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MELAS and l -arginine therapy
Mitochondrion 7 (2007) 133–139 www.elsevier.com/locate/mito
MELAS and L-arginine therapy Yasutoshi Koga a,*, Yukihiro Akita a, Junko Nishioka a, Shuichi Yatsuga a, Nataliya Povalko a,b, Koujyu Katayama a, Toyojiro Matsuishi a a
Department of Pediatrics and Child Health, Kurume University School of Medicine, 67 Asahi Machi, Kurume, Fukuoka 830-0011, Japan b Research Centre for Medical Genetics RAMNS, Moskvorechie St. 1, Moscow 115478, Russia Received 8 August 2006; accepted 24 October 2006 Available online 5 December 2006
Abstract We investigated the endothelial function in MELAS patients and also evaluated the therapeutic effects of L-arginine. Concentrations of L-arginine during the acute phase of MELAS were significantly lower than in control subjects. L-arginine infusions significantly improved all symptoms suggesting stroke within 30 min, and oral administration significantly decreased frequency and severity of stroke-like episodes. Flow-mediated dilation (FMD) in patients showed a significant decrease than those in the controls. Two years of oral supplementation of L-arginine significantly improved endothelial function to the control levels and was harmonized with the normalized plasma levels of L-arginine in patients. L-arginine therapy showed promise in treating stroke-like episodes in MELAS. Ó 2007 Elsevier B.V. and Mitochondria Research Society. All rights reserved. Keywords: MELAS; Clinical trial; Endothelial function; L-arginine; Stroke-like episodes
1. Introduction Mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) is a maternally inherited multisystem mitochondrial disorder (Pavlakis et al., 1984). Mitochondrial angiopathy, with degenerative changes in small arteries and arterioles, has been reported in many MELAS patients (Ohama et al., 1987; Kishi et al., 1988); these blood vessels have been designated strongly succinate dehydrogenase-reactive vessels (SSVs) (Hasegawa et al., 1991). However, the primary cause for stroke-like episodes in young MELAS patients – whether mitochondrial cytopathy, angiopathy, or both – remains controversial. Although many therapeutic trials have been undertaken to cure mitochondrial disorders, few trials have succeeded in reversing the enzymatic abnormality (Manfredi et al., 2002). Among the successes, several pharmacologic agents have been reported to be effective in MELAS (Kuroda et al., 1997; Penn et al., 1992; Ikejiri et al., 1996; *
Corresponding author. Tel.: +81 942 31 7565; fax: +81 942 38 1792. E-mail address: [email protected] (Y. Koga).
Napolitano et al., 2000). However, no effective therapeutic strategies have focused on the acute stroke phase in MELAS. Based on a hypothesis that stroke-like episodes in MELAS are caused by segmental impairment of vasodilation in intracerebral arteries, we infused L-arginine in MELAS patients during the acute phase of stroke (Koga et al., 2002, 2005). We reported that L-arginine therapy quickly decreased severity of stroke-like symptoms in MELAS, enhanced dynamics of the microcirculation, and also reduced tissue injury from ischemia. Recently, cardioprotective effects of L-arginine and nitric oxide (NO) have been ascribed to endothelial cell preservation (Shiono et al., 2002), decreased neutrophil activation (Kubes et al., 1991), improved coronary blood flow, and reduced free radical-mediated injury (Radomski and Moncada, 1993). Since mitochondria are an important cellular source of reactive oxygen species (ROS), MELAS patients are always at high risk of oxidative stress (Oexle and Zwirner, 1997; Filosto et al., 2002), as they often have respiratory chain dysfunction caused by the genetic abnormality. Many human disorders associated with endothelial dysfunction ultimately lead to arterial occlusive disorders,
1567-7249/$ - see front matter Ó 2007 Elsevier B.V. and Mitochondria Research Society. All rights reserved. doi:10.1016/j.mito.2006.11.006
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Y. Koga et al. / Mitochondrion 7 (2007) 133–139
including hypercholesterolemia (Ross, 1993), hypertension (Vanhoutte, 1996), diabetes mellitus (Tilton et al., 1997), cardiovascular diseases (Drexler et al., 1992), and homocystinuria (de Groot et al., 1983). Blood concentrations of L-arginine, a precursor of NO in the reaction catalyzed by endothelial nitric oxide synthase (NOS), and/or those of asymmetrical dimethylarginine (ADMA), an endogenous competitive inhibitor of NO synthase, recently have been found to importantly influence endothelial function in hypercholesterolemia (Boger et al., 1998), or ischemic heart diseases (Maxwell et al., 2002), hyperglycemia (Giugliano et al., 1997), pulmonary hypertension (Pearson et al., 2001), and necrotizing enterocolitis (Zamora et al., 1997). In the present study, we examined how L-arginine therapy during the acute phase of stroke-like episodes determines how plasma concentrations of endothelial regulatory factors including L-arginine, NOx (metabolites serving as markers for NO), cyclic GMP, and ADMA differ in patients with MELAS from concentrations in normal subjects. In MELAS patients, we also compared concentrations during the acute stroke phase with those during the interictal phase. In addition, we administered L-arginine intravenously to the patients in a therapeutic trial aiming to improve endothelial-dependent vasodilation, cerebral blood flow as evident by ECD single-photon emission computed tomography (ECD-SPECT), and symptoms suggesting the acute phase of stroke. Finally we evaluated long-term oral administration of L-arginine (6–24 g/day for more than 6 months) for effects on incidence and outcome of stroke-like attacks, cerebral blood flow, and on the endothelial function by FMD analysis. 2. Methods 2.1. Patients Among patients referred to our hospital in Kurume, Japan between April 1, 1992 and April 30, 2003, 24 were diagnosed with MELAS according to clinical findings, muscle histopathologic, and genetic studies. All patients were Japanese who had an A3243G mutation in the mitochondrial tRNALeu(UUR) gene in muscle homogenate as well as in lymphocytes. In 18 of 24 patients, basal growth hormone secretion was low, and growth hormone deficiency was diagnosed by a 0.5 g/kg of L-arginine loading test. Patients with congenital anomalies, sepsis, intravenous hyperalimentation, diabetes mellitus, cardiac failure, or a bedridden state were excluded from this study. 2.2. L-arginine therapy at acute episodes of stroke Twenty-four MELAS patients or patients’ parents gave informed consent, and the L-arginine study protocol was approved by the University Ethics Committee (Kurume University IRB#9715). The study design was chosen because patient availability and finances did not permit a balanced randomized design involving multiple centers.
Among 34 times for stroke-like episodes, patients took part in this study of L-arginine vs. placebo. Beginning within 3 h of the onset of stroke-like symptoms, patients were administered L-arginine (Arugi U, Ajinomoto Pharma, Tokyo, Japan; 0.5 g/kg/dose) as a 10% solution in 24 separate episodes; a placebo (5% dextrose, 0.5 g/kg/dose) in 6 other episodes; and D-arginine (Wako Pure Chemical Industries, Tokyo, Japan) in a 10% solution in the remaining 4 episodes. Each treatment was given intravenously over 15 min during the acute phase of stroke. The following symptoms were evaluated before and at 15 min, 30 min, and 24 h after administration: headache (scored on a scale from 0 for no pain to 3, severe pain), clinical disability (scored from 0 for no disability to 3, severe disability), nausea (present or absent), vomiting (present or absent), and teichopsia (present or absent), as described elsewhere (Lassen et al., 1997). Biochemical determinations in plasma included concentrations of L-arginine, L-citrulline, ADMA, pyruvate, and lactate, as well as nitric oxide metabolites (NOx) and cGMP. In several episodes intracranial hemodynamics were measured using ECD-SPECT (approximate total radioactivity, up to 740 MBq) before and after L-arginine administration. 2.3. Long-term health of MELAS patients with oral supplementation
L-arginine
Six patients were treated by oral administration of to prevent stroke-like episodes (i.e., to maintain the interictal condition). Four to 24 g of L-arginine (Arugi U, Ajinomoto Pharma; 0.15–0.3 g/kg/day) was given orally for 12–18 months. Patients were monitored clinically and biochemically as described above once every 2 weeks. When patients admitted to the hospital for a stroke-like episode, following symptoms were scored: headache (present 1, none 0), vomiting (present 1, none 0), teichopsia (present 1, none 0), convulsion (present 1, none 0) and hemiparesis (present 1, none 0). For each admission during the study periods, these scores were summed as the severity score for the stroke. Frequency of admission was taken to be the frequency of stroke-like episodes. Severity and frequency were related to time as number/month and were compared between periods 18 months before and 12–18 months after oral administration of L-arginine.
L-arginine
2.4. Laboratory measurements Analysis of amino acids was performed on protein-free extracts of fresh plasma using described methods. Plasma concentrations of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO synthase, were measured by high-performance liquid chromatography (HPLC) as previously described (Boger et al., 1997). Plasma or urine concentrations of NO metabolites were detected spectrophotometrically by measuring the final stable equimolar degradation products, nitrite and nitrate (NOx) as described previously (Nims et al., 1996). Plasma cGMPs
Y. Koga et al. / Mitochondrion 7 (2007) 133–139
was measured by radioimmunoassay using a cGMP assay kit (Yamasa, Tokyo, Japan) (Honma et al., 1977). Plasma samples obtained from patients and control subjects were frozen immediately at 20 °C; cGMP was succinylated directly without prior deproteinization and then allowed to be bound by the antibody in an imidazole buffer. 2.5. Endothelial function analysed by FMD FMD analysis was performed in the morning after overnight fasting. With subjects in a supine position, we measured FMD by brachial-artery diameter responses to hyperemic flow (endothelium-dependent), before and 2-h after IV infusion of L-arginine (0.5 g/kg of 10% solution), or placebo administration, with high resolution ultrasonography (SSA-380A, Toshiba, Tokyo), as described previously. The patients’ endothelial function was also measured after 2 years of treatment by oral administration of L-arginine to prevent stroke-like episodes (i.e., to maintain the interictal condition). Four to 24 g of L-arginine (Arugi U, Ajinomoto Co.; 0.15–0.3 g/kg/day) was given orally for at least 24 months. The improvements of distribution of cerebral blood circulation (CBF) were measured by SPMSPECT after IV administration or oral supplementation of L-arginine. We compared FMD measurements in MELAS patients with those of controls using unpaired ttests. We also compared the dates before and after the oral L-arginine supplementation with paired t-tests. All data are presented as means + SD. P values of 0.05 or less were considered significant. 2.6. Intracranial hemodynamics according to SPECT In 6 patients, intracranial hemodynamics were measured using ECD-SPECT (approximate total radioactivity where appropriate, 740 MBq) before and after L-arginine administration. Regional cerebral blood flow (rCBF) was measured in the appropriate region of interest (ROI) compared with the same region on the healthy contra lateral side. 2.7. Statistical analysis Plasma concentrations of amino acids, NOx, and ADMA in patients in the acute stroke phase or interictal phase of MELAS were compared with those in controls using unpaired t-tests, with Bonferroni corrections for outlying values. All data are presented as means + SD. P values of 0.025 or less were considered to indicate statistical significance. Concentrations of L-arginine, L-citrulline, NOx, ADMA, and cGMP in plasma obtained before, 30 min after, and 24 h after L-arginine infusion in patients during the acute phase of stroke were compared with those in controls using paired t-tests with Bonferroni corrections for outlying values. All data are presented as means + SD. P values of 0.025 or less were considered to indicate statistical significance. Statistical analysis of clinical improvement was per-
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formed using Fisher’s exact test; for this the level of significance was set at p < 0.05. Frequency and severity of stroke-like episodes in six MELAS patients after long-term oral L-arginine supplementation were compared with those in patients without supplementation using a nonparametric Mann–Whitney U test. All data are presented as means + SD. Here, P values of 0.05 or less were considered to indicate statistical significance. 3. Results 3.1. Characteristics of patients and controls Ninety-six children, all Japanese were enrolled. Twentyfour were MELAS patients, while 72 were free from neuromuscular and metabolic disorders, serving as controls. All MELAS patients had an A3243G mutation in the mitochondrial tRNALeu(UUR) gene. Clinical onset of disease was at 6.0 ± 4.2 years (means + SD). Of 24 patients, 22 showed dwarfism (stature below 2.5 SD); 15 of these patients had received growth hormone supplementation therapy (0.175 mg/kg/week) at some time before the study. None of the MELAS patients received insulin injection therapy. No significant differences were noted in baseline characteristics of patients and control groups except for height, body mass index (BMI), and biochemical parameters such as amino acids, pyruvate, lactate, and L/P ratio. 3.2. L-arginine, citrulline, NOx, and ADMA in acute and interictal phases of MELAS and controls Concentrations of amino acids were significantly altered in MELAS from those in controls (Table 1). Mean plasma concentrations of L-arginine and citrulline were significantly lower in both acute and interictal phases of MELAS than in controls. Concentrations of L-arginine in the acute phase also were significantly lower than in the interictal phase, while those of citrulline did not show a significant phase-related change. NOx concentrations were significantly lower in the acute phase of MELAS than in controls, while in Table 1 L-arginine, citrulline, NOx, and ADMA in the acute and interictal phase of MELAS and in controlsa L-arginine
Control MELAS Interictal phase Acute phase
Citrulline
NOx
ADMA
108.1 ± 27.6 34.6 ± 8.8
45.4 ± 30.1 0.53 ± 0.15
83.6 ± 25.8 P < 0.01b 46.6 ± 12.7 P < 0.01b P < 0.01c
91.4 ± 44.4 P < 0.01b 24.0 ± 9.8 P < 0.01b P < 0.01c
26.2 ± 9.6 P < 0.01b 23.2 ± 10.2 P < 0.01b NSc
0.42 ± 0.10 P < 0.01b 0.45 ± 0.10 NSb NSc
NS, not significant; NOx, nitric oxide metabolites; ADMA, asymmetrical dimethylarginine. a Plus-minus values are means ± SD. Values are expressed as lmol/l of plasma. b Compared with control. c Compared with interictal phase.
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the interictal phase they were significantly higher than in controls. On the other hand, concentrations of ADMA did not significantly differ between controls and acute-phase patients, although the ADMA/L-arginine ratio was significantly higher in the acute phase than in the controls or in the interictal phase (0.011 ± 0.004, vs. 0.005 ± 0.001, and 0.005 ± 0.001, respectively). 3.3. Effects of L-arginine therapy in the acute phase of stroke-like episodes in MELAS After administration of L-arginine, all symptoms suggesting stroke dramatically improved. Effects on headache, clinical disability, nausea, vomiting, and teichopsia were marked by 30 min after L-arginine administration (Koga et al., 2005). No adverse effects occurred except for headache when L-arginine was infused too rapidly in two patients. Mean arterial pressure after L-arginine treatment reached a minimum (representing a decreased of 10 mm Hg) at 30 min after administration, coinciding with the L-citrulline peak (45.6 ± 14.4 lmol/l). Lactate and pyruvate in plasma were significantly normalized at 24 h after treatment, and becoming comparable to amount present during the interictal phase. Concentrations of NOx and cGMP in treated episodes reached a maximum 30 min after L-arginine administration (55.9 ± 20.4, and 0.86 ± 0.16 lmol/l, respectively), and were significantly greater than with placebo. With treatment, concentrations of L-arginine, citrulline, NOx, cGMP, and ADMA returned to interictal-phase concentrations by 24 h. 3.4. Effects of L-arginine therapy on cerebral blood flow during acute stroke episodes At 60 min after L-arginine infusion, the focal decrease in rCBF in the ischemic region had become less marked severe moderate mild severe moderate mild severe moderate mi l d severe moderate mild sev er e m oderate mild seve re moderate mild
according to SPECT, but the percent increase was less than 13% (12.8 ± 2.9%, means + SD, n = 6) that seen contralaterally. 3.5. Long-term outcome of stroke-like episodes after oral supplementation
L-arginine
After oral L-arginine supplementation, the frequency and severity of symptoms caused by the stroke were decreased dramatically (Fig. 1). Frequency of stroke-like episodes after treatment (0.09 ± 0.09) was significantly decreased than that before supplementation (0.78 ± 0.42) (p = 0.03). Severity score after treatment (0.17 ± 0.18) was also significantly decreased than that before supplementation (2.04 ± 0.34) (p = 0.03). After L-arginine supplementation therapy, no MELAS patient had a major stroke-like attack including hemiconvulsion or hemiparesis, but only headache or teichopsia. Plasma concentrations of L-arginine in MELAS patients ranged from 81.6 to 120.1 lmol/l (91.6 ± 16.8; mean ± SD) after initiation of L-arginine supplementation. 3.6. Endothelial functions in MELAS There was a decrease of FMD in the MELAS patients (104.7 ± 1.8) compared to the controls (112.2 ± 1.7) (p < 0.05). Two hours after L-arginine loading, FMD values in the controls showed no improvement compared to the levels before (112.5 ± 1.2), however, those in MELAS patients (108.1 ± 2.6) improved compared to the levels before (104.7 ± 1.8) (p < 0.05). FMD values after 2 years supplementation of L-arginine (113.1 ± 2.4) were improved compared to the original values (104.7 ± 1.8) (p < 0.05), and became control levels (112.2 ± 1.7) in all MELAS patients (Koga et al., 2006).
Case 1
0.17 0.33/kg
Case 2
0.44/kg
Case 3
0.04 0.09/kg
Case 4
0.08 0.24/kg
Case 5
0.16/kg
Case 6
0.19/kg ARG-U Dose
-24M
-12M
0
12M
24M
Fig. 1. Oral L-arginine supplementation improve the severity and frequency of stroke-like episodes in MELAS. Frequency of stroke-like episodes after treatment (0.09 ± 0.09) was significantly decreased than that before supplementation (0.78 ± 0.42) (p = 0.03). Severity score after treatment (0.17 ± 0.18) was also significantly decreased than that before supplementation (2.04 ± 0.34) (p = 0.03). Plasma concentrations of L-arginine in MELAS patients ranged from 81.6 to 120.1 lmol/l (91.6 ± 16.8; mean ± SD) after initiation of L-arginine supplementation.
Y. Koga et al. / Mitochondrion 7 (2007) 133–139
4. Discussion MELAS, a mitochondrial myopathy, is a progressive disease for which adequate treatment is not available. We investigated the therapeutic effects of L-arginine by intravenous administration during the acute phase of stroke-like episodes and by oral administration during the interictal phase. L-arginine, which plays an important role in endothelialdependent vascular relaxation (Moncada and Higgs, 1993), was significantly lowered in both acute and interictal phases of MELAS than in control subjects (Fig. 2). Plasma L-arginine homeostasis is a complex process affected by arginine intake, arginine consumption, and endogenous arginine production. The latter occurs principally in the kidneys (conversion of citrulline to L-arginine), in the gut (production of citrulline and direct conversion of glutamine to L-arginine), and in the liver (arginine-urea cycles). The significant decrease in plasma L-arginine and NOx in the acute phase of MELAS may be partially explained by limited arginine intake, but, the decreased concentration of L-arginine in the interictal phase cannot be explained solely in this manner. Reduced availability of L-arginine may be induced by reduced activity of the cationic amino acid transporter, increased activity of arginase, and/or reduced activity of argininosuccinate synthase (Cooke and Dzau, 1997). Why plasma L-arginine is decreased in the acute phase of MELAS remains to be elucidated. Considering significantly increased NOx together with significantly decreased L-arginine and citrulline in the interictal phase compared with control subjects, limited L-arginine consumption, increased metabolic demand for arginine, and/ or limited endogenous synthesis may be related to the pathogenesis of MELAS. Concentrations of ADMA, an endogenous competitive inhibitor of NO synthase, were not
significantly different in the acute phase of MELAS from concentrations in controls. ADMA recently has been reported to be a novel risk factor for endothelial dysfunction in humans (Ito et al., 1999). A putative endogenous regulator of NOS activity, ADMA inhibits NO-dependent endothelially mediated vasodilation, this inhibition has been reversed by exogenous arginine in hypercholesterolemia (Ito et al., 1999) and cardiac syndrome X (Piatti et al., 2003). Normally, ADMA is degraded by dimethylarginine dimethyl-aminohydrolase (DDAH) to L-citrulline and dimethylamine, which activity in human endothelial cells was affected by oxidized low-density lipoprotein (oxLDL) or tumor necrosis factor (TNF-a). Since concentrations of total cholesterol and LDL cholesterol did not differ between MELAS and control subjects, the increased ADMA in MELAS is unlikely to be caused by induction of DDAH. However, ADMA and DDAH may provide a mechanism for control of NO synthesis in physiologic or pathologic states. The ADMA/L-arginine ratio was significantly greater in the acute phase than in control subjects or in the interictal phase, suggesting that endothelial-dependent vascular relaxation is impaired in the acute phase of MELAS. A low L-arginine concentration and a relatively high ADMA concentration may predispose to stroke-like episodes in MELAS. Impairment of endothelial function associated with increased ADMA concentrations is reversed by intravenous L-arginine (Piatti et al., 2003). Consistent with these data, we ameliorated the ischemic process during acute phase of MELAS by L-arginine infusion. The primary cause of stroke-like episodes in MELAS remains unknown. Focal cerebral hyperemia has been reported in MELAS by several investigators (Gropen et al., 1994). Although the underlying mechanisms is incompletely understood, the hyperemia is thought to reflect vasodilation caused by local metabolic acidosis in
L-arginine
cytokine glucocorticoids NADPH/NADP
induction
endothelium
RC deficiency
137
L-arginine
NOS
NO
Superoxide
cytokine glucocorticoids
smooth muscle
induction
ADMA NOS
+ NADPH/NADP
L-arginine
Relaxation
L-arginine
sGC NO
+
GTP
Cyclic GMP
Fig. 2. Endothelial function improved by L-arginine supplementation.
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the area of the infarct. Another explanation is that the focal hyperperfusion corresponds to foci of periodic epileptiform discharge, gradually dissipating along with the periodic discharges (Iizuka et al., 2002). However, most cerebral blood flow studies in MELAS patients are performed at several days or several weeks after onset of a stroke-like episode. Several days after onset of stroke, secondarily induced NO production generated by inducible NOS in the region injured may alter evidence of the primary pathophysiologic abnormality. In an analysis of SPECT findings in young MELAS patients at a very early stage of infarction (within 3 h after onset), we invariably found hypoperfusion in the region affected by the strokelike episode (Koga et al., 2005). We cannot explain conclusively why our findings differ from those reported by neurologists treating adults. First, differences may result from timing of examinations. We performed SPECT very early following stroke-like episodes in MELAS, before NO secondarily induced by inducible NO synthase would create further ischemic brain damage. Second, pediatric MELAS patients appear to have more severe and more uniform symptoms than in adult patients. Heteroplasmy of the A3243G mutation in the mitochondrial tRNALeu(UUR) gene is much greater in early-onset MELAS patients (before 15 years) than in late-onset MELAS (late 30 s or older). Third, adult MELAS patients usually acquire additional risk factors for atherosclerosis, such as hypercholesterolemia, hyperglycemia, and hypertension which usually are not seen in young MELAS patients. If the sites of angiopathy in MELAS most likely include small cerebral arteries, arterioles, and capillaries, small infarcts would be expected rather than the large confluent region of infarction described in many reports of MELAS. Yoneda et al. (1999) recently reported finding a contribution from vasogenic edema in stroke-like lesions in a 10-yearold patient with MELAS. Thirty minutes after administration of L-arginine during the acute phase of MELAS, we observed that symptoms suggesting stroke decreased dramatically. Since citrulline, lactate, NOx, cGMP, and ADMA had increased significantly at 30 min after treatment, these increases are considered to be pharmacologic effects of L-arginine treatment. Alleviation of symptoms at 30 min after treatment correlated well with peaks for NOx, cGMP, and citrulline. On SPECT, decreased cerebral blood flow (rCBF) in the ischemic region showed improvement; however, the percent increase was less than 13% (12.9 ± 2.6%: mean ± SD) of the increase on the contralateral side. Since L-arginine has not been found to produce cerebral vasodilation in the region of an old infarct (Sporer et al., 1997), we could not reliably interpret the percent increase in rCBF because the areas studied often included old infarcts. In this study we attempted to prevent or limit ischemic brain damage in the acute phase (within 3 h) of stroke-like episodes in MELAS by giving L-arginine to induce vasodilation. L-arginine is an important precursor of NO, which may reduce ischemic damage in the acute phase of focal brain
ischemia by increasing microcirculation in the cerebral blood flow (Morikawa et al., 1994), inhibiting post-ischemic leukocyte-endothelial adhesion (Gidday et al., 1998), decreasing quantities of hydroxyl radical (Wink et al., 1993), and reducing brain damage in early stages of cerebral ischemia (Zhang et al., 1994). However, details of mechanisms involving L-arginine and NO are not yet fully known. We evaluated long-term outcome of stroke-like episodes after oral L-arginine supplementation. After this therapy, the frequency and severity of clinical symptoms caused by stroke were dramatically decreased without any serious adverse effects (Fig. 1). No prophylactically treated MELAS patients have had major stroke-like attacks such as hemiconvulsion and hemiparesis, but only headache and teichopsia. We concluded that L-arginine supplementation currently is the best interictal therapy for preventing stroke-like episodes in MELAS. Our data indicated that L-arginine infusion during the acute phase significantly enhanced the microcirculation and reduced tissue injury from ischemia, while oral L-arginine supplementation during the interictal phase significantly decreased the frequency and severity of stroke-like attacks in MELAS. Such treatment shows promise in the acute stroke-like phase and in the interictal phase of MELAS. Acknowledgements This work was supported in part by grants #13670853 (Y.K.), and #16390308 (Y.K.) from the Ministry of Culture and Education in Japan, #H14-006 (Y.K.), and #H14-team(syouni)-005 (Y.K.) from Evidence-based Medicine, Ministry of Health, Labor and Welfare in Japan, The research Grant (17A-10) for Nervous and Mental Disorders from the Ministry of Health, Labour ad Welfare in Japan; and Uehara Memorial Foundation. Y.A. is a recipient of grant in aid for young investigator #17790733 from the Ministry of Culture and Education in Japan. References Boger, R.H., Bode-Boger, S.M., Thiele, W., Junker, W., Alexander, K., Frolich, J.C., 1997. Biochemical evidence for impaired nitric oxide synthesis in patients with peripheral arterial occlusive diseases. Circulation 95, 2068–2074. Boger, R.H., Bode-Boger, S.M., Szuba, A., Tsao, P.S., Chan, J.R., Tangphao, O., Blaschke, T.F., Cooke, J.P., 1998. Asymmetric dimethylarginine (ADMA): a novel risk factor for endothelial dysfunction. Its role in hypercholesterolemia. Circulation 98, 1842–1847. Cooke, J.P., Dzau, V.J., 1997. Derangements of the nitric oxide synthase pathway, L-arginine, and cardiovascular diseases. Circulation 96, 379– 382. de Groot, P.G., Willems, C., Boers, G.H., Gonsalves, M.D., van Aken, W.G., van Mourik, J.A., 1983. Endothelial cell dysfunction in homocystinuria. Eur. J. Clin. Invest. 13 (5), 405–410. Drexler, H., Hayoz, D., Munzel, T., Hornig, B., Just, H., Brunner, H.R., Zelis, R., 1992. Endothelial function in chronic congestive heart failure. Am. J. Cardiol. 69, 1596–1601. Filosto, M., Tonin, P., Vattemi, G., Spagnolo, M., Rizzuto, N., Tomelleri, G., 2002. Antioxidant agents have a different expression pattern in
Y. Koga et al. / Mitochondrion 7 (2007) 133–139 muscle fibers of patients with mitochondrial diseases. Acta Neuropathol. 103, 215–220. Gidday, J.M., Park, T.S., Shah, A.R., Gonzales, E.R., 1998. Modulation of basal and post-ischemic leukocyte-endothelial adherence by nitric oxide. Stroke 29, 1423–1430. Giugliano, D., Marfella, R., Coppola, L., Verrazzo, G., Acampora, R., Giunta, R., Nappo, F., Lucarelli, C., D’Onofrio, F., 1997. Vascular effects of acute hyperglycemia in humans are reversed by L-arginine. Evidence for reduced availability of nitric oxide during hyperglycemia. Circulation 95, 1783–1790. Gropen, T.I., Prohovnik, I., Tatemichi, T.K., Hirano, M., 1994. Cerebral hyperemia in MELAS. Stroke 25, 1873–1876. Hasegawa, H., Matsuoka, T., Goto, Y., Nonaka, I., 1991. Strongly succinate dehydrogenase-reactive blood vessels in muscles from patients with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes. Ann. Neurol. 29, 601–605. Honma, M., Satoh, T., Takezawa, J., Ui, M., 1977. An ultrasensitive method for the simultaneous determination of cyclic GMP in smallvolume samples from blood and tissue. Biochem. Med. 18, 257–273. Iizuka, T., Sakai, F., Suzuki, N., Hata, T., Tsukahara, S., Fukuda, M., Takiyama, Y., 2002. Neuronal hyperexcitability in stroke-like episodes of MELAS syndrome. Neurology 59, 816–824. Ikejiri, Y., Mori, E., Ishii, K., Nishimoto, K., Yasuda, M., Sasaki, M., 1996. Idebenone improves cerebral mitochondrial oxidative metabolism in a patient with MELAS. Neurology 47, 583–585. Ito, A., Tsao, P.S., Adimoolam, S., Kimoto, M., Ogawa, T., Cooke, J.P., 1999. Novel mechanism for endothelial dysfunction dysregulation of dimethylarginine dimethylaminohydrolase. Circulation 99, 3092– 3095. Kishi, M., Yamamura, Y., Kurihara, T., Fukuhara, N., Tsuruta, K., Matsukura, S., Hayashi, T., Nakagawa, M., Kuriyama, M., 1988. An autopsy case of mitochondrial encephalomyopathy: biochemical and electron microscopic studies of the brain. J. Neurol. Sci. 86, 31–40. Koga, Y., Ishibashi, M., Ueki, I., Yatsuga, S., Fukiyama, R., Akita, Y., Matsuishi, T., 2002. Effects of L-arginine on the acute phase of strokes in three patients with MELAS. Neurology 58, 827–828. Koga, Y., Akita, Y., Nishioka, J., Yatsuga, S., Povalko, N., Tanabe, Y., Fujimoto, S., Matsuishi, T., 2005. L-arginine improves the symptoms of stroke-like episodes in MELAS. Neurology 64, 710–712. Koga, Y., Akita, Y., Nishioka, J., Yatsuga, S., Povalko, N., Fukiyama, R., Ishii, M., Matsuishi, T., 2006. Endothelial dysfunction in MELAS was improved by L-arginine supplementation. Neurology 66, 1766– 1769. Kubes, P., Suzuki, M., Granger, D.N., 1991. Nitric oxide:An endogeneous modulator of leukocyte adhesion. Proc. Natl. Acad. Sci. USA 88, 4651–4655. Kuroda, Y., Ito, M., Naito, E., Yokota, I., Matsuda, J., Saijo, T., Kondo, S., Yoneda, Y., Miyazaki, M., Mori, K., Iwamoto, H., 1997. Concomitant administration of sodium dichloroacetate and vitamin B1 for lactic acidemia in children with MELAS syndrome. J. Pediatr. 131, 450–452. Lassen, L.H., Ashina, M., Christiansen, I., Ulrich, V., Olesen, J., 1997. Nitric oxide synthase inhibition in migraine. Lancet 349, 401–402. Manfredi, G., Fu, J., Ojaimi, J., Sadlock, J.E., Kwong, J.Q., Guy, J., Schon, E.A., 2002. Rescue of a deficiency in ATP synthesis by transfer of MTATP6, a mitochondrial DNA-encoded gene, to the nucleus. Nat. Genet. 30 (4), 394–399. Maxwell, A.J., Zapien, M.P., Pearce, G.L., MacCallum, G., Stone, P.H., 2002. Randomized trial of a medical food for the dietary management of chronic, stable angina. J. Am. Coll. Cardiol. 39, 37–45. Moncada, S., Higgs, A., 1993. The L-arginine-nitric oxide pathway. N. Engl. J. Med. 329, 2002–2012. Morikawa, E., Moskowitz, M.A., Huang, Z., Yoshida, T., Irikura, K., Dalkara, T., 1994. L-arginine infusin promotes nitric oxide-dependent vasodilation, increases regional cerebral blood flow, and reduces infarction volume in the rat. Stroke 25, 429–435.
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Napolitano, A., Salvetti, S., Vista, M., Lombardi, V., Siciliano, G., Giraldi, C., 2000. Long-term treatment with idebenone and riboflavin in a patient with MELAS. Neurol. Sci. 21, S981–S982. Nims, R.W., Cook, J.C., Krishna, M.C., Christodoulou, D., Poore, C.M., Miles, A.M., Grisham, M.B., Wink, D.A., 1996. Colorimetric assays for nitric oxide and nitrogen oxide species formed from nitric oxide stock solutions and donor compounds. Meth. Enzymol. 268, 93–105. Oexle, K., Zwirner, A., 1997. Advanced telomere shortening in respiratory chain disorders. Hum. Mol. Genet. 6, 905–908. Ohama, E., Ohara, S., Ikuta, F., Tanaka, K., Nishizawa, M., Miyatake, T., 1987. Mitochondrial angiopathy in cerebral blood vessels of mitochondrial encephalomyopathy. Acta Neuropathol. 74, 226–233. Pavlakis, S.G., Phillips, P.C., DiMauro, S., De Vivo, D.C., Rowland, L.P., 1984. Mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes: a distinctive clinical syndrome. Ann. Neurol. 16, 481–488. Pearson, D.L., Dawling, S., Walsh, W.F., Haines, J.L., Christman, B.W., Bazyk, A., Scott, N., Summar, M.L., 2001. Neonatal pulmonary hypertension. Urea-cycle-intermediates, nitric oxide production, and carbamoyl-phosphate synthetase function. N. Engl. J. Med. 344, 1832– 1838. Penn, A.M., Lee, J.W., Thuillier, P., Wagner, M., Maclure, K.M., Menard, M.R., Hall, L.D., Kennaway, N.G., 1992. MELAS syndrome with mitochondrial tRNALeu(UUR) mutation: correlation of clinical state, nerve conduction, and muscle 31P magnetic resonance spectroscopy during treatment with nicotinamide and riboflavin. Neurology 42, 2147–2152. Piatti, P., Fragasso, G., Monti, L.D., Setola, E., Lucotti, P., Fermo, I., Paroni, R., Galluccio, E., Pozza, G., Chierchia, S., Margonato, A., 2003. Acute intravenous L-arginine infusion decreases endothelin-1 levels and improves endothelial function in patients with angina pectoris and normal coronary arteriograms. Circulation 107, 429–436. Radomski, M.W., Moncada, S., 1993. The biological and pharmacological role of nitric oxide in platelet function. Adv. Exp. Med. Biol. 344, 251–264. Ross, R., 1993. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 362 (6423), 801–809. Shiono, N., Rao, V., Weisel, R.D., Kawasaki, M., Li, R.K., Mickle, D.A., Fedak, P.W., Tumiati, L.C., Ko, L., Verma, S., 2002. L-arginine protects human heart cells from low-volume anoxia and reoxygenation. Am. J. Physiol. 282, H805–H815. Sporer, B., Martens, K.H., Koedel, U., Haberl, R.L., 1997. L-arginineinduced regional cerebral blood flow increase is abolished after transient focal cerebral ischemia in the rat. J. Cereb. Blood Flow. Met. 17, 1074–1080. Tilton, R.G., Kawamura, T., Chang, K.C., Ido, Y., Bjercke, R.J., Stephan, C.C., Brock, T.A., Williamson, J.R., 1997. Vascular dysfunction induced by elevated glucose levels in rats is mediated by vascular endothelial growth factor. J. Clin. Invest. 99, 2192–2202. Vanhoutte, P.M., 1996. Endothelial dysfunction in hypertension. J. Hypertens. (Suppl. 1), S83–S93. Wink, D.A., Hanbauer, I., Krishna, M.C., DeGraff, W., Gamson, J., Mitchell, J.B., 1993. Nitric oxide protects against cellular damage and cytotoxicity from reactive oxygen species. Proc. Natl. Acad. Sci. USA 90, 9813–9817. Yoneda, M., Maeda, M., Kimura, H., Fujii, A., Katayama, K., Kuriyama, M., 1999. Vasogenic edema on MELAS: A serial study with diffusion-weighted MR imaging. Neurology 53, 2182–2184. Zamora, S.A., Amin, H.J., McMillan, D.D., Kubes, P., Fick, G.H., Butzner, J.D., Parsons, H.G., Scott, R.B., 1997. Plasma L-arginine concentrations in premature infants with necrotizing enterocolitis. J. Pediatr. 131, 226–232. Zhang, F., White, J.G., Iadecola, C., 1994. Nitric oxide donors increase blood flow and reduce brain damage in focal ischemia: evidence that nitric oxide is beneficial in the early stages of cerebral ischemia. J. Cereb. Blood Flow Metab. 14, 217–226.
MELAS and L-arginine therapy Yasutoshi Koga a,*, Yukihiro Akita a, Junko Nishioka a, Shuichi Yatsuga a, Nataliya Povalko a,b, Koujyu Katayama a, Toyojiro Matsuishi a a
Department of Pediatrics and Child Health, Kurume University School of Medicine, 67 Asahi Machi, Kurume, Fukuoka 830-0011, Japan b Research Centre for Medical Genetics RAMNS, Moskvorechie St. 1, Moscow 115478, Russia Received 8 August 2006; accepted 24 October 2006 Available online 5 December 2006
Abstract We investigated the endothelial function in MELAS patients and also evaluated the therapeutic effects of L-arginine. Concentrations of L-arginine during the acute phase of MELAS were significantly lower than in control subjects. L-arginine infusions significantly improved all symptoms suggesting stroke within 30 min, and oral administration significantly decreased frequency and severity of stroke-like episodes. Flow-mediated dilation (FMD) in patients showed a significant decrease than those in the controls. Two years of oral supplementation of L-arginine significantly improved endothelial function to the control levels and was harmonized with the normalized plasma levels of L-arginine in patients. L-arginine therapy showed promise in treating stroke-like episodes in MELAS. Ó 2007 Elsevier B.V. and Mitochondria Research Society. All rights reserved. Keywords: MELAS; Clinical trial; Endothelial function; L-arginine; Stroke-like episodes
1. Introduction Mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) is a maternally inherited multisystem mitochondrial disorder (Pavlakis et al., 1984). Mitochondrial angiopathy, with degenerative changes in small arteries and arterioles, has been reported in many MELAS patients (Ohama et al., 1987; Kishi et al., 1988); these blood vessels have been designated strongly succinate dehydrogenase-reactive vessels (SSVs) (Hasegawa et al., 1991). However, the primary cause for stroke-like episodes in young MELAS patients – whether mitochondrial cytopathy, angiopathy, or both – remains controversial. Although many therapeutic trials have been undertaken to cure mitochondrial disorders, few trials have succeeded in reversing the enzymatic abnormality (Manfredi et al., 2002). Among the successes, several pharmacologic agents have been reported to be effective in MELAS (Kuroda et al., 1997; Penn et al., 1992; Ikejiri et al., 1996; *
Corresponding author. Tel.: +81 942 31 7565; fax: +81 942 38 1792. E-mail address: [email protected] (Y. Koga).
Napolitano et al., 2000). However, no effective therapeutic strategies have focused on the acute stroke phase in MELAS. Based on a hypothesis that stroke-like episodes in MELAS are caused by segmental impairment of vasodilation in intracerebral arteries, we infused L-arginine in MELAS patients during the acute phase of stroke (Koga et al., 2002, 2005). We reported that L-arginine therapy quickly decreased severity of stroke-like symptoms in MELAS, enhanced dynamics of the microcirculation, and also reduced tissue injury from ischemia. Recently, cardioprotective effects of L-arginine and nitric oxide (NO) have been ascribed to endothelial cell preservation (Shiono et al., 2002), decreased neutrophil activation (Kubes et al., 1991), improved coronary blood flow, and reduced free radical-mediated injury (Radomski and Moncada, 1993). Since mitochondria are an important cellular source of reactive oxygen species (ROS), MELAS patients are always at high risk of oxidative stress (Oexle and Zwirner, 1997; Filosto et al., 2002), as they often have respiratory chain dysfunction caused by the genetic abnormality. Many human disorders associated with endothelial dysfunction ultimately lead to arterial occlusive disorders,
1567-7249/$ - see front matter Ó 2007 Elsevier B.V. and Mitochondria Research Society. All rights reserved. doi:10.1016/j.mito.2006.11.006
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including hypercholesterolemia (Ross, 1993), hypertension (Vanhoutte, 1996), diabetes mellitus (Tilton et al., 1997), cardiovascular diseases (Drexler et al., 1992), and homocystinuria (de Groot et al., 1983). Blood concentrations of L-arginine, a precursor of NO in the reaction catalyzed by endothelial nitric oxide synthase (NOS), and/or those of asymmetrical dimethylarginine (ADMA), an endogenous competitive inhibitor of NO synthase, recently have been found to importantly influence endothelial function in hypercholesterolemia (Boger et al., 1998), or ischemic heart diseases (Maxwell et al., 2002), hyperglycemia (Giugliano et al., 1997), pulmonary hypertension (Pearson et al., 2001), and necrotizing enterocolitis (Zamora et al., 1997). In the present study, we examined how L-arginine therapy during the acute phase of stroke-like episodes determines how plasma concentrations of endothelial regulatory factors including L-arginine, NOx (metabolites serving as markers for NO), cyclic GMP, and ADMA differ in patients with MELAS from concentrations in normal subjects. In MELAS patients, we also compared concentrations during the acute stroke phase with those during the interictal phase. In addition, we administered L-arginine intravenously to the patients in a therapeutic trial aiming to improve endothelial-dependent vasodilation, cerebral blood flow as evident by ECD single-photon emission computed tomography (ECD-SPECT), and symptoms suggesting the acute phase of stroke. Finally we evaluated long-term oral administration of L-arginine (6–24 g/day for more than 6 months) for effects on incidence and outcome of stroke-like attacks, cerebral blood flow, and on the endothelial function by FMD analysis. 2. Methods 2.1. Patients Among patients referred to our hospital in Kurume, Japan between April 1, 1992 and April 30, 2003, 24 were diagnosed with MELAS according to clinical findings, muscle histopathologic, and genetic studies. All patients were Japanese who had an A3243G mutation in the mitochondrial tRNALeu(UUR) gene in muscle homogenate as well as in lymphocytes. In 18 of 24 patients, basal growth hormone secretion was low, and growth hormone deficiency was diagnosed by a 0.5 g/kg of L-arginine loading test. Patients with congenital anomalies, sepsis, intravenous hyperalimentation, diabetes mellitus, cardiac failure, or a bedridden state were excluded from this study. 2.2. L-arginine therapy at acute episodes of stroke Twenty-four MELAS patients or patients’ parents gave informed consent, and the L-arginine study protocol was approved by the University Ethics Committee (Kurume University IRB#9715). The study design was chosen because patient availability and finances did not permit a balanced randomized design involving multiple centers.
Among 34 times for stroke-like episodes, patients took part in this study of L-arginine vs. placebo. Beginning within 3 h of the onset of stroke-like symptoms, patients were administered L-arginine (Arugi U, Ajinomoto Pharma, Tokyo, Japan; 0.5 g/kg/dose) as a 10% solution in 24 separate episodes; a placebo (5% dextrose, 0.5 g/kg/dose) in 6 other episodes; and D-arginine (Wako Pure Chemical Industries, Tokyo, Japan) in a 10% solution in the remaining 4 episodes. Each treatment was given intravenously over 15 min during the acute phase of stroke. The following symptoms were evaluated before and at 15 min, 30 min, and 24 h after administration: headache (scored on a scale from 0 for no pain to 3, severe pain), clinical disability (scored from 0 for no disability to 3, severe disability), nausea (present or absent), vomiting (present or absent), and teichopsia (present or absent), as described elsewhere (Lassen et al., 1997). Biochemical determinations in plasma included concentrations of L-arginine, L-citrulline, ADMA, pyruvate, and lactate, as well as nitric oxide metabolites (NOx) and cGMP. In several episodes intracranial hemodynamics were measured using ECD-SPECT (approximate total radioactivity, up to 740 MBq) before and after L-arginine administration. 2.3. Long-term health of MELAS patients with oral supplementation
L-arginine
Six patients were treated by oral administration of to prevent stroke-like episodes (i.e., to maintain the interictal condition). Four to 24 g of L-arginine (Arugi U, Ajinomoto Pharma; 0.15–0.3 g/kg/day) was given orally for 12–18 months. Patients were monitored clinically and biochemically as described above once every 2 weeks. When patients admitted to the hospital for a stroke-like episode, following symptoms were scored: headache (present 1, none 0), vomiting (present 1, none 0), teichopsia (present 1, none 0), convulsion (present 1, none 0) and hemiparesis (present 1, none 0). For each admission during the study periods, these scores were summed as the severity score for the stroke. Frequency of admission was taken to be the frequency of stroke-like episodes. Severity and frequency were related to time as number/month and were compared between periods 18 months before and 12–18 months after oral administration of L-arginine.
L-arginine
2.4. Laboratory measurements Analysis of amino acids was performed on protein-free extracts of fresh plasma using described methods. Plasma concentrations of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO synthase, were measured by high-performance liquid chromatography (HPLC) as previously described (Boger et al., 1997). Plasma or urine concentrations of NO metabolites were detected spectrophotometrically by measuring the final stable equimolar degradation products, nitrite and nitrate (NOx) as described previously (Nims et al., 1996). Plasma cGMPs
Y. Koga et al. / Mitochondrion 7 (2007) 133–139
was measured by radioimmunoassay using a cGMP assay kit (Yamasa, Tokyo, Japan) (Honma et al., 1977). Plasma samples obtained from patients and control subjects were frozen immediately at 20 °C; cGMP was succinylated directly without prior deproteinization and then allowed to be bound by the antibody in an imidazole buffer. 2.5. Endothelial function analysed by FMD FMD analysis was performed in the morning after overnight fasting. With subjects in a supine position, we measured FMD by brachial-artery diameter responses to hyperemic flow (endothelium-dependent), before and 2-h after IV infusion of L-arginine (0.5 g/kg of 10% solution), or placebo administration, with high resolution ultrasonography (SSA-380A, Toshiba, Tokyo), as described previously. The patients’ endothelial function was also measured after 2 years of treatment by oral administration of L-arginine to prevent stroke-like episodes (i.e., to maintain the interictal condition). Four to 24 g of L-arginine (Arugi U, Ajinomoto Co.; 0.15–0.3 g/kg/day) was given orally for at least 24 months. The improvements of distribution of cerebral blood circulation (CBF) were measured by SPMSPECT after IV administration or oral supplementation of L-arginine. We compared FMD measurements in MELAS patients with those of controls using unpaired ttests. We also compared the dates before and after the oral L-arginine supplementation with paired t-tests. All data are presented as means + SD. P values of 0.05 or less were considered significant. 2.6. Intracranial hemodynamics according to SPECT In 6 patients, intracranial hemodynamics were measured using ECD-SPECT (approximate total radioactivity where appropriate, 740 MBq) before and after L-arginine administration. Regional cerebral blood flow (rCBF) was measured in the appropriate region of interest (ROI) compared with the same region on the healthy contra lateral side. 2.7. Statistical analysis Plasma concentrations of amino acids, NOx, and ADMA in patients in the acute stroke phase or interictal phase of MELAS were compared with those in controls using unpaired t-tests, with Bonferroni corrections for outlying values. All data are presented as means + SD. P values of 0.025 or less were considered to indicate statistical significance. Concentrations of L-arginine, L-citrulline, NOx, ADMA, and cGMP in plasma obtained before, 30 min after, and 24 h after L-arginine infusion in patients during the acute phase of stroke were compared with those in controls using paired t-tests with Bonferroni corrections for outlying values. All data are presented as means + SD. P values of 0.025 or less were considered to indicate statistical significance. Statistical analysis of clinical improvement was per-
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formed using Fisher’s exact test; for this the level of significance was set at p < 0.05. Frequency and severity of stroke-like episodes in six MELAS patients after long-term oral L-arginine supplementation were compared with those in patients without supplementation using a nonparametric Mann–Whitney U test. All data are presented as means + SD. Here, P values of 0.05 or less were considered to indicate statistical significance. 3. Results 3.1. Characteristics of patients and controls Ninety-six children, all Japanese were enrolled. Twentyfour were MELAS patients, while 72 were free from neuromuscular and metabolic disorders, serving as controls. All MELAS patients had an A3243G mutation in the mitochondrial tRNALeu(UUR) gene. Clinical onset of disease was at 6.0 ± 4.2 years (means + SD). Of 24 patients, 22 showed dwarfism (stature below 2.5 SD); 15 of these patients had received growth hormone supplementation therapy (0.175 mg/kg/week) at some time before the study. None of the MELAS patients received insulin injection therapy. No significant differences were noted in baseline characteristics of patients and control groups except for height, body mass index (BMI), and biochemical parameters such as amino acids, pyruvate, lactate, and L/P ratio. 3.2. L-arginine, citrulline, NOx, and ADMA in acute and interictal phases of MELAS and controls Concentrations of amino acids were significantly altered in MELAS from those in controls (Table 1). Mean plasma concentrations of L-arginine and citrulline were significantly lower in both acute and interictal phases of MELAS than in controls. Concentrations of L-arginine in the acute phase also were significantly lower than in the interictal phase, while those of citrulline did not show a significant phase-related change. NOx concentrations were significantly lower in the acute phase of MELAS than in controls, while in Table 1 L-arginine, citrulline, NOx, and ADMA in the acute and interictal phase of MELAS and in controlsa L-arginine
Control MELAS Interictal phase Acute phase
Citrulline
NOx
ADMA
108.1 ± 27.6 34.6 ± 8.8
45.4 ± 30.1 0.53 ± 0.15
83.6 ± 25.8 P < 0.01b 46.6 ± 12.7 P < 0.01b P < 0.01c
91.4 ± 44.4 P < 0.01b 24.0 ± 9.8 P < 0.01b P < 0.01c
26.2 ± 9.6 P < 0.01b 23.2 ± 10.2 P < 0.01b NSc
0.42 ± 0.10 P < 0.01b 0.45 ± 0.10 NSb NSc
NS, not significant; NOx, nitric oxide metabolites; ADMA, asymmetrical dimethylarginine. a Plus-minus values are means ± SD. Values are expressed as lmol/l of plasma. b Compared with control. c Compared with interictal phase.
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the interictal phase they were significantly higher than in controls. On the other hand, concentrations of ADMA did not significantly differ between controls and acute-phase patients, although the ADMA/L-arginine ratio was significantly higher in the acute phase than in the controls or in the interictal phase (0.011 ± 0.004, vs. 0.005 ± 0.001, and 0.005 ± 0.001, respectively). 3.3. Effects of L-arginine therapy in the acute phase of stroke-like episodes in MELAS After administration of L-arginine, all symptoms suggesting stroke dramatically improved. Effects on headache, clinical disability, nausea, vomiting, and teichopsia were marked by 30 min after L-arginine administration (Koga et al., 2005). No adverse effects occurred except for headache when L-arginine was infused too rapidly in two patients. Mean arterial pressure after L-arginine treatment reached a minimum (representing a decreased of 10 mm Hg) at 30 min after administration, coinciding with the L-citrulline peak (45.6 ± 14.4 lmol/l). Lactate and pyruvate in plasma were significantly normalized at 24 h after treatment, and becoming comparable to amount present during the interictal phase. Concentrations of NOx and cGMP in treated episodes reached a maximum 30 min after L-arginine administration (55.9 ± 20.4, and 0.86 ± 0.16 lmol/l, respectively), and were significantly greater than with placebo. With treatment, concentrations of L-arginine, citrulline, NOx, cGMP, and ADMA returned to interictal-phase concentrations by 24 h. 3.4. Effects of L-arginine therapy on cerebral blood flow during acute stroke episodes At 60 min after L-arginine infusion, the focal decrease in rCBF in the ischemic region had become less marked severe moderate mild severe moderate mild severe moderate mi l d severe moderate mild sev er e m oderate mild seve re moderate mild
according to SPECT, but the percent increase was less than 13% (12.8 ± 2.9%, means + SD, n = 6) that seen contralaterally. 3.5. Long-term outcome of stroke-like episodes after oral supplementation
L-arginine
After oral L-arginine supplementation, the frequency and severity of symptoms caused by the stroke were decreased dramatically (Fig. 1). Frequency of stroke-like episodes after treatment (0.09 ± 0.09) was significantly decreased than that before supplementation (0.78 ± 0.42) (p = 0.03). Severity score after treatment (0.17 ± 0.18) was also significantly decreased than that before supplementation (2.04 ± 0.34) (p = 0.03). After L-arginine supplementation therapy, no MELAS patient had a major stroke-like attack including hemiconvulsion or hemiparesis, but only headache or teichopsia. Plasma concentrations of L-arginine in MELAS patients ranged from 81.6 to 120.1 lmol/l (91.6 ± 16.8; mean ± SD) after initiation of L-arginine supplementation. 3.6. Endothelial functions in MELAS There was a decrease of FMD in the MELAS patients (104.7 ± 1.8) compared to the controls (112.2 ± 1.7) (p < 0.05). Two hours after L-arginine loading, FMD values in the controls showed no improvement compared to the levels before (112.5 ± 1.2), however, those in MELAS patients (108.1 ± 2.6) improved compared to the levels before (104.7 ± 1.8) (p < 0.05). FMD values after 2 years supplementation of L-arginine (113.1 ± 2.4) were improved compared to the original values (104.7 ± 1.8) (p < 0.05), and became control levels (112.2 ± 1.7) in all MELAS patients (Koga et al., 2006).
Case 1
0.17 0.33/kg
Case 2
0.44/kg
Case 3
0.04 0.09/kg
Case 4
0.08 0.24/kg
Case 5
0.16/kg
Case 6
0.19/kg ARG-U Dose
-24M
-12M
0
12M
24M
Fig. 1. Oral L-arginine supplementation improve the severity and frequency of stroke-like episodes in MELAS. Frequency of stroke-like episodes after treatment (0.09 ± 0.09) was significantly decreased than that before supplementation (0.78 ± 0.42) (p = 0.03). Severity score after treatment (0.17 ± 0.18) was also significantly decreased than that before supplementation (2.04 ± 0.34) (p = 0.03). Plasma concentrations of L-arginine in MELAS patients ranged from 81.6 to 120.1 lmol/l (91.6 ± 16.8; mean ± SD) after initiation of L-arginine supplementation.
Y. Koga et al. / Mitochondrion 7 (2007) 133–139
4. Discussion MELAS, a mitochondrial myopathy, is a progressive disease for which adequate treatment is not available. We investigated the therapeutic effects of L-arginine by intravenous administration during the acute phase of stroke-like episodes and by oral administration during the interictal phase. L-arginine, which plays an important role in endothelialdependent vascular relaxation (Moncada and Higgs, 1993), was significantly lowered in both acute and interictal phases of MELAS than in control subjects (Fig. 2). Plasma L-arginine homeostasis is a complex process affected by arginine intake, arginine consumption, and endogenous arginine production. The latter occurs principally in the kidneys (conversion of citrulline to L-arginine), in the gut (production of citrulline and direct conversion of glutamine to L-arginine), and in the liver (arginine-urea cycles). The significant decrease in plasma L-arginine and NOx in the acute phase of MELAS may be partially explained by limited arginine intake, but, the decreased concentration of L-arginine in the interictal phase cannot be explained solely in this manner. Reduced availability of L-arginine may be induced by reduced activity of the cationic amino acid transporter, increased activity of arginase, and/or reduced activity of argininosuccinate synthase (Cooke and Dzau, 1997). Why plasma L-arginine is decreased in the acute phase of MELAS remains to be elucidated. Considering significantly increased NOx together with significantly decreased L-arginine and citrulline in the interictal phase compared with control subjects, limited L-arginine consumption, increased metabolic demand for arginine, and/ or limited endogenous synthesis may be related to the pathogenesis of MELAS. Concentrations of ADMA, an endogenous competitive inhibitor of NO synthase, were not
significantly different in the acute phase of MELAS from concentrations in controls. ADMA recently has been reported to be a novel risk factor for endothelial dysfunction in humans (Ito et al., 1999). A putative endogenous regulator of NOS activity, ADMA inhibits NO-dependent endothelially mediated vasodilation, this inhibition has been reversed by exogenous arginine in hypercholesterolemia (Ito et al., 1999) and cardiac syndrome X (Piatti et al., 2003). Normally, ADMA is degraded by dimethylarginine dimethyl-aminohydrolase (DDAH) to L-citrulline and dimethylamine, which activity in human endothelial cells was affected by oxidized low-density lipoprotein (oxLDL) or tumor necrosis factor (TNF-a). Since concentrations of total cholesterol and LDL cholesterol did not differ between MELAS and control subjects, the increased ADMA in MELAS is unlikely to be caused by induction of DDAH. However, ADMA and DDAH may provide a mechanism for control of NO synthesis in physiologic or pathologic states. The ADMA/L-arginine ratio was significantly greater in the acute phase than in control subjects or in the interictal phase, suggesting that endothelial-dependent vascular relaxation is impaired in the acute phase of MELAS. A low L-arginine concentration and a relatively high ADMA concentration may predispose to stroke-like episodes in MELAS. Impairment of endothelial function associated with increased ADMA concentrations is reversed by intravenous L-arginine (Piatti et al., 2003). Consistent with these data, we ameliorated the ischemic process during acute phase of MELAS by L-arginine infusion. The primary cause of stroke-like episodes in MELAS remains unknown. Focal cerebral hyperemia has been reported in MELAS by several investigators (Gropen et al., 1994). Although the underlying mechanisms is incompletely understood, the hyperemia is thought to reflect vasodilation caused by local metabolic acidosis in
L-arginine
cytokine glucocorticoids NADPH/NADP
induction
endothelium
RC deficiency
137
L-arginine
NOS
NO
Superoxide
cytokine glucocorticoids
smooth muscle
induction
ADMA NOS
+ NADPH/NADP
L-arginine
Relaxation
L-arginine
sGC NO
+
GTP
Cyclic GMP
Fig. 2. Endothelial function improved by L-arginine supplementation.
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the area of the infarct. Another explanation is that the focal hyperperfusion corresponds to foci of periodic epileptiform discharge, gradually dissipating along with the periodic discharges (Iizuka et al., 2002). However, most cerebral blood flow studies in MELAS patients are performed at several days or several weeks after onset of a stroke-like episode. Several days after onset of stroke, secondarily induced NO production generated by inducible NOS in the region injured may alter evidence of the primary pathophysiologic abnormality. In an analysis of SPECT findings in young MELAS patients at a very early stage of infarction (within 3 h after onset), we invariably found hypoperfusion in the region affected by the strokelike episode (Koga et al., 2005). We cannot explain conclusively why our findings differ from those reported by neurologists treating adults. First, differences may result from timing of examinations. We performed SPECT very early following stroke-like episodes in MELAS, before NO secondarily induced by inducible NO synthase would create further ischemic brain damage. Second, pediatric MELAS patients appear to have more severe and more uniform symptoms than in adult patients. Heteroplasmy of the A3243G mutation in the mitochondrial tRNALeu(UUR) gene is much greater in early-onset MELAS patients (before 15 years) than in late-onset MELAS (late 30 s or older). Third, adult MELAS patients usually acquire additional risk factors for atherosclerosis, such as hypercholesterolemia, hyperglycemia, and hypertension which usually are not seen in young MELAS patients. If the sites of angiopathy in MELAS most likely include small cerebral arteries, arterioles, and capillaries, small infarcts would be expected rather than the large confluent region of infarction described in many reports of MELAS. Yoneda et al. (1999) recently reported finding a contribution from vasogenic edema in stroke-like lesions in a 10-yearold patient with MELAS. Thirty minutes after administration of L-arginine during the acute phase of MELAS, we observed that symptoms suggesting stroke decreased dramatically. Since citrulline, lactate, NOx, cGMP, and ADMA had increased significantly at 30 min after treatment, these increases are considered to be pharmacologic effects of L-arginine treatment. Alleviation of symptoms at 30 min after treatment correlated well with peaks for NOx, cGMP, and citrulline. On SPECT, decreased cerebral blood flow (rCBF) in the ischemic region showed improvement; however, the percent increase was less than 13% (12.9 ± 2.6%: mean ± SD) of the increase on the contralateral side. Since L-arginine has not been found to produce cerebral vasodilation in the region of an old infarct (Sporer et al., 1997), we could not reliably interpret the percent increase in rCBF because the areas studied often included old infarcts. In this study we attempted to prevent or limit ischemic brain damage in the acute phase (within 3 h) of stroke-like episodes in MELAS by giving L-arginine to induce vasodilation. L-arginine is an important precursor of NO, which may reduce ischemic damage in the acute phase of focal brain
ischemia by increasing microcirculation in the cerebral blood flow (Morikawa et al., 1994), inhibiting post-ischemic leukocyte-endothelial adhesion (Gidday et al., 1998), decreasing quantities of hydroxyl radical (Wink et al., 1993), and reducing brain damage in early stages of cerebral ischemia (Zhang et al., 1994). However, details of mechanisms involving L-arginine and NO are not yet fully known. We evaluated long-term outcome of stroke-like episodes after oral L-arginine supplementation. After this therapy, the frequency and severity of clinical symptoms caused by stroke were dramatically decreased without any serious adverse effects (Fig. 1). No prophylactically treated MELAS patients have had major stroke-like attacks such as hemiconvulsion and hemiparesis, but only headache and teichopsia. We concluded that L-arginine supplementation currently is the best interictal therapy for preventing stroke-like episodes in MELAS. Our data indicated that L-arginine infusion during the acute phase significantly enhanced the microcirculation and reduced tissue injury from ischemia, while oral L-arginine supplementation during the interictal phase significantly decreased the frequency and severity of stroke-like attacks in MELAS. Such treatment shows promise in the acute stroke-like phase and in the interictal phase of MELAS. Acknowledgements This work was supported in part by grants #13670853 (Y.K.), and #16390308 (Y.K.) from the Ministry of Culture and Education in Japan, #H14-006 (Y.K.), and #H14-team(syouni)-005 (Y.K.) from Evidence-based Medicine, Ministry of Health, Labor and Welfare in Japan, The research Grant (17A-10) for Nervous and Mental Disorders from the Ministry of Health, Labour ad Welfare in Japan; and Uehara Memorial Foundation. Y.A. is a recipient of grant in aid for young investigator #17790733 from the Ministry of Culture and Education in Japan. References Boger, R.H., Bode-Boger, S.M., Thiele, W., Junker, W., Alexander, K., Frolich, J.C., 1997. Biochemical evidence for impaired nitric oxide synthesis in patients with peripheral arterial occlusive diseases. Circulation 95, 2068–2074. Boger, R.H., Bode-Boger, S.M., Szuba, A., Tsao, P.S., Chan, J.R., Tangphao, O., Blaschke, T.F., Cooke, J.P., 1998. Asymmetric dimethylarginine (ADMA): a novel risk factor for endothelial dysfunction. Its role in hypercholesterolemia. Circulation 98, 1842–1847. Cooke, J.P., Dzau, V.J., 1997. Derangements of the nitric oxide synthase pathway, L-arginine, and cardiovascular diseases. Circulation 96, 379– 382. de Groot, P.G., Willems, C., Boers, G.H., Gonsalves, M.D., van Aken, W.G., van Mourik, J.A., 1983. Endothelial cell dysfunction in homocystinuria. Eur. J. Clin. Invest. 13 (5), 405–410. Drexler, H., Hayoz, D., Munzel, T., Hornig, B., Just, H., Brunner, H.R., Zelis, R., 1992. Endothelial function in chronic congestive heart failure. Am. J. Cardiol. 69, 1596–1601. Filosto, M., Tonin, P., Vattemi, G., Spagnolo, M., Rizzuto, N., Tomelleri, G., 2002. Antioxidant agents have a different expression pattern in
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