Restoration of impaired nitric oxide production in MELAS syndrome with citrulline and arginine supplementation
Highlights
► Using stable isotope infusion we assessed NO metabolism in subjects with MELAS. ► Subjects with MELAS had lower NO synthesis rate. ► Arginine and citrulline supplementation increased NO production in MELAS subjects. ► Citrulline was superior to arginine in increasing NO production. ► Citrulline may have a better therapeutic effect than arginine.
Introduction
Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is one of the most common mitochondrial disorders, with an estimated prevalence of 60:100,000 [1]. MELAS syndrome is primarily caused by mutations in mitochondrial DNA, with the most frequent being the m.3243A>G mutation in the MTTL1 gene that encodes tRNALeu/(UUR) [2]. It has a broad spectrum of manifestations, including stroke-like episodes, exercise intolerance, muscle weakness, epilepsy, dementia, migraine headaches, short stature, sensorineural hearing loss, lactic acidosis, and diabetes [3]. MELAS syndrome typically affects a young population, with 75% of cases presenting before 20 years of age, and causes significant morbidity, with the life-time prevalence of stroke-like episodes approaching 99% [3]. However, no specific consensus approach to its treatment has been established [4].
When stroke-like episodes occur in MELAS syndrome, the affected areas do not correspond to classical vascular territories but rather have an irregular distribution, suggesting that events reflect a small-vessel etiology [5]. The pathogenesis of these stroke-like episodes is not clear; however, it has been suggested that these events are due to ischemic insults, based upon SPECT (single photon emission computed tomography) scanning studies showing hypoperfusion in the affected regions during early stages of stroke-like episodes [6].
Energy depletion due to mitochondrial dysfunction in MELAS syndrome can stimulate mitochondrial proliferation in various tissues, including vascular endothelial cells [7], [8]. It has been suggested that endothelial dysfunction, resulting from mitochondrial proliferation, can lead to impaired cerebral blood flow and stroke-like episodes in MELAS syndrome [5], [9]. One of the functions of the vascular endothelium is to produce nitric oxide (NO), which, amongst a host of properties, plays a major role in smooth muscle relaxation that is needed to maintain the patency of small blood vessels [10], [11]. Thus, endothelial dysfunction may result in impaired NO production. Furthermore, mitochondrial proliferation in endothelial cells is associated with increased cytochrome c oxidase (COX) activity, which can bind to and sequester NO. Thus, decreased NO availability, either by impaired production or sequestration by COX, can potentially play a major role in the mechanism of stroke-like episodes observed in MELAS syndrome [5], [9]. This hypothesis is supported by the observation of lower NO metabolite concentrations during stroke-like episodes in subjects with MELAS syndrome [6].
NO is formed from arginine via the enzyme nitric oxide synthase (NOS), which catalyzes the conversion of arginine to citrulline. The NO is rapidly oxidized to nitrite and nitrate, which are more stable molecules. Citrulline is a nonessential amino acid for which the main source is the de novo synthesis in small intestine enterocytes through a number of mitochondrial enzymes. Citrulline can be converted to arginine via argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL). Arginine is derived from the diet, protein breakdown, and the de novo synthesis from citrulline, the latter constituting 5–15% of arginine production (Fig. 1) [12], [13], [14]. Along with arginine availability, NO synthesis is also regulated by asymmetric dimethylarginine (ADMA) which is an endogenous inhibitor of NOS. ADMA is generated by the methylation of arginine residues in proteins and subsequently released by protein hydrolysis within cells to be transported in plasma. ADMA enters cells by the same cationic amino acid transporters (CATs) used by arginine, and thus can also limit intracellular arginine availability [15]. Elevated ADMA levels can potentially contribute to endothelial dysfunction in a number of human diseases, including chronic renal disease, hypertension, and diabetes [16], [17], [18], [19], [20].
Both arginine and citrulline potentially act as NO precursors, and it has been proposed that their administration may result in increased NO availability and hence can have therapeutic effects in stroke-like episodes in MELAS syndrome [9]. This hypothesis is supported by the demonstration that oral and intravenous arginine administration to subjects with MELAS syndrome led to improvement in the clinical symptoms associated with stroke-like episodes and a decrease in the frequency and severity of these episodes [6], [21] No previous clinical studies have been performed to assess NO production and the effect of arginine on NO production in MELAS syndrome. Likewise, no studies have been conducted to evaluate the effect of citrulline, which can theoretically be a more effective NO precursor than arginine, as arginase may compete with NOS for the intracellular arginine pool. In addition, there is evidence that ASS and ASL co-localize and are co-induced with inducible NOS (iNOS) in various cell types [22]. Such co-localization can result in substrate channeling of citrulline to more efficiently produce NO in certain sub-cellular compartments.
We hypothesized that subjects with MELAS syndrome have impaired NO production, administration of l-arginine or l-citrulline would improve NO production, and l-citrulline would improve NO production to a higher degree than l-arginine supplementation. To test these hypotheses we measured whole body NO synthesis rates, among other variables, in control subjects and subjects with MELAS syndrome before and after arginine or citrulline oral supplementation. In this study, we have used well-established stable isotope infusion protocols to provide a quantitative assessment of whole body in vivo arginine, citrulline, and NO metabolism. Such methods have not been previously employed in this syndrome or other mitochondrial diseases. Therefore, this is the first study to assess the in vivo NO metabolism in subjects with MELAS syndrome.
Section snippets
Subjects
Ten subjects with MELAS syndrome and ten control subjects participated in this study. Subjects with MELAS syndrome were adults aged 18–57 years, diagnosed clinically with MELAS syndrome and harbored the m.3243A>G mutation in the MTTL1 gene (Supplementary Table 1). Control subjects were healthy adults aged 20–46 years. We aimed to obtain a control cohort that was age-, gender, and BMI-matched to the cohort of subjects with MELAS. The study was approved by the Institutional Review Board (IRB) at
Results
Subjects with MELAS syndrome had a lower mean body weight and height (p < 0.05), but no significant differences in age, gender, and body mass index (Table 1). Subjects with MELAS syndrome were found to have lower plasma arginine (p < 0.001) and citrulline (p < 0.05) concentrations. They also had higher concentrations of plasma alanine (p < 0.001) and branched-chain amino acids (BCAA) valine (p < 0.005), leucine (p < 0.05), and isoleucine (p < 0.05). Other amino acids did not show significant differences
Discussion
This study aimed to compare NO synthesis rates, amongst other related variables, between subjects with MELAS syndrome and control subjects, and to assess the effect of arginine and citrulline supplementation. The use of stable isotope infusion protocols in subjects with this syndrome provided a quantitative assessment of whole body arginine, citrulline, and NO metabolism.
Both impaired NO production due to endothelial dysfunction and post-production sequestration by increased COX have been
Conclusions
In conclusion, lower NO production in subjects with MELAS syndrome may result from several mechanisms, including higher plasma ADMA concentrations and lower arginine availability due to increased arginine clearance and decreased de novo arginine synthesis. Based on our finding that citrulline supplementation results in higher NO production than arginine supplementation, we propose that a major determinant of NO production is the de novo arginine synthesis, which is substantially increased with
Acknowledgment
This study was funded by the Society for Inherited Metabolic Disorders (SIMD) through the SIMD/Hyperion fellowship award that Ayman El-Hattab has received. We also acknowledge the patients and their families for their participation, the National Institutes of Health for funding the General Clinical Research Center (GCRC) (M01-RR0188), the GCRC staff at Texas Children's Hospital, the United Mitochondrial Disease Foundation (UMDF), and the Pfeiffer Foundation.
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