J Neural Transm (2000) 107: 1021–1026
Serum levels of coenzyme Q10 in patients with amyotrophic lateral sclerosis J. A. Molina1, F. de Bustos2, F. J. Jiménez-Jiménez3, C. Gómez-Escalonilla1, A. García-Redondo2, J. Esteban1, A. Guerrero-Sola4, P. del Hoyo2, A. Martínez-Salio1, C. Ramírez-Ramos4, G. Ruiz Indurain1, and J. Arenas2 1 Department of Neurology and Department of Biochemistry, Hospital Universitario Doce de Octubre, 3 Department of Medicine-Neurology, University of Alcalá, Alcalá de Henares, 4 Service of Neurology, Hospital Universitario San Carlos, Madrid, Spain 2
Received September 1, 1999; accepted January 4, 2000
Summary. To elucidate whether serum coenzyme Q10 levels are related with the risk for amyotrophic lateral sclerosis (ALS), we compared serum levels of coenzyme Q10 and the coenzyme Q10/cholesterol ratio, in 30 patients with ALS and 42 matched controls using a high performance liquid chromatography technique. The mean serum coenzyme Q10 levels and the coenzyme Q10/ cholesterol ratio did not differ significantly between the 2 study groups. These values were not influenced by the clinical form (spinal vs. bulbar) of ALS, and they did not correlate with age, age at onset, and duration of the disease. These results suggest that serum coenzyme Q10 concentrations are unrelated with the risk for ALS. Keywords: Amyotrophic lateral sclerosis, motoneuron disease, coenzyme Q10, serum levels. Introduction
The etiology and pathogenesis of amyotrophic lateral sclerosis (ALS) is unknown. It has been suggested a role of genetic and environmental factors, autoimmune mechanisms, oxidative stress, excitotoxicity, viral infection, cytoskeletal abnormalities, and loss of trophic support (Coria and Cuadrado, 1996; Louvel et al., 1998). Rosen et al. (1993) described mutations in the Cu/Zn superoxide dismutase (SOD-1) gene in a subgroup of patients with familial ALS (FALS). Since then, there has been identified a number of mutations in the SOD gene which could explain around 20% of FALS (Coria and Cuadrado, 1996; Louvel et al., 1998). These findings suggested a possible role of oxidative stress in the pathogenesis of ALS (Coria and Cuadrado, 1996; Bergeron, 1995).
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Recently, Wiedemann et al. (1998) reported impairment of NADH:CoQoxidoreductase in skeletal muscle from patients with ALS. Coenzyme Q10 (CoQ10) is the electron acceptor for mitochondrial complexes I and II and a powerful antioxidant (Ernster and Dallner, 1995). Oteiza et al. (1997) reported similar plasma coenzyme Q10 levels in 13 patients with sporadic ALS (SALS) compared with 11 controls. The aim of this study was to assess the serum levels CoQ10 in a large series of patients with SALS compared to a control population, in order to elucidate whether low serum levels of this antioxidant could play a role as risk factor for ALS. Patients and methods We studied 30 patients with SALS, according to the criteria developed by the World Federation of Neurology Subcommitte on Motor Neuron Disease at EI Escorial (World Federation of Neurology Research Group on Neuromuscular Disorders, 1994), and 42 age and sex-matched controls. Unselected SALS patients were recruited from outpatients making the first visit to the department of neurology or three hospitals. Informed consent was obtained in each case. The clinical features of both study groups are summarized in Table 1. The following exclusion criteria were applied both to patients and controls: A) Ethanol intake higher than 80 g/day in the last 6 months. B) Previous history of chronic hepatopathy or diseases causing malabsorption. C) Previous history of severe systemic disease. D) Atypical dietary habits (diets constituted exclusively by one type of foodstuff, such as vegetables, fruits, meat, or others, special diets because of religious reasons, etc). F) Intake of drugs which modify lipid absorption. G) Therapy with vitamin supplements in the last 6 months. Venous blood samples were taken from each fasted patient or control between 8.00 and 10.00 a.m. The blood samples were collected on ice and centrifuged. The serum specimens were frozen at 230°C and protected from light exposure with aluminum foil until analysis. The determinations were performed blindly. Serum levels of CoQ10 were determined by high performance liquid chromatography with electrochemical detection. The method used was that of Langedijk et al. (1996) with Table 1. Clinical data and results of ALS patient and control groups ALS-patients n 5 30 Clinical data Age (years) Female Male Age at onset of ALS (years) Duration of ALS (years) Analitical data Coenzyme Q10 levels (nmol/l) Spinal form (n 5 20) Bulbar form (n 5 10) Coenzyme Q10/cholesterol ratio Spinal form (n 5 20) Bulbar form (n 5 10)
61.8 6 14.8 16 14 58.8 6 14.1 2.0 6 1.7 1,098 6 1,211 6 1,043 6 0.21 6 0.23 6 0.19 6
346 368 421 0.07 0.08 0.05
Controls n 5 42 59.7 6 11.0 24 18
1,215 6 376 0.23 6 0.05
Data of quantitative variables are expressed as mean 6 SD. ALS Amyotrophic lateral sclerosis
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some modifications. Since CoQH2 (reduced form of CoQ10) is easily oxidized to CoQ10 (ubiquinone) from the moment of the sample extraction, we evaluated the total CoQ10 (sume of CoQ10 and CoQH2). When samples were extracted, precautions were not taken to prevent oxidation of CoQH2 to CoQ10, so the ratio CoQH2/CoQ10, could not be determined. Sample preparation was the same as Langedijk et al. (1996). The stationary phase was a reverse phase column (HR-80 RP-C18, 80 3 4,6 mm. ESA Inc). The mobile phase was prepared dissolving 7 gr. of NaClO4·H2O in 1,000 ml of methanol/propanol/ HClO4 70%, 700.8:200:0.2 (vol/vol), and the flow rate was set at 0.8 ml/min. The column eluate was monitored with a Coulochem II electrochemical detector fitted to a conditioning cell (model 5010, ESA, Bedford, USA) and a analytical cell (model 5010, ESA, Bedford, USA). The first electrode was set at a potential of 10.5V, while the second and third electrodes were set at 20.5V and 10.3V. Using the electrochemical detector in this mode, we get a high specifity of the detection system and only those hydrophobic compounds that were able to reduce and oxidize at low potentials were detected at the third electrode. The programe and the post-column valve were the same too. The system was enterely controlled by a computer (Kromasystem 2000, Kontron Instruments). Injections were made in a 50 µl injection valve (Model 7161, Rheodyne, Cotaty, USA) with a 100 µl syringe from Hamilton (Bonaduz, Switzerland). In this conditions, the retention times for CoQ10H2 and CoQ10 were 4.1 and 8.3 minutes respectively, but total chromatogram time was 12 minutes because an interfering compound appears at minute 11. The calibration method used ubiquinone as external standard. The within-run coefficients of variation for CoQ10 and CoQH2 were, respectively, 5 and 3.2%, and the day to day precisions were 9.2 and 6.3%. CoQ10 recovery ranged between 88 and 93%. The measurements of CoQ10 were expressed in nmol/l. The determinations of serum cholesterol were carried out in a Hitachi 717 autoanalyzer with in vitro diagnostic kits (Boehringer Mannhein, Mannhein, Germany). The results were expressed as mean 6 SD. The statistical analysis used the Biostatistical Packet of “R-Sigma Data Base” (Horus Hardware) (Moreu et al., 1990), and included the two-tailed student’s t test, ANOVA, and calculation of Pearson’s correlation coefficient when appropriate.
Results
The results are summarized in Table 1. The mean serum levels of coenzyme Q10 and the coenzyme Q10/cholesterol ratio did not differ significantly between SALS-patient and control groups. These values were not influenced by the clinical form (spinal versus bulbar) of ALS. There was no significant correlation in ALS patients between the serum levels of coenzyme Q10 and coenzyme Q10/cholesterol and the following variables: age, age at onset, and duration of the disease. Discussion
Several data suggested a possible role of oxidative stress hypothesis in the pathogenesis of ALS. SOD-1 activity is decreased in brain and erythrocytes of patients with FALS; and it has been reported to be normal or decreased in the brain, normal or decreased in the spinal cord, normal in erythrocytes and decreased in the CSF of patients with SALS (reviewed by Jiménez-Jiménez et al., 1996). Bergeron et al. (1994) found increased SOD-1 mRNA levels in motorneurons. [35S]Glutathione binding sites in spinal cord are increased (Lanius et al., 1993), and basal comsumption rate (QO2), cytochrome c oxidase and catalase activities, and lactate production in lymphocytes of patients with SALS are normal (Curti et al., 1993).
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Serum levels of vitamine E (De Bustos et al., 1998; Iwasaki et al., 1995), beta-carotene, alpha-carotene and retinol (Molina et al., 1999; Oteiza et al., 1997) are normal. Toghi et al. (1995) found decreased cerebrospinal levels of α-tocopherol, and specially of its oxidized form α-tocopherol quinone; however our group found normal cerebrospinal levels of α-tocopherol (De Bustos et al., 1998). Chronic pharmacologic inhibition of SOD1 can produce apoptotic death of spinal motor neurons, but this effect is partially blocked by antioxidants (Rothstein et al., 1994). In addition, vitamin E partially blocks the toxicity of CSF from patients with ALS against neurons in culture (Terro et al., 1996), and delays onset, but does not prolong survival in a transgenic animal model of FALS (Gurney et al., 1996). CoQ10 is well known for its role as electron carrier in the lipid phase of mitochondrial membrane. The low potential required for its oxidation or reduction makes it possible to fulfil its pivotal role in the mitochondrial electron transport chain (Langedijk et al., 1996), particularly in complexes I and II. For this reason it performs a crucial role in the energetic metabolism. CoQ10 is present in all human tissues, it is transported in the circulation by lipoproteins and, as cholesterol, is synthesized by the mevalonate pathway. Plasma cholesterol concentrations are well correlated with CoQ10 (Langedijk et al., 1996). Although the use of lipidlowering drugs was not included among exclusion criteria, only 4 patients and 6 controls were using these drugs. In addition, serum cholesterol concentrations were measured, and the CoQ10/ cholesterol ratio was calculated. The data of the present study show that, when compared with controls, SALS patients had similar serum CoQ10 levels and CoQ10/cholesterol ratios. CoQ10 was not related with the analized clinical features of SALS. Nevertheless, these results do not necessarily reflect the antioxidant state of SALS patients, since serum CoQ10 levels are rather reflecting CoQ synthesis than oxidative state of the body. The normality of serum levels of CoQ10 and CoQ10/cholesterol in SALS patients found in the present study is in agreement with the previous report by Oteiza et al. (1997), and suggests that serum CoQ10 levels are apparently unrelated with the risk for SALS. Acknowledgements This work was supported in part by a grant of the Comunidad de Madrid (Exp. 08.5/0005/ 1997), by the “Fundación Neurociencias y Envejecimiento” and Rhone-Poulenc.
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World Federation of Neurology Research Group on Neuromuscular Disorders (1994) EI Escorial WFN Criteria for the Diagnosis of Amyotrophic Lateral Sclerosis. J Neurol Sci 124 [Suppl]: 96–107 Authors’ address: F. J. Jiménez Jiménez, C/ Corregidor José de Pasamonte, 24 3° D, E-28030 Madrid, Spain, e-mail:
[email protected]
