
be effective [
12
]. Thus, the minimum effective dose is ~3 g/day, whereas the maximal effective dose
may be as high as 10 g/day.
The metabolism of orally ingested L-citrulline is mainly confined to the biosynthesis of L-arginine.
Researchers previously thought that most dietary L-citrulline was synthesized via the consumption
of L-glutamine through a transamination reaction in the enterocyte of the gastrointestinal tract [
37
].
However, a recent study using a labeled 2,3,3,4,4 [
2
H
5
] glutamine tracer provided evidence that the
contribution of L-glutamine to L-citrulline may be rather modest [
28
]. Since L-citrulline metabolism in
the liver is somewhat compartmentalized to the urea cycle, exogenous L-citrulline typically bypasses
hepatic metabolism. Circulating L-citrulline, released from the gut, is absorbed by the proximal tubular
cells of the kidney [
38
]. L-Citrulline is rapidly acted upon by cytosolic arginosuccinate synthase
and converted into arginosuccinate which is then converted into L-arginine by arginosuccinate lyase
(see Figure 1). This partial urea cycle meets the demand of the body’s L-arginine requirement. De novo
synthesis of L-arginine from L-citrulline is essential for downregulating urea formation in the liver
during periods of low protein intake to increase nitrogen retention [39].
Another potential source of L-citrulline is its synthesis via the NO cycle (Figure 2). L-arginine is
required for NO formation via eNOS, iNOS, and nNOS. Hydrolysis of the intermediate nitrosoarginine
from L-arginine yields NO and L-citrulline [
15
]. This is particularly useful in endothelial cells via
eNOS [
40
,
41
] and activated macrophages via iNOS [
42
] to sustain L-citrulline as a precursor to
L-arginine to produce NO [
36
] (Figure 1). However, the recycling of L-citrulline to L-arginine does not
appear as viable in cell types that have suboptimal uptake of L-citrulline from plasma, such as aortic
smooth muscle cells. Indeed, aortic smooth muscle cells take up L-citrulline at a relatively slow rate
compared to L-arginine, due in part to its transport through a low affinity (K
m
1.6 mM) transporter [
36
].
Consistent with this notion, Hattori and colleagues reported that physiologically high concentrations
of L-citrulline are necessary to maximally stimulate iNOS activity in cultured smooth muscle cells [
43
].
Nutrients 2018, 10, 921 5 of 24
Under inflammatory conditions (Lipopolysaccharides and Interferon-
γ
stimulation), L-arginine itself
is capable of inducing NOS via its increased transport.
Nutrients 2018, 10, x FOR PEER REVIEW 5 of 23
Figure 2. Nitric Oxide (NO) Cycle. Schematic representation of the NO cycle.
4. Vasoprotective Effects of
L-Citrulline
4.1. Endothelial Vasodilator Function
L-citrulline increases NO biosynthesis indirectly by increasing L-arginine synthesis, which in
turn may lead to improved endothelial vasodilator function [12,24,44]. The reduction in synthesis of
eNOS is thought to play a major role in the endothelial dysfunction associated with aging,
menopause, and cardiometabolic disease [1,45,46]. Work in rodent models of reduced
L-arginine
bioavailability has demonstrated that
L-citrulline, but not L-arginine supplementation, increased NO
synthesis and microcirculatory (gut villi) blood flow [47]. Furthermore, in a rodent model of
spontaneous hypertension and chemical disruption of NO bioavailability,
L-citrulline restored levels
of NO by increases
L-arginine/asymmetrical dymethylarginine (ADMA) ratio [48–50]. These pre-
clinical studies support the hypothesis that endothelial function may be enhanced by the capability
of
L-citrulline supplementation to increase L-arginine levels.
In healthy, young participants increased levels of
L-arginine, nitrate/nitrite, and cGMP activities
have been consistently demonstrated after
L-citrulline supplementation [11,12,51]. However,
improvement in endothelial function, as measured by brachial artery flow mediated dilation (FMD),
have not been reported with acute or short-term (~7 days) administration of
L-citrulline, despite
significantly increased
L-arginine bioavailability and increased urinary nitrate/nitrite (NOX)
[12,24,44]. One study that provided young, healthy subjects with 10 g of
L-citrulline (i.e., the
maximally clinically effective dose) demonstrated increased de novo
L-arginine and NO synthesis
but did not observe increased forearm blood flow during reactive hyperemia measured by
plethysmography [24]. Both the acute time course of these studies and the healthy state of the
participants may explain the lack of benefit of
L-citrulline supplementation under these conditions.
Age-related endothelial dysfunction is associated with a reduced bioavailability of
L-arginine
and reduction in eNOS synthesis [1,41,46]. Older adults with heart failure increased de novo
L-
arginine and NO synthesis after acute ingestion of
L-citrulline (10 g), but these synthesis rates were
still low when compared to young subjects [24]. When compared to the placebo conditions,
L-
citrulline supplementation also did not improve forearm blood flow during reactive hyperemia
measured by plethysmography in these subjects. Likewise, another study in elderly male subjects
measured blood flow at rest and post-exercise by providing
L-citrulline combined with whey protein,
compared to whey protein alone or whey protein with additional non-essential amino acids [44]. No
increases in plasma nitrate/nitrite or limb blood flow were observed during resting or exercise
postprandial periods [31]. As is the case with young healthy participants, the length of treatment may
be a mediating factor in determining
L-citrulline’s lack of efficacy in improving FMD. As an example,
Figure 2. Nitric Oxide (NO) Cycle. Schematic representation of the NO cycle.
4. Vasoprotective Effects of L-Citrulline
4.1. Endothelial Vasodilator Function
L-citrulline increases NO biosynthesis indirectly by increasing L-arginine synthesis, which in
turn may lead to improved endothelial vasodilator function [
12
,
24
,
44
]. The reduction in synthesis of
eNOS is thought to play a major role in the endothelial dysfunction associated with aging, menopause,
and cardiometabolic disease [
1
,
45
,
46
]. Work in rodent models of reduced L-arginine bioavailability
has demonstrated that L-citrulline, but not L-arginine supplementation, increased NO synthesis
and microcirculatory (gut villi) blood flow [
47
]. Furthermore, in a rodent model of spontaneous
hypertension and chemical disruption of NO bioavailability, L-citrulline restored levels of NO by
increases L-arginine/asymmetrical dymethylarginine (ADMA) ratio [
48
–
50
]. These pre-clinical studies
support the hypothesis that endothelial function may be enhanced by the capability of L-citrulline
supplementation to increase L-arginine levels.
In healthy, young participants increased levels of L-arginine, nitrate/nitrite, and cGMP activities
have been consistently demonstrated after L-citrulline supplementation [
11
,
12
,
51
]. However,
improvement in endothelial function, as measured by brachial artery flow mediated dilation (FMD),
have not been reported with acute or short-term (~7 days) administration of L-citrulline, despite
significantly increased L-arginine bioavailability and increased urinary nitrate/nitrite (NOX) [
12
,
24
,
44
].
One study that provided young, healthy subjects with 10 g of L-citrulline (i.e., the maximally clinically
effective dose) demonstrated increased de novo L-arginine and NO synthesis but did not observe
increased forearm blood flow during reactive hyperemia measured by plethysmography [
24
]. Both the
acute time course of these studies and the healthy state of the participants may explain the lack of
benefit of L-citrulline supplementation under these conditions.
Age-related endothelial dysfunction is associated with a reduced bioavailability of L-arginine and
reduction in eNOS synthesis [
1
,
41
,
46
]. Older adults with heart failure increased de novo L-arginine
and NO synthesis after acute ingestion of L-citrulline (10 g), but these synthesis rates were still
low when compared to young subjects [
24
]. When compared to the placebo conditions, L-citrulline
supplementation also did not improve forearm blood flow during reactive hyperemia measured by
plethysmography in these subjects. Likewise, another study in elderly male subjects measured blood
Nutrients 2018, 10, 921 6 of 24
flow at rest and post-exercise by providing L-citrulline combined with whey protein, compared to
whey protein alone or whey protein with additional non-essential amino acids [
44
]. No increases
in plasma nitrate/nitrite or limb blood flow were observed during resting or exercise postprandial
periods [
31
]. As is the case with young healthy participants, the length of treatment may be a
mediating factor in determining L-citrulline’s lack of efficacy in improving FMD. As an example,
in a separate study, L-citrulline supplementation at 800 mg/day for 8 weeks was necessary for
elevating plasma L-arginine levels and improving FMD [
52
]. However, in patients with vasospastic
angina, 8 weeks of L-citrulline (800 mg/day) was sufficient to improve L-arginine/ADMA levels and
FMD [
52
]. The variability in changes in FMD in response to L-citrulline supplementation reflect similar
investigations into L-arginine supplementation [
53
]. There are several possible explanations for the
lack of consistent observations in this field of study. Both animal and human studies demonstrate a
significant relationship between improved FMD and increase L-arginine/ADMA ratio. There is also the
possibility that age-associated endothelial dysfunction is not mediated by reduced NO production due
to high ADMA levels and therefore, L-citrulline or L-arginine supplementation may not be expected to
improve endothelial function under these conditions [54].
4.2. Protection against Endothelial Damage
Endothelial dysfunction, often associated with obesity-induced insulin resistance, is thought to be
a major factor in the development of cardiovascular disease [
45
]. Dietary factors, such as high fat/high
cholesterol diet, adipocyte derived factors, and aging have also been implication in the promotion
of low-grade inflammation and further exacerbated endothelial dysfunction, contributing to the
development of cardiovascular disease [
45
,
55
–
58
]. Studies in rats fed a high fat/high cholesterol
diet observed an elevation of the liver enzymes aspartate aminotransferase (AST) and alanine
aminotransferase (ALT), indicative of liver stress/damage [
59
]. Subsequent oral L-citrulline or
L-arginine treatment increased high density lipoprotein (HDL) levels, reduced serum AST/ALT,
and confirmed some modest but favorable, structural changes in the endothelial structure of the
thoracic aorta [
59
]. These data support the notion that L-citrulline may protect against the liver damage
and endothelial dysfunction induced by chronic exposure to a high fat/high cholesterol diet.
Reversal of deficient L-arginine levels via L-citrulline supplementation has been investigated as
a therapeutic strategy for preserving endothelial function in rodents consuming an atherogenic diet.
Both wild type and apoE
−
/
−
mice fed a high fat/high cholesterol diet exhibited increased plasma and
tissue arginase activity, leading to a reduction in L-arginine bioavailability [
60
]. In rats fed a Western
diet (high fat/high fructose), enrichment with L-citrulline (1 g/kg) reduced hepatic microvesicular lipid
droplets and circulating triacylglycerol [
61
].
In vitro
studies in porcine coronary artery endothelium
demonstrated that L-citrulline (100
µ
mol/L) treatment preserved eNOS production following ADMA
treatment [
62
]. L-citrulline preserves endothelial function in rabbits during exposure to a high
cholesterol diet by decreasing the production of superoxide and associated oxygen-sensitive proteins
ELK-1 and p-CREB [
63
]. Collectively, these studies in diverse animal models have demonstrated the
critical role of NO in endothelial protection against atherogenic dietary conditions and the viable
role of L-citrulline in promoting increased endogenous NO production, as well as in reducing the
deleterious impact of oxidative stress on NO bioavailability.
4.3. Antioxidant and Anti-Inflammatory Effects
In vascular tissues the generation of ROS is driven by nicotinamide adenine dinucleotide
phosphate oxidases, which promote platelet aggregation, reduce endothelium-mediated relaxation, and
induce pathological vascular remodeling [
64
]. The mechanisms of L-citrulline to improve endothelial
dysfunction, in conditions such as atherosclerosis, are most likely mediated via direct reduction
of hydroxyl radical formation, direct action on vascular smooth muscle, and indirect action of NO
synthesis. The antioxidant effects of L-citrulline can be viewed in two different ways: NO-dependent
and NO-independent pathways. As previously mentioned, L-citrulline is capable of increasing eNOS in
Nutrients 2018, 10, 921 7 of 24
endothelial cells, which in turn reduces ROS formation [
65
]. Nonetheless, L-citrulline possess qualities,
as an amino acid, which make it a functional antioxidant under certain physiological conditions.
According to the Haber-Weiss and Fenton reactions, the production of superoxide is coupled
with the formation of hydroxyl radicals (HO
−
) [
66
]. Hydroxyl radical formation is highly upregulated
during ischemia/low oxygen availability in tissues supplied by atherosclerotic blood vessels [
62
].
L-citrulline can reduce hydroxyl radical formation, independent of NO, by directly interacting
with hydroxyl radicals via the alpha-amino acids in its protonated NH
3
state leading to water
formation [67,68]
. Interestingly, L-citrulline, under ex vivo conditions, performs as a more effective
scavenger of ROS mediated endothelial dysfunction at concentrations between 100
µ
M and 3 mM [
68
].
However, the effects of L-citrulline on endothelial protection at higher concentrations (30 mM)
were absent.
The source of the oxidative stress (superoxide vs. hydroxyl radicals) will likely determine
the effectiveness of L-citrulline to attenuate endothelial damage and dysfunction. Therefore, the
antioxidant capacity of L-citrulline, and L-arginine, are dependent on the nature of the oxidative stress.
The therapeutic actions of L-citrulline are likely mediated by its relative concentration
in vivo
, especially
considering evidence that L-citrulline at higher concentration did not impart protective effects.
L-citrulline supplementation is also likely to provide indirect benefits to vascular health by
modulating chronic low-grade inflammation. Indeed, oral L-citrulline ingestion has been shown to
reduce serum inflammatory cytokine concentrations, such as IL-6, tumor necrosis factor (TNF)-alpha,
and C-reactive protein in both aged animals [
69
] and humans [
70
]. Although the exact mechanisms
underlying the citrulline-mediated improvements in systemic inflammation remain unknown,
Breuilard et al. (2015) recently advocated that serum L-citrulline may exert its health benefits
by dampening macrophage cytokine production [
71
]. This elegant study showed that peritoneal
macrophages isolated from Zucker Diabetic Fatty (ZDF) rats secreted significantly less TNF-alpha
when stimulated with increasing concentrations of L-citrulline
in vitro
[
71
]. This is further supported
by
in vivo
models of sepsis where L-citrulline supplementation induces selective reductions in
pro-inflammatory cytokines (IL-6) but preserves anti-inflammatory cytokine (IL-10) production [
72
]
and NO production [73].
In addition to its impact on innate immunity, L-citrulline supplementation appears to regulate
adaptive immunity and further reduce cytokine-induced low-grade inflammation. Emerging research
on immune energetics has highlighted the importance of metabolic regulation on adaptive immune
function [
74
], and recent studies have placed L-citrulline at the center of CD4+ T-cell metabolism,
function, and survival [
75
]. Considering the equal importance of L-arginine in CD8+ T-cell metabolic
profile
in vivo
[
76
], it can be hypothesized that L-citrulline supplementation will directly and
indirectly modulate the metabolism of innate and adaptive immune cells, improve the efficiency
of immune responses to pathogen and reduce both the magnitude and duration of inflammatory
responses in response to antigenic challenges. Taken together, these studies suggest that L-citrulline
supplementation is likely to have great clinical benefits, especially in the context of inflamm-aging, the
chronic low-grade inflammation associated with aging [
77
], which is a well-known aggravating factor
for cardiometabolic disorders [
78
], poor vascular health [
79
], and overall morbidity and mortality [
80
].
4.4. Effects on Basal and Hyperemic Limb Blood Flow
Although L-citrulline is an effective inducer of L-arginine, NO, and cGMP levels, there are
conflicting reports regarding its effectiveness on tissue perfusion during rest or exercise in
healthy volunteers [
11
,
12
,
24
,
81
]. In a study of healthy young men, no differences between an
acute combined nitrate-citrulline supplement and placebo were observed in post-ischemia vascular
responses, as measured by near-infrared spectroscopy (NIRS) [
82
]. Other studies in healthy young
participants have provided evidence of increased muscle blood flow during moderate intensity
exercise after short-term (7 days) L-citrulline supplementation [
81
,
83
,
84
]. It has been speculated
that the lack of significant improvement in blood flow and proxy measurements of endothelial
Nutrients 2018, 10, 921 8 of 24
function (reactive hyperemic forearm blood flow) in healthy volunteers is likely due to the
physiological limits of vessel compliance [
24
]. Moreover, during (forearm) exercise sympathetic
activation increases NO production in an eNOS dependent fashion in vascular endothelial cells,
locally overriding systemic vasoconstriction within the arterioles feeding the exercising muscles [
85
].
This auto-regulatory mechanism is likely intact in healthy volunteers, and thus does not benefit from
L-citrulline supplementation.
5. Anti-Hypertensive Effects of L-Citrulline
5.1. Resting Blood Pressure and Arterial Stiffness
L-Citrulline has been investigated as a potential therapeutic agent to reduce resting
blood pressure by increasing endogenous synthesis of L-arginine and ultimately, NO levels.
In spontaneously hypertensive rats (SHR), L-citrulline treatment prevent hypertension by increasing
the L-arginine/ADMA ratio [
48
]. Rats and their offspring treated with a selective NOS inhibitor,
N
G
-nitro- L-arginine-methyl ester (L-NAME), develop hypertension. L-NAME treatment reduces
the bioavailability of NO by first direct inhibition of the NOS enzyme, thus decreasing NO level
and lowering L-Arginine/ADMA ratio in the kidney [
49
]. L-NAME treated rats, and their offspring,
eventually develop hypertension. L-citrulline supplementation protect against hypertension in the
offspring of L-NAME treated dams by increasing the L-arginine/ADMA ratio in the kidney [
50
].
These pre-clinical studies suggest that L-citrulline increases renal NO levels, contributing to the
prevention of hypertension.
Both pharmaceutical/nutraceutical grade L-citrulline and watermelon extract have demonstrated
some efficacy in reducing blood pressure in both pre-hypertensive and hypertensive patients.
Compared to placebo, treatment with watermelon extract containing 6 g/day of L-citrulline/L-arginine
for 6 weeks exhibited reduced ankle and brachial systolic blood pressure (
−
12
±
4 and
−15 ± 3 mmHg
,
respectively), ankle and brachial diastolic blood pressure (
−
8
±
2 and
−
8
±
2 mmHg, respectively),
as well as reducing the carotid augmentation index in obese pre- and hypertensive men [
86
]. Short-term
treatment for 7–14 days with L-citrulline (5.6 g/day) reduced arterial stiffness in healthy and
overweight middle-aged men [
87
,
88
]. Obese post-menopausal women with hypertension treated
with 6 weeks of watermelon extract (6 g/day L-citrulline) showed evidence of reduced arterial stiffness
and aortic systolic blood pressure, as indicated by a reduction in pressure pulse wave reflection [
89
].
In a group of middle-aged, pre-hypertensive, men and women, 6 weeks of watermelon extract
supplementation (2.7 g/1.3 g L-citrulline/L-arginine) improved peripheral vascular tone (decrease
augmentation index and pulse wave velocity), and lead to a significant reduction in aortic systolic blood
pressure (
−
9
±
3 vs.
−
2
±
3 mmHg, p = 0.01), and non-significant reductions in brachial artery systolic
blood pressure (
−
9
±
7 vs.
−
3
±
7 mmHg, p = 0.10) compared to placebo controls [
90
]. L-citrulline
has also been shown to promote favorable adaptations in blood vessel wall stiffness measured by
pulse wave velocity and hypertensive responses to cold [86–88]. Reduced arterial stiffness and aortic
systolic blood pressure was observed in obese post-menopausal women with hypertension after
6-week supplementation with watermelon extract (6 g/day citrulline) [
89
]. Collectively, the current
evidence supports L-citrulline and watermelon extract as viable nutritional supplements to improve
resting aortic hemodynamics in individuals with prehypertension and hypertension. Although these
studies demonstrate the potential for L-citrulline and watermelon extract to improve resting blood
pressure and arterial stiffness, the patient population, dose, and duration of treatment appear to impact
the magnitude of these effects and warrant further investigation. Table 1 provides a list of studies
examining the effectiveness of L-citrulline (via watermelon extract) on reducing blood pressure in
normotensive, pre-hypertensive, and hypertensive men and women.
Nutrients 2018, 10, 921 9 of 24
Table 1.
A series of human clinical trials that investigated changes, after L-citrulline or watermelon extract supplementation, in blood pressure and associated indices
of blood vessel function under resting and physiologically stressful conditions.
Reference
Population
BP Status
Formulation
Dose
Duration
Resting Function
Results
Cardiovascular Reactivity
Figueroa et al. (2010) [91] 17 M Normotensive L-Citrulline 6 g/day 4 weeks ↓ bSBP, aSBP, aPP
Orozco-Gutierrez et al.
(2010) [92]
9 M
6 F
Heart failure w/
preserved EF
L-Citrulline-
Malate
3 g/day 8 weeks ↓bSPB, bDBP, ↑ RVEF during exercise
Figueroa et al. (2011) [90]
4 M
5 W
Pre-hypertensive
Watermelon
Extract
2.7 g/day 6 weeks ↓bPP, aSBP, aPP, AIx
Figueroa et al. (2012) [86]
3 M
11 W
Pre-hypertensive
Watermelon
Extract
2.7 g/day 6 weeks
↓ ankle SBP, DBP, MAP,
↓ bSBP, bDBP, bMAP
↓ carotid AIx
Figueroa et al. (2013) [89] 12 W
Hypertensive
Post-menopausal
Watermelon
Extract
6 g/day 6 weeks
↓
b-aPWV, aSBP, aDBP, aSBP2
↔ AIx
Sanchez-Gonzalez et al.
(2013) [88]
16 M Normotensive L-Citrulline 100 mg/kg 2 weeks
↓ CI and IHG increases in bSBP, aSBP
and AIx
Alsop et al. (2016) [93]
4 M
8 F
Normotensive L-Citrulline 3 d/day 1 week
↓ bSBP, bDBP, MAP, pulse
interval
↓ Pulse interval, Pulse Amplitude
Ratio,
↑ HRV post 30% MVC exercise
Figueroa et al. (2016) [94] 16 M
Normotensive
Overweight/obese
L-Citrulline 6 d/day 2 weeks
Attenuated the increase in
aSBP and AIx during IHG
and reduced MAP aDBP
↓ aSBP, aPP, AIx during IHG
↓ aDBP, MAP, AIx during PEMI
↓ aSBP, DBP, aPP, an baPWV during
PEMI + CPT
Bailey et al. (2016) [81] 8 M Normotensive
Watermelon
Juice
~3.4 g/day 2 weeks ↑ aSBP and MAP
Massa et al. (2016) [95]
10 M
10 W
Pre-hypertensive
Watermelon
Extract
6 g/day 6 weeks
↓ bSBP and bDBP
↔ cardiac autonomic
function
Wong et al. (2016)
[96]
25 F *
Normotensive/
Pre-hypertensive
L-Citrulline 6 g/day 8 weeks
↓bSBP, bDBP, and nLF (SNS
activity), LnLF/LnHF
(sympathovagal balance)
Gonzales et al. (2017) [97]
12M
13W
Normotensive/
Pre-hypertensive
L-Citrulline 6 g/day 2 weeks ↓ seated bSBP
↑muscle blood flow during
submaximal exercise in men
Abbreviations: M: male, F: female, bSBP: brachial systolic blood pressure, aSBP: aortic systolic blood pressure, aPP: aortic pulse pressure, AIx: augmentation index, DBP: diastolic blood
pressure, cAIx: carotid augmentation index, CI: cold induced, CPT: cold pressor test, EF: ejection fraction IHG: intermittent hand grip exercise, MAP: mean arterial pressure, aDBP: aortic
diastolic blood pressure, MVC: maximal voluntary contraction, LnLF: natural log low frequency from heart rate variability test and LnHF: natural log high frequency from heart rate
variability test, ↓ decrease, ↑ increase, ↔ no significant change. * Post-menopausal women 50–65 years of age.