Citrulline and arginine utility in treating nitric oxide deficiency in mitochondrial disorders

Molecular Genetics and Metabolism 107 (2012) 247–252

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Molecular Genetics and Metabolism

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Citrulline and arginine utility in treating nitric oxide deficiency inmitochondrial disorders

Ayman W. El-Hattab a, Lisa T. Emrick b, William J. Craigen b, Fernando Scaglia b,⁎a Medical Genetics Section, Department of Pediatrics, The Children's Hospital at King FahadMedical City and King Saud bin Abdulaziz University for Health Science, Riyadh, Kingdomof Saudi Arabiab Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA

⁎ Corresponding author at: Department of Molecu825 4294.

E-mail address: [email protected] (F. Scaglia).

1096-7192/$ – see front matter © 2012 Elsevier Inc. Alldoi:10.1016/j.ymgme.2012.06.018

a b s t r a c t

Article history:Received 26 May 2012Received in revised form 30 June 2012Accepted 30 June 2012Available online 6 July 2012

Keywords:Nitric oxide deficiencyNitric oxide synthase (NOS)Endothelial dysfunctionStroke-like episodesMyopathyLactic acidosis

Mitochondrial diseases arise as a result of dysfunction of the respiratory chain, leading to inadequate ATPproduction required to meet the energy needs of various organs. On the other hand, nitric oxide (NO)deficiency can occur in mitochondrial diseases and potentially play major roles in the pathogenesis of severalcomplications including stroke-like episodes, myopathy, diabetes, and lactic acidosis. NO deficiency in mito-chondrial disorders can result from multiple factors including decreased NO production due to endothelialdysfunction, NO sequestration by cytochrome c oxidase, NO shunting into reactive nitrogen species forma-tion, and decreased availability of the NO precursors arginine and citrulline. Arginine and citrulline supple-mentation can result in increased NO production and hence potentially have therapeutic effects on NOdeficiency-related manifestations of mitochondrial diseases. Citrulline is a more efficient NO donor thanarginine as it results in a greater increase in de novo arginine synthesis, which plays a major role in drivingNO production. This concept is supported by the observation that the three enzymes responsible for recyclingcitrulline to NO (argininosuccinate synthase and lyase, and nitric oxide synthase) function as a complex thatcan result in compartmentalizing NO synthesis and channeling citrulline efficiently to NO synthesis. Clinicalresearch evaluating the effect of arginine and citrulline in mitochondrial diseases is limited to uncontrolledopen label studies demonstrating that arginine administration to subjects with MELAS syndrome results inimprovement in the clinical symptoms associated with stroke-like episodes and a decrease in the frequencyand severity of these episodes. Therefore, controlled clinical studies of the effects of arginine or citrullinesupplementation on different aspects of mitochondrial diseases are needed to explore the potentialtherapeutic effects of these NO donors.

© 2012 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2482. Arginine, citrulline, and nitric oxide metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248

2.1. Arginine metabolism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2482.2. Citrulline metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2482.3. Nitric oxide synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248

3. Nitric oxide deficiency in mitochondrial diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2493.1. Mechanisms of NO deficiency in mitochondrial diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249

3.1.1. Decreased NO production due to endothelial dysfunction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2503.1.2. Post-production NO scavenging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2503.1.3. Decreased NO precursors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2503.1.4. Increased ADMA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250

3.2. Potential consequences of NO deficiency in mitochondrial diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2504. The utility of arginine and citrulline in treating NO deficiency in mitochondrial diseases . . . . . . . . . . . . . . . . . . . . . . . . . . 251

4.1. Arginine supplementation in MELAS syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2514.2. Citrulline supplementation as a NO donor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251

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248 A.W. El-Hattab et al. / Molecular Genetics and Metabolism 107 (2012) 247–252

4.2.1. Citrulline is more efficient in increasing plasma arginine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2514.2.2. Citrulline is more efficient in increasing intracellular arginine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2514.2.3. The role of intracellular de novo-synthesized arginine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251

4.3. Potential therapeutic benefits of arginine and citrulline supplementation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2515. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252

Fig. 1. Schematic presentation of arginine metabolism (NO: nitric oxide).

1. Introduction

Mitochondrial diseases are a heterogeneous group of disorders thatresult fromdysfunction of themitochondrial respiratory chain. Dysfunc-tional mitochondria are unable to generate sufficient ATP to meet theenergy needs of various tissues and organs, particularly those withhigh energy demand such as the central nervous system, skeletal andcardiac muscles, kidneys, liver, and endocrine systems. Other aspectsof mitochondrial function such as calcium buffering, apoptosis, and ret-rograde signaling are also likely perturbed. Disturbed mitochondrialfunction in various tissues and organs can explain themulti-organman-ifestations of mitochondrial diseases including epilepsy, intellectual dis-ability, skeletal and cardiac myopathy, diabetes, sensorineural hearingloss, and renal impairment [1]. In addition to reduced energy produc-tion, there is growing evidence that nitric oxide (NO) deficiency occursin mitochondrial diseases and can play a major role in the pathogenesisof several complications observed in mitochondrial diseases includingstroke-like episodes, myopathy, diabetes, and lactic acidosis [2–7]. Theamino acids arginine and citrulline act as NO precursors and can beused to restore NO production and may be of therapeutic utility intreating NO deficiency-related manifestations of mitochondrial diseases[7].

In this article, we review the metabolic pathways of arginine, citrul-line, and NO, present evidence for NO deficiency in mitochondrial dis-eases, discuss the possible causes of NO deficiency, review the clinicalmanifestations that can result from NO deficiency, and, finally, evaluatethe role of arginine and citrulline supplementation in treating NO defi-ciency in mitochondrial disorders.

2. Arginine, citrulline, and nitric oxide metabolism

2.1. Arginine metabolism

L-Arginine (2-amino-5-guanidinopentanoic acid) was first isolatedfrom lupin seedlings in 1886, and in 1895 was identified as a compo-nent of animal proteins [8]. It is an essential amino acid for youngmam-mals, and a conditionally essential amino acid for adult humans andother animals during catabolic stress (e.g. infection or trauma) or inthose with diseases involving the kidneys or small intestine (e.g. renalfailure or massive small bowel resection). It is a non-essential (dispens-able) amino acid in healthy human adults [9,10]. Arginine is derivedfrom the diet, as a result of protein turnover, and from endogenous(de novo) synthesis from citrulline. Arginine is utilized in protein syn-thesis as well as for the synthesis of urea, NO, creatine, agmatine, andpolyamines [11] (Fig. 1).

2.2. Citrulline metabolism

L-Citrulline (2-amino-5-carbamoylaminopentanoic acid) is namedafter Citrullus vulgaris (Latin for watermelon) from which it was firstisolated in 1930 [12]. Citrulline functions as an intermediate in theurea cycle and as a precursor of arginine. Citrulline is nonessentialunder physiological conditions but considered a conditionally essen-tial amino acid in situations where intestinal function is compromised[13]. Citrulline is a non-protein amino acid for which the main sourceis de novo synthesis in the small intestine. Most circulating citrulline

derives from glutamine conversion in the enterocyte cells. Glutamineis first converted to glutamate via the enzyme glutaminase. Subse-quently, pyrroline-5-carboxylate (P5C) synthase, which is locatedalmost exclusively in the intestinal mucosa, converts glutamate toP5C, which is converted into ornithine via ornithine aminotransferase(OAT). Ornithine is then converted to citrulline via the enzyme orni-thine transcarbamylase (OTC), which is expressed only in the liverand intestine. Arginine and proline can also act as intestinal citrullineprecursors. All of the enzymes involved in citrulline synthesis arelocated in the mitochondria of enterocytes [14–16] (Fig. 2). The activ-ities of the two enzymes that catabolize citrulline, argininosuccinatesynthase (ASS) and argininosuccinate lyase (ASL), are very low in theintestine. Therefore, citrulline cannot be catabolized in enterocytesand is released into the circulation [17].

The majority of citrulline released by the intestine (~70%) is metab-olized within the kidney, where it is converted into arginine by theenzymes ASS and ASL present in the proximal convoluted tubule ofthe nephron. Therefore, endogenous arginine synthesis involves aninter-organ pathway known as intestinal-renal axis, with de novo argi-nine synthesis from citrulline representing 5–15% of arginine produc-tion. Circulating arginine can be catabolized by liver arginase I to beutilized to fuel the urea cycle via ornithine production [8,18,19] (Fig. 2).

2.3. Nitric oxide synthesis

Arginine and citrulline act as NO precursors in a wide variety ofcells including vascular endothelium, neurons, and macrophages.NO is synthesized from arginine by three NO synthase (NOS)isoforms: neuronal NOS (nNOS) primarily present in neuronal cells,endothelial NOS (eNOS) primarily present in endothelial cells, andcytokine-inducible NOS (iNOS) present in various cell types includingmacrophages, hepatocytes, muscles, and chondrocytes. The eNOSplays a role in regulating the physiological vascular tone whereasiNOS produces NO under pathological conditions e.g. infection[20,21]. In the presence of tetrahydrobiopterin, NADPH, and oxygen,

Fig. 2. Schematic presentation of arginine, citrulline, and NO metabolic pathways (Arg I: arginase I, Arg II: arginase II, ASL: argininosuccinate lyase, ASS: argininosuccinate synthase,Glu: glutaminase, NOS: nitric oxide synthase, OAT: ornithine aminotransferase, OTC: ornithine transcarbamylase, P5CS: pyrroline 5 carboxylate synthase, PO: proline oxidase). Notethat this diagram does not show all substrates and products in these pathways.

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NOS catalyzes the conversion of arginine to NO and citrulline. Citrullinecan be recycled to arginine by the combined action of ASS and ASL,which are expressed to some degree in nearly all cell types. Therefore,both arginine and citrulline support NO synthesis in a variety of tissues[8,22]. The three enzymes responsible for recycling citrulline to produceNO(ASS, ASL, andNOS)have an interesting relationship. It has beendem-onstrated that ASS andASL are co-inducedwith iNOS in various cell types[22,23]. Furthermore, ASS and ASL have been shown to interact andco-localize with the different NOS isoforms, suggesting that theseproteins function as a complex [24–26]. It has been proposed that theformation of this complex (the ASL–ASS–NOS complex) is needed forNO production based on the observation that the loss of ASL results indecreased abundance of the ASL–ASS–NOS complex and NO synthesis[26]. The ASL–ASS–NOS complex is speculated to function in the cellularcompartmentalization of NO synthesis under physiological and patholog-ical conditions based on the above-mentioned cellular studies [22–26].

NO synthesis is largely dependent upon the availability of intracellulararginine which is affected by: a) transport of extracellular arginine, b)intracellular synthesis of arginine from citrulline, which is dependenton citrulline availability, and c) the activity of arginase [22]. Arginine istransported into the cells via cationic amino acid transporter (CAT)isoforms CAT-1, CAT-2, and CAT-3. CAT-1 is expressed ubiquitously,CAT-2 is highly expressed in the liver and at less abundant levels in vari-ous tissues,while CAT-3 expression is limited to the brain. The expressionof these transporters has been shown to be co-induced with iNOS in awide variety of cells, indicating that arginine transport capacity increasesto support elevated rates of NO synthesis [8,22]. Arginase exists in twoknown isoforms: arginase I, which is highly expressed in the liver, andarginase II, which is expressed in non-hepatic organs including thekidney, brain, and small intestine. Both NOS and arginase use arginineas a common substrate, and arginase may reduce NO production bycompeting with NOS for arginine [8,22].

3. Nitric oxide deficiency in mitochondrial diseases

NO deficiency may occur in mitochondrial diseases and can playa major role in the pathogenesis of several complications, includingstroke-like episodes, myopathy, diabetes, and lactic acidosis. It has beenshown that patients with MELAS (mitochondrial encephalomyopathy,lactic acidosis, and stroke-like episodes) syndrome have lower concen-trations of NO metabolites (nitrite and nitrate, NOx) during stroke-likeepisodes [3,5]. Furthermore, the NO synthesis rate as measured by stableisotope infusion techniques has been shown to be lower in patientswith MELAS syndrome who are not experiencing acute stroke-likeepisodes [7]. Another study has demonstrated that sarcoplasmic NOSactivity is reduced in cytochrome c oxidase (COX) deficient fibers ofmuscle biopsies obtained from patients with chronic progressive ex-ternal ophthalmoplegia (CPEO), mitochondrial myopathy, and MELASsyndrome [2]. Flow-mediated vasodilation (FMD), which is a functionof NO synthesized by endothelial cells in response to re-perfusion,was found to be impaired in patients with mitochondrial myopathy,MELAS, MERRF (myoclonic epilepsy with ragged red fibers), MIDD(maternally inherited diabetes and deafness), and CPEO, providing fur-ther evidence of NO deficiency in mitochondrial diseases [4,6].

3.1. Mechanisms of NO deficiency in mitochondrial diseases

NO deficiency in mitochondrial disorders is believed to bemulti-factorial in origin with the following factors playing roles: a)impaired NO production due to a generalized impairment of endothelialfunction (endothelial dysfunction), b) NO sequestration by COX inCOX-positive sites, c) NO shunting into reactive nitrogen species forma-tion, d) decreased availability of theNOprecursors arginine and citrulline,and e) increased asymmetric dimethylarginine (ADMA) concentrations.

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3.1.1. Decreased NO production due to endothelial dysfunctionNO deficiency has been suggested to be the main etiology of

stroke-like episodes observed in subjectswithMELAS syndrome. Energydepletion due tomitochondrial dysfunction can stimulatemitochondrialproliferation in various tissues, including vascular endothelial cells[27,28]. Such proliferation can result in impaired normal endothelialfunction (endothelial dysfunction), including the reduced NO produc-tion necessary for smooth muscle relaxation that is needed to maintainthe patency of small blood vessels [29–31]. Therefore, decreased endo-thelial NO synthesis reflects one aspect of endothelial dysfunction.

3.1.2. Post-production NO scavengingMitochondrial proliferation in endothelial cells in MELAS syndrome

can be associated with increased COX activity, which can react withand thus sequester NO [31,32]. In addition to ATP production,mitochon-dria also generate reactive oxygen species (ROS) as a by-product ofoxidative phosphorylation [33]. Respiratory chain impairment canpotentially result in increased ROS generation (oxidative stress).Increased protein nitration has been reported to occur in endothelialcells of small blood vessels inmuscle tissues from subjectswith differentmitochondrial diseases including mitochondrial myopathy, MELAS,MERRF, MIDD, and CPEO [6]. Based on these findings it was suggestedthat oxidative stress in mitochondrial diseases results in decreased NOavailability by shunting NO into reactive nitrogen species formation[6]. Therefore, NO deficiency can result from post-production scaveng-ing of NO by COX and/or the diversion of NO to reactive nitrogen speciesformation.

3.1.3. Decreased NO precursorsDecreased availability of the NO precursors arginine and citrulline

can contribute to NO deficiency in mitochondrial diseases. Lowplasma arginine has been reported in patients with MELAS syndromein both the acute phase of stroke like episodes [5] and the interictalphase (periods without stroke-like episodes) [5,7]. Similarly, lowplasma citrulline was observed in MELAS syndrome in both acute[5] and interictal phase [5,7,31]. Low plasma citrulline was alsofound in subjects with NARP (neurogenic weakness, ataxia and retini-tis pigmentosa) syndromes [34]. It was suggested that lower plasmaarginine may result from increased renal losses based on the findingof high arginine clearance rate in patients with MELAS syndrome[7]. Low plasma citrulline in mitochondrial diseases may result fromdecreased citrulline synthesis in the mitochondria of enterocytesdue to mitochondrial dysfunction leading to impaired ATP productionneeded for citrulline synthesis [31,34]. This potential mechanism issupported by the finding that patients with MELAS syndrome havea lower citrulline flux, which is a reflection of the de novo citrullinesynthesis rate [7]. Most of the citrulline flux is directed towards argi-nine synthesis [35]; therefore lower citrulline availability can result indecreased de novo arginine synthesis and lower intracellular arginineavailability. De novo arginine synthesis has been reported to bedecreased in patients with MELAS syndrome [7]. Therefore, decreasedarginine availability for NO synthesis can be due to two factors: i)increased arginine clearance resulting in a lower plasma arginine con-centration, and ii) decreased citrulline synthesis, leading to decreasedcitrulline availability for de novo arginine synthesis and hence lowerintracellular arginine.

3.1.4. Increased ADMAAlong with arginine availability, NO synthesis is also regulated by

ADMA, which is an endogenous inhibitor of NOS. ADMA is generatedby the methylation of arginine residues in proteins and subsequentrelease by protein hydrolysis within cells and transported into the plas-ma. ADMA enters cells by the same CAT used by arginine, and thus canalso limit intracellular arginine availability by competing for transport[36]. However, such competition may not have a significant impact onarginine transport due to the fact that the plasma arginine concentration

is more than 100-fold than that of ADMA even when ADMA levels areincreased in mitochondrial disease [7]. It was reported that patientswith MELAS syndrome have high plasma ADMA concentrations [7].This elevation may be secondary to mitochondrial dysfunction, leadingto increased ROS production and oxidative stress that may impair theactivity of dimethylarginine dimethylaminohydrolase (DDAH), theenzyme metabolizing ADMA. Alternatively, oxidative stress can stimu-late cell injury and increased proteolysis, resulting in increased ADMAproduction [7,35].

3.2. Potential consequences of NO deficiency in mitochondrial diseases

NO deficiency has been suggested to be the main cause of stroke-likeepisodes observed in subjects with MELAS syndrome [3,7,31,32]. NOdeficiency can result in decreased blood perfusion in the microvascula-ture of various tissues that can potentially contribute to many of thecomplications observed in mitochondrial diseases, including stroke-likeepisodes, myopathy, and diabetes.

Dysfunctionalmitochondria are unable to generate sufficient ATP viathe oxidative phosphorylation pathway to meet the energy needs ofmuscle tissue, resulting in themyopathy that can be seen inmanymito-chondrial diseases and that manifests as muscle weakness, exerciseintolerance, wasting, and histological changes in the muscle biopsy.NO deficiencymay also play a significant role in themyopathic manifes-tations of mitochondrial diseases. Endothelial cells release basal andstimulated NO. During physical activity increased muscular blood flowstimulates endothelial NO production that contributes significantly toexercise-induced hyperemia in muscular tissue [30]. This concept issupported by the demonstration of increased plasma and urinary nitritelevels in response to prolonged aerobic exercise [37]. By studying theeffect of infusing eNOS inhibitors such as N-monomethyl-L-arginine(L-NMMA) and N-nitro-L-arginine methyl ester (L-NAME) duringexercise, the investigators were able to demonstrate a significant reduc-tion in exercise-induced hyperemia [38]. Decreased NO availability canpotentially lead to impaired muscle exercise-induced hyperemia andthus contribute significantly to the exercise intolerance and decreasedphysical fitness in those with mitochondrial diseases. It is also possiblethat NO deficiency may result in decreased basal muscular perfusion,leading to limited availability of nutrients such as amino acids, andthus decreased muscle protein synthesis that may contribute to themyopathy and muscle wasting observed in mitochondrial diseases.

Diabetes mellitus (DM) can be a clinical feature of mitochondrialdiseases. The pathogenesis of DM in mitochondrial disease is notclear. Different pathophysiological mechanisms have been postulat-ed, including decreased glucose utilization by muscle tissues,increased hepatic gluconeogenesis (secondary to lactic acidosis thatmay lead to increased lactate flux into the liver, fueling gluconeogen-esis), and failure of insulin secretion by pancreatic beta cells second-ary to inactivation of cellular potassium channels due to ATPdepletion [32,39]. End-organ insulin resistance is another possiblemechanism, with NO deficiency having a role in this context. Amajor action of insulin in muscle tissue involves the translocation ofglucose transporters to the plasma membrane and activation ofdownstream pathways of glucose metabolism (glycolytic and oxida-tive metabolism and glycogen synthesis). However, before insulininteracts with the receptor on the plasma membrane, insulin andglucose must be adequately delivered to the muscle cells [40,41]. Ithas been shown that insulin causes an NO-mediated vasodilation,leading to an increase skeletal muscle blood flow [42,43]. Thevasodilatory action of insulin is in part due to stimulation of proteinkinase B (Akt), which directly increases eNOS activity, leading toincreased NO production [44,45]. Therefore, NO deficiency in mito-chondrial disease may lead to impaired insulin-induced vasodilatoryaction, resulting in insulin resistance and the development of DM.

Lactic acidosis is a common finding in many mitochondrial diseases.Lactic acidosis results from an inability of a dysfunctional mitochondrial

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respiratory chain to adequately oxidize glucose, leading to the accumu-lation of pyruvate and shunting of pyruvate to lactate [32]. Moreover,hypoperfusion may result in lactic acidosis due to decreased oxygendelivery to peripheral tissues and a shift to anaerobic glycolysis. NOdeficiency in mitochondrial diseases can result in decreased bloodperfusion, which may further aggravate lactic acidosis.

4. The utility of arginine and citrulline in treating NO deficiency inmitochondrial diseases

Arginine supplementation, an essential component in the treat-ment of urea cycle defects, has also been examined in several otherdisorders, including vascular diseases, glutaric aciduria type I, andcreatine transporter deficiency [46]. Citrulline supplementation hasbeen used in OTC deficiency and short bowel syndrome [12]. Formitochondrial diseases, arginine and citrulline supplementation hasonly been evaluated in MELAS syndrome [3,5,7].

4.1. Arginine supplementation in MELAS syndrome

Both arginine and citrulline act as NO precursors; therefore it hasbeen proposed that their administration can result in increased NOavailability and hence have therapeutic benefits in stroke-like epi-sodes in MELAS syndrome [31]. This hypothesis was supported byclinical studies showing that the administration of intravenous argi-nine to subjects with MELAS syndrome during stroke-like episodesled to improvement in the clinical symptoms associated with theseepisodes, and oral arginine supplementation at the interictal phasedecreased frequency and severity of stroke-like episodes [3,5]. Thetherapeutic effect of arginine in stroke-like episodes in MELAS isproposed to be due to increased NO availability leading to improvingintra-cerebral vasodilation and blood flow. This has been supportedby the demonstration that arginine supplementation to subjectswith MELAS results in increased NO production rate [7] and improvedflow-mediated dilation [4].

4.2. Citrulline supplementation as a NO donor

Although the clinical effects of citrulline administration in mito-chondrial diseases have not been studied, a stable isotope study hasdemonstrated that citrulline supplementation to subjects withMELAS syndrome leads to increased NO production [7]. Interestingly,citrulline supplementation induces a greater increase in the NO syn-thesis rate than that associated with arginine supplementation, indi-cating that citrulline is a more effective NO precursor than arginine[7]. This can be due to the superiority of citrulline in raising plasmaand intracellular arginine levels, as discussed in the following Sec-tions (4.2.1–4.2.3).

4.2.1. Citrulline is more efficient in increasing plasma arginineIt has been demonstrated that oral citrulline supplementation

increases plasma arginine levels more than supplementation of thesame dose of arginine in patients with MELAS syndrome [7]. Thismay be due to higher intestinal absorption of citrulline than that ofarginine due to the action of intestinal arginase II on ingested arginine[22]. Another potential cause of this finding is the fact that citrullinebypasses the liver, whereas arginine is converted to ornithine in theliver through the action of arginase I [12].

Pharmacokinetics (PK) studies have demonstrated that both argi-nine and citrulline have similar PK parameters except for Cmax (maxi-mum plasma concentration) that is several-folds higher with citrullinethan with arginine indicating that citrulline has better absorption andsystemic bioavailability than arginine [47,48]. Another PK study hasshown that citrulline supplementation increased AUC (area under theconcentration-time curve) and Cmax of plasma argininemore effectivelythan arginine supplementation [49].

4.2.2. Citrulline is more efficient in increasing intracellular arginineIt has been proposed that citrulline is superior to arginine in increas-

ing the intracellular arginine pool because of the action of arginase andthe tight regulation of extracellular arginine transport by CAT [22],whereas citrulline acts as a precursor for intracellular arginine synthesis.The de novo arginine synthesis rate has been shown to increase ten-foldafter citrulline supplementation in patients with MELAS syndrome [7].Therefore, it is expected that citrulline results in higher intracellulararginine, explaining the higher NO synthesis rate associated withcitrulline supplementation.

4.2.3. The role of intracellular de novo-synthesized arginineAs we have described earlier, citrulline supplementation to patients

with MELAS syndrome results in a higher NO synthesis rate than thatassociated with arginine supplementation (8 fold vs. 2 fold). Thisincrease in NO production was accompanied by increased de novo argi-nine synthesis and an increased plasma arginine concentration,suggesting that the increase in NO production is driven by increasedarginine availability. Although citrulline supplementation results in ahigher plasma arginine than arginine supplementation (3-fold vs.2.5-fold), an even more robust effect of citrulline supplementation wasobserved with regard to the de novo arginine synthesis rate (10-foldvs. 1.3-fold). Therefore, it was suggested that the de novo-synthesizedarginine plays a more important role in driving NO synthesis [7]. Thisproposal is further supported with the findings suggesting that thethree enzymes responsible for recycling citrulline to NO (ASS, ASL, andNOS) function as a complex [24–26]. Such a complex may lead to NOsynthesis in certain subcellular compartments that utilize citrulline asthe primary substrate, resulting in channeling citrulline to NO synthesismore efficiently than arginine [7].

4.3. Potential therapeutic benefits of arginine and citrullinesupplementation

Increasing NO availability with arginine or citrulline supplementa-tionwill potentially improve perfusion in all microvasculature compart-ments. Therefore, the effect of arginine and citrulline supplementationmay not be limited to improving stroke-like episodes, but may alsolead to improvements in other clinical features of mitochondrialdiseases, including muscle weakness, exercise intolerance, diabetes,and lactic acidosis. Interestingly, arginine and citrulline supplementa-tion has been reported to result in a reduction in plasma alanineconcentrations, suggesting that such supplementations may improvelactic acidemia in MELAS syndrome by increasing NO production andimproving perfusion and oxygen delivery [7]. Based on the finding thatcitrulline supplementation can result in a higher NO production thanarginine supplementation, it was proposed that citrulline may have abetter therapeutic effect than arginine [7].

Clinical research evaluating the effect of arginine and citrulline inmitochondrial diseases is very limited, with the effect of arginine onstroke-like episodes in MELAS syndrome being the only field studiedby uncontrolled open label clinical studies [3,5]. Therefore, additionalmeasures of the clinical effects of arginine and citrulline supplementa-tion on different aspects of mitochondrial diseases are warranted todetermine the potential therapeutic effect of such supplementation.

5. Conclusions

NO deficiency in mitochondrial disorders can result from multipleinterrelated factors. Both arginine and citrulline act as NO precursors;therefore their administration can result in increased NO availabilityand hence may have therapeutic effects on NO deficiency-relatedmanifestations of mitochondrial disease. Citrulline supplementationcan raise NO production to a greater extent than that associatedwith arginine due to the substantial increase in the de novo argininesynthesis rate associated with citrulline supplementation. Therefore,

252 A.W. El-Hattab et al. / Molecular Genetics and Metabolism 107 (2012) 247–252

citrulline supplementation may have a better therapeutic effect thanarginine. Controlled studies assessing the clinical effects of arginineor citrulline supplementation on different aspects of mitochondrialdiseases are needed to support the use of such supplementations asa treatment modality for mitochondrial diseases.

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